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ArticleJune 16, 2020

Myelin Basic Protein Phospholipid Complexation Likely Competes with Deimination in Experimental Autoimmune Encephalomyelitis Mouse Model
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Anddre Osmar Valdivia
Anddre Osmar Valdivia
Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miami, Florida 33136, United States
Neuroscience Graduate Program, University of Miami, Miami, Florida 33136, United States
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http://orcid.org/0000-0002-8425-9102
Pratul K. Agarwal
Pratul K. Agarwal
Department of Biochemistry & Cell and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996, United States
Department of Physiological Sciences and High Performance Computing Center and, Oklahoma State University, Stillwater, 106 Math Sciences, Stillwater, Oklahoma 74078-1010, United States
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http://orcid.org/0000-0002-3848-9492
Sanjoy K. Bhattacharya*
Sanjoy K. Bhattacharya
Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miami, Florida 33136, United States
Neuroscience Graduate Program, University of Miami, Miami, Florida 33136, United States
*Email: [email protected]. Tel: (305) 482-4103. Fax: (305) 326-6547. Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami, 1638 NW 10th Avenue, Room 707A, Miami, Florida 33136, United States.
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http://orcid.org/0000-0003-3759-647X

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Cite this: ACS Omega 2020, 5, 25, 15454–15467

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https://doi.org/10.1021/acsomega.0c01590

Published June 16, 2020

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Abstract

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Multiple sclerosis has complex pathogenesis encompassing a variety of components (immunologic, genetic, and environmental). The autoimmunogenicity against the host’s myelin basic protein is a major contributor. An increase in myelin basic protein deimination (a post-translational modification) and a change in phospholipid composition have been associated with multiple sclerosis. The interaction of myelin basic protein with phospholipids in the myelin membrane is an important contributor to the stability and maintenance of proper myelin sheath function. The study of this aspect of multiple sclerosis is an area that has yet to be fully explored and that the present study seeks to understand. Several biochemical methods, a capillary electrophoresis coupled system and mass spectrometry, were used in this study. These methods identified four specific phospholipids complexing with myelin basic protein. We show that lysophosphatidylcholine 18:1 provides a robust competitive effect against hyper-deimination. Our data suggest that lysophosphatidylcholine 18:1 has a different biochemical behavior when compared to other phospholipids and lysophosphatidylcholines 14:0, 16:0, and 18:0.

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1. Introduction

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Multiple sclerosis (MS) is a demyelinating disease that manifests with neurological disabilities that include impaired muscle control, tremors, and visual impairments. (1) This is mainly due to the interruption of signal propagation in different neuronal circuits through the disruption of the myelin sheath. (1) MS is a complex disease with various components contributing to pathogenesis; however, immune reactivity against myelin components seems to be a major contributor. (2−5) Early efforts sought to identify myelin components associated with MS. It was found that interferences in the interaction of myelin basic protein (MBP) with the rest of the myelin components produce immune-reactive epitopes. (2,5−7)

The myelin sheath allows for rapid transduction of nerve impulses through the electrical insulating properties of the plasma membrane. (8) Myelin sheath is composed of two major constituents: proteins and lipids. Among proteins, MBP is the second-most abundant component, whose primary role is to maintain a compact myelin sheath, while lipids provide electrical insulation. There are 14 distinct isoforms of MBP that have been reported in the murine central nervous system (CNS), all of which are the results of different exon compositions of a single MBP gene. (9) The classic family of MBP is the one predominantly expressed in the major dense line of the myelin sheath, and isoform 5 (18.5 kDa) is the isoform primarily associated with MS. The most prominent immune-reactive epitopes found in the cerebrospinal fluid of MS patients are against MBP peptides. (10,11) These peptides have been documented to be selectively produced by proteolytic cleavage by a number of metalloproteases. (12−14) The rate of digestion at which metalloproteases produce these immune-reactive peptides has been directly linked to the levels of deimination (a post-translational modification) in MBP.

Deimination is a post-translational modification mediated by the family of peptidyl arginine deiminases (PADs) that converts protein-bound arginine to protein-bound citrulline. (15) This modification effectively neutralizes the positively charged guanidinium group found in arginine by replacing it with a neutral ureido group. (15−17) Deimination is considered to be an irreversible post-translational modification given that the enzyme responsible for the reverse reaction has not been found. (18) In addition, due to the longevity of deimination, this poses the potential for long-lasting effects on protein regulation.

Studies focused on the structure of deiminated MBP have documented that deimination renders MBP to an open structure, which allows proteases access to internal residues, increasing the rate of digestion and production of immune-reactive peptides. (10,19−22) The elevated levels of MBP deimination have been documented to positively correlate with the severity of MS in human subjects, as well as in the experimental autoimmune encephalomyelitis (EAE) mouse. (6,11,23−29) Subsequent studies demonstrated that native MBP takes a C-shape folded structure upon lipid binding, which resulted in an increase in compaction and stability. (30−32) This highlights the importance that MBP–phospholipid complexation has in maintaining myelin stability.

Lipids in the myelin sheath are arranged in an asymmetric pattern. Sphingolipids are the major class of lipids that face the extracellular surface, and phospholipids are the major class facing the cytoplasmic space. (33) Changes in lipid composition have been documented in human MS brains. Members of phospholipid classes [phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), phosphatidylinositols (PIs), and phosphatidylserines (PSs)] have been reported to increase in relative amounts (relative to the total) for specific lipid species and decrease for others. (34−42) MBP is a cytoplasmic side peripheral membrane protein likely to complex with phospholipids (6,43) and help maintain an ordered myelin sheath.

Although studies have demonstrated that MS pathogenicity is associated with changes in lipid profiles and disruption of MBP structural roles, a study exploring the specific phospholipid interactions that are native to MBP remains to be performed. The effects of MBP–phospholipid complexation on protein deimination also remain to be studied. The aim of the study is to address this gap in the scientific literature, that is, to understand the individual phospholipid that complexes with MBP and plays a potential role toward influencing protein deimination.

2. Results and Discussion

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Demyelinating diseases such as multiple sclerosis converge diverse components resulting in pathogenicity. The immunological events, despite being most well studied, still warrant further intense investigation. To contribute to our understanding of disease pathogenicity, it is crucial to understand the changes in the basic biochemical conditions associated with the myelin sheath. Autoimmunogenicity against host’s MBP is a hallmark of multiple sclerosis. Understanding the native biochemistry can shed light on how the state of antigenicity becomes perturbed in disease conditions to result in autoimmunogenicity.

The animal models available for the study of MS include the toxin-induced models (cuprizone and lysolecithin, useful for understanding demyelination and remyelination processes), viral-induced model (Theiler’s murine encephalomyelitis, useful for understanding axonal damage and inflammation), and chronic EAE model. (44) The EAE mouse model is one of the most characterized models in the literature. The benefits of this model include chronic demyelination and its effects on the central nervous system, allowing for the study of immunological and neuroinflammatory processes.

We present findings that (1) PAD-mediated hyper-deimination is associated with EAE (Figure 1B), (2) there are broad changes of phospholipid composition in EAE (30 days post injection; Figure 2C), (3) there is a differential complexation of phospholipids with MBP in EAE (Figure 3B,C), and (4) lysophosphatidylcholine (LPC) 18:1 likely competes for hyper-deimination of MBP (Figures 4A,B, 5A, and 7A). Our findings in the EAE model are parallel to the reports of phospholipid deficiency in human multiple sclerosis subjects only in the PC class. (34−42) Although there were overall changes in the general classes of phospholipids, we focused on the specific phospholipids that were deficient in the EAE group, thus enabling the selection of a finite number of species that can be pursued with ease. Specific phospholipid species have been reported to be deficient in human MS samples despite increases in the overall class of phospholipids. (34−42)

Figure 1. Myelin basic protein in EAE is deiminated. (A) Representative sucrose density ultracentrifugation fractions of mouse brains probing for myelin basic protein (Abcam-ab7349). *Sucrose fraction 32 (SF 32) from each group was used for further analysis (coomassie, Western blot, mass spectrometry, and capillary electrophoresis (CE)). EAE, n = 15; sham-injection control, n = 10; and no-injection control, n = 10. Each immunoblot was repeated three times. (B) Coomassie blue stain and Western blot for sucrose fraction 32. Bands were excised from coomassie gel and analyzed by mass spectrometry. Western blot probed for levels of deimination (citrulline, Millipore-MABS54887). (C) Mass spectrometry analysis of excised bands from coomassie blue stain gel. PSM = peptide-spectrum match.

Figure 2. Myelin basic protein isolated from EAE has different phospholipid compositions than controls. (A) Representative capillary electrophoresis (CE) chromatogram of sucrose fraction 32 (SF 32). Arrow points toward the peak collected for protein and phospholipid analysis. (B) Dot immunoblot for MBP in CE fractions, 10 min before the peak, peak (as noted by the arrow), and 10 min after the peak. MBP was probed with an anti-MBP antibody (Abcam, ab7349). (C) Phospholipid analysis of CE peak fractions, three replicates, and bars correspond to standard error mean (SEM) (*p < 0.05).

Figure 3. Confirmation of MBP–phospholipid complexation. (A) Structure of candidate lipids: PC 16:0/22:6, 1-((4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoyl)-2-hexadecanoyl-sn-glycero-3-phosphocholine, LMGP01011116; PC 16:1/16:1, 1,2-di-((9Z)-hexadecanoyl)-sn-glycero-3-phosphocholine, LMGP01010684; PI 18:0/20:4, 1-octadecanoyl-2-((5Z,8Z,11Z,14Z)-eicosatetraenoyl)-sn-glycero-3-phospho-(1′-myo-inositol), LMGP06010010; PS 18:0/18:1, 1-octadecanoyl-2-((9Z)-octadecanoyl)-sn-glycero-3-phosphoserine, LMGP03010025; LPC 18:1, 2-((9Z)-octadecanoyl)-sn-glycero-3-phosphocholine, LMGP01050082. (B) Liposome flotation assay (LFA) of candidate phospholipids demonstrating MBP complexation with phospholipids. B, bottom fraction; M, middle fraction; and T, top fraction (MBP, Abcam, ab7349). (C) Protein–lipid overlay assay (PLOA) of candidate phospholipids demonstrating MBP complexation with phospholipids (MBP, Abcam, ab7349).

Figure 4. Complexation of LPC 18:1 with MBP is competitive for MBP hyper-deimination. (A) Dot blot analysis of deimination reaction for MBP (MBP, Abcam-ab7349) and deiminated MBP (citrulline, Millipore-MABS54887) in the presence of candidate phospholipids. PAD, peptidyl arginine deiminase. (B) Deimination fold change analysis of Western blot, three replicates, and densitometry using ImageJ was normalized to MBP (***p < 0.0005, comparison to MBP–PAD). (C) Representative circular dichroism (CD) spectra of deimination reaction for MBP in the presence of candidate phospholipids, three replicates per spectra. (D) CD spectra analysis of secondary structure probability, utilizing CD analysis and plotting tool (CAPITO) software (mean with SEM).

Figure 5. LPC 18:1 competes with hyper-deimination of MBP arginine residues and has differential complexation with MBP. (A) Mass spectrometry analysis of MBP arginine residues that are being deiminated in in vitro deimination assay (Millipore, 13-104, Burlington, MA). The deiminated Myelin basic protein (UniProt accession: P02687) peptides were identified by mass spectrometry (see Table 3). Gray highlight, deiminated arginine; red bold square, arginine not deiminated in LPC 18:1 reaction (residues 63, 112, and 129). (B) Protein–lipid overlay assay of LPC 18:1, LPC 14:0, LPC 16:0, and LPC 18:0 MBP was probed with an anti-MBP antibody (Abcam, ab7349).

2.1. Confirmation of the Experimental Autoimmune Encephalomyelitis (EAE) Mouse Model

To confirm that the multiple sclerosis mouse model utilized for this study (EAE) exhibited its characteristic traits, animals were clinically scored to monitor the progression of EAE (Figure S1A). The clinical score graph demonstrated that animals maintained an elevated clinical score and corresponded to the chronic phase of disease progression (day 30). Additionally, visual impairment was monitored by measuring the inner retinal function (retinal ganglion cell activity) using pattern electroretinogram (PERG). The PERG measurements demonstrated that 30 days post immunization, EAE mice exhibited a decrease in amplitude when compared to the no-injection control and sham-injection control groups (Figure S1B). Flash electroretinogram (FERG) was utilized to determine if the photoreceptor activity was contributing to visual impairment. The FERG demonstrated no change in any of the groups, suggesting that visual impairment was mediated through the inner retina (data not shown). To eliminate the possibility that an aberrant retinal morphology was contributing to visual decline, optical coherence tomography (OCT) was utilized and demonstrated no change in retinal thickness (Figure S1C). Immunohistochemical analysis of optic nerves demonstrated that EAE mice were suffering from optic nerve demyelination as it was observed by a decrease in the MBP signal and topographical distribution (Figure S1D). The blood antibody analysis specific for myelin oligodendrocyte glycoprotein (MOG) further corroborated that experimental animals exhibited characteristic traits of the EAE mouse model (Figure S1E). Thus, our EAE animals demonstrated all clinical neurological deficits, as well as visual function impairment, in parallel to that in human MS.

2.2. Isolation of the MBP–Phospholipid Complex and Deimination in EAE

Several studies have highlighted the importance of MBP–phospholipid complexation; however, the specific lipid species that complex with MBP have not been explored in detail. (10,19−22,30−32) The isolation of intact complex using inert biochemical methods is the first critical step toward the identification of native MBP–phospholipid complex constituents. The inert methods preserve the intermolecular interactions within the complex. From confirmed EAE mice, we collected neuronal tissue (brain) and subjected the sample to homogenization and fractionation through sucrose gradient ultracentrifugation. Sucrose fractions were then evaluated using immunoblotting for the identification of the fractions containing MBP (Figure 1A). The distribution of MBP was rather uniform throughout many different fractions (fractions 14–36; Figure 1A). Sucrose fraction 32, taken as a representative, was analyzed for verification of the presence of MBP and levels of deimination. Coomassie staining demonstrated the same pattern of MBP within the gel as observed in the Western blot analysis (Figure 1B, upper panel); the latter also showed an increase in levels of deimination in the EAE sucrose fraction (Figure 1B, lower panel). Bands from coomassie gel were excised and analyzed, verifying the presence of MBP in the fractions (Figure 1C). Sucrose fraction 32 (SF 32) from all groups was further fractionated through capillary electrophoresis (CE). The CE electropherogram displayed large peaks, as indicated by the arrows. The peaks did not demonstrate a constant retention time; however, they typically eluted between 55 and 65 min (Figure 2A). Peaks were collected as well as the eluents 10 min before and after the peak (each for 10 min duration). They were subjected to dot immunoblot analysis using the antibody against MBP. Results demonstrated the presence of MBP in the peak but not in the other fractions, confirming the presence of MBP and its constituents in the peaks (Figure 2B). We therefore collected MBP complexes at the end of CE fractionation.

2.3. MBP–Phospholipid Complexation

Mass spectrometric lipidomic analysis of the CE peak fractions demonstrated a decrease in phosphatidylcholines (PCs), no change in phosphatidylethanolamines (PEs), and an increase in phosphatidylinositols (PIs) and phosphatidylserines (PSs) in the EAE group when compared to that of control groups (Figure 2C). The following criteria were used to identify candidate phospholipids that potentially complex with MBP: (1) phospholipids had to be present in both the no-injection control and sham-injection control groups but (2) absent in the EAE group. We found five candidate lipids that met these criteria (Table 1 and Figure 3A).

Table 1. Candidate Phospholipids after CE^a

lipid molecule	calculated mass
PC 16:0/22:6	805.5622
PC 16:1/16:1	729.5309
PI 18:0/20:4	858.5258
PS 18:0/18:1	789.552
LPC 18:1	521.3481

^{^a}

Candidate phospholipids that are present in the noninject and sham groups but absent in the EAE group.

To corroborate their direct complexation with MBP, the candidate phospholipids were analyzed using the liposome flotation assay (LFA) and protein–lipid overlay assay (PLOA) (Figure 3B,C). For this purpose, liposomes/micelles were prepared using extrusion and were confirmed for being within the appropriate size range, using nanoparticle tracking analysis. (45) The assay revealed that there are various degrees of complexation that exist between MBP and candidate phospholipids, as revealed by the movement of MBP to either the middle or top layer (Figure 3B).

The PLOA analysis demonstrated and confirmed the same pattern observed in the LFA (Figure 3C). However, PC 16:0/22:6 consistently presented difficulties adhering to the membrane during dot immunoblot in the PLOA and was not pursued for further analysis (Table 2). The PLOA demonstrated that LPC 14:0, LPC 16:0, and LPC 18:0 had equal complexation to MBP between the control groups and EAE group. This contrasts to the complexation of LPC 18:1, which showed a difference between MBP derived from controls and EAE (Figure 5B).

Table 2. Curated Phospholipids after LFA and PLOA^a

lipid molecule	calculated mass
PC 16:1/16:1	729.5309
PI 18:0/20:4	858.5258
PS 18:0/18:1	789.552
LPC 18:1	521.3481

^{^a}

Lipids demonstrating direct complexation with MBP. Liposome flotation assay (LFA) and protein–lipid overlay assay (PLOA).

Comparison of phospholipids deficient in the EAE with the control groups helped identify and validate four phospholipids that complex with MBP, of which PC 16:1/16:1 and LPC 18:1 have been reported to be deficient in humans (Figure 3 and Table 2). (40,42)

Lipids in an aqueous environment can attain different geometries due to their hydrophobic tails and hydrophilic head groups. The packing parameters (P) dictate that the geometry lipids will undertake in an aqueous environment. (46,47) Phospholipids (P ∼ 1) and lysophospholipids (P < 1) exhibit a cylindrical and an inverted cone geometry, respectively. In both of these species, the hydrophilic head group will face the outer shell of the liposome (phospholipids) or micelle (lysophospholipids). The myelin sheath mimics the same geometry, that is, the head group is facing the cytoplasmic space and interacting with MBP. Only lipids with a P > 1 (for example, dioleoylphosphocholine) will exhibit an inverted micellar geometry (our study did not identify any such lipid species).

The variable degree of travel observed in the LFA for specific MBP–phospholipid complexes can be accounted for by either the strength of the MBP–phospholipid complexation or the presence of liposomes/micelles of smaller sizes. The smaller-sized liposomes/micelles may fail to move MBP to the top layer due to a lack of attaining sufficient buoyancy. PC 16:0/20:4 was used as a control phospholipid to PC 16:0/22:6. This control was used to determine if reducing the length and saturation of the hydrocarbon chain affected complexation with MBP (Figure S2A,B). LFA and PLOA for PC 16:0/20:4 demonstrated the same pattern observed with PC 16:0/22:6, suggesting that small variations in the length and saturation of the hydrocarbon chain do not affect MBP complexation. Similarly, PC 18:1/18:1 and LPCs (14:0, 16:0, and 18:0) were used as controls for LPC 18:1. These controls were used to determine if the absence of a second hydrocarbon chain and variations in the hydrocarbon chain length affected MBP complexation (Figures S2C,D and 5B). LFA and PLOA demonstrated that PC 18:1/18:1 did not complex with MBP, suggesting that the number of hydrocarbon chains has an effect in complexation with MBP. PLOA of other LPCs demonstrated no preference for MBP complexation derived from controls or EAE group.

2.4. LPC 18:1 Likely Competes for Hyper-deimination of MBP

EAE sucrose fraction demonstrated an increase in deimination (Figure 1B, lower panel); hence, the effects of phospholipid complexation with MBP were analyzed for their competitive effects against hyper-deimination. We present evidence that LPC 18:1 competes for PAD-catalyzed hyper-deimination of arginine residues in MBP when compared to the other phospholipids (Figures 4A,B and 5A). In the present study, LPC 18:1 was identified to be present in healthy, but absent in chronic EAE mice. The absence in EAE suggests that endogenous LPC 18:1 can potentially have important biological functions in healthy animals. The complexation of LPC 18:1 is likely to be beneficial for MBP stability, preventing MBP from further deimination when challenged with PAD. It is likely that LPC 18:1 complexation occurs prior to deimination and thus prevents hyper-deimination. In normal humans, MBP is known to be deiminated in 6 of a total of 19 arginine residues (UniProt accession: P02686-5, in MBP isoform 5; Figure S5). However, deimination in MBP isoform 5 goes beyond these six sites in MS and/or in demyelinating diseases. (48) In other words, hyper-deimination is prevented in normal conditions but occurs only in demyelinating diseases/MS. (6,10,24,28,49) The more severe the demyelinating disease, the more intense the hyper-deimination. (23) In mouse and bovine, four and eight residues are found deiminated in normal or wild-type animals, respectively. The number of sites (beyond four and eight, respectively) increases in mouse and bovine in demyelinating disease conditions consistent with hyper-deimination in humans (Figure S5).

The complexation of LPC 18:1 with MBP is partial in vitro as demonstrated by LFA of MBP in the middle layer and the partial signal in the PLOA, but more complete complexation in vivo cannot be ruled out. When LPC 18:1–MBP complex is challenged by PAD, only nine arginine residues are deiminated (one more residue compared to eight endogenous residues in MBP). In contrast, all other LPCs (14:0, 16:0, and even 18:0) and other phospholipids demonstrated deimination of 13 and 11–13 arginine residues, respectively (Figure 5A and Table 3). This supports the hypothesis that MBP–LPC 18:1 complexation prevents hyper-deimination of arginine residues by PAD (Figure 7B,C). LPC 18:1 complexation with MBP derived from the EAE group (Figure 3B,C) is consistent with the availability of strong binding sites (vacant sites) for LPC 18:1 in hyper-deiminated MBP. In vivo, the EAE animals (or human MS CNS samples) are deficient in LPC 18:1, leaving such binding sites vacant in MBP derived from these sources. This is consistent with our CE experiments, where we found a lack of bound LPC 18:1 with the EAE-derived MBP (Table 1). LPC 18:1 confers protection against hyper-deimination, contrasting other LPCs (14:0, 16:0, and 18:0). These LPCs are the ones frequently used for demyelination (at or beyond 100 μM, they induce toxicity). (50−53) A comparative study of the degree of demyelination caused by various LPC species remains to be performed.

Table 3. Arginine Residues Deiminated in MBP–Lipid Challenge Assay^a

lipid molecule	total deiminated cites	protein coverage (%)	arginine residue deiminated^b
MBP + PAD + PC 16:1/16:1	13	85.80	41, 47, 52, 63, 78, 96, 106, 112, 129, 158, 161, 168, 169
MBP + PAD + PI 18:0/20:4	12	85.80	47, 52, 63, 78, 96, 106, 112, 129, 158, 161, 168, 169
MBP + PAD + PS 18:0/18:1	11	85.80	52, 63, 78, 96, 106, 112, 129, 158, 161, 168, 169
MBP + PAD + LPC 18:1	9	94.08	47, 52, 78, 96, 106, 158, 161, 168, 169
MBP + PAD + LPC 18:0	13	92.90	23, 47, 52, 63, 78, 96, 106, 112, 129, 158, 161, 168, 169
MBP + PAD + LPC 16:0	13	92.90	23, 47, 52, 63, 78, 96, 106, 112, 129, 158, 161, 168, 169
MBP + PAD + LPC 14:0	13	92.90	23, 47, 52, 63, 78, 96, 106, 112, 129, 158, 161, 168, 169
MBP + PAD + no lipid	12	94.08	47, 52, 63, 78, 96, 106, 112, 129, 158, 161, 168, 169
MBP + No PAD + no lipid	8	94.08	52, 78, 96, 106, 158, 161, 168, 169

^{^a}

Analysis of arginine deimination of myelin basic protein (MBP). For chymotrypsin-digested MBP (UniProt accession number: P02687; Millipore, 13-104, Burlington, MA), the total number of spectrum matches were 1756 and 20 unique peptides, which were identified by mass spectrometry. The deimination cites had a 99–100% confidence. LPC 18:1 incubation protected residues 63, 112, and 129 from deimination.

^{^b}

The sequence positions are in reference to canonical MBP isoform 1 (UniProt accession number: P02687). See Table S1 for more details.

2.5. Structural Analysis of LPC 18:1–MBP Complex

Sequence analysis-based computer methods and full-atomic-level molecular dynamics (MD) simulations are consistent with MBP being an intrinsically disordered protein (IDP). MBP exhibiting a disordered structure (Movie S1 and Figure S3) is supported by previous literature reports. (43,54,55) This was further corroborated by circular dichroism (CD) studies, which demonstrated that despite MBP being an intrinsically disordered protein (IDP), it retains a partial secondary structure. The CD showed altered spectra when MBP was denatured at 105 °C (Figure 4C, top left panel) compared to that at 20 °C. The LPC 18:1 binding does not cause any change in the secondary structure (Figure 4C, bottom left panel). In hyper-deiminated MBP, the loss of secondary structure (Figure 4C, top right panel) is quite pronounced (Figure 4C, top right panel), as expected. However, the loss of secondary structure in hyper-deiminated MBP is somewhat protected (not statistically significant) when complexed with LPC 18:1 (Figure 4C, top right panel and bottom left panel, respectively).

Computer simulations indicated that MBP is a highly flexible protein that shows a variable conformational landscape, a behavior characteristic of IDPs (see Movies S1–S3 in the Supporting Information). MD simulations indicated that the presence of lipids could change the level of deimination of arginine residues due to accessibility by PAD. This change could be due to a combination of two possible factors. The first factor is the presence of the arginine residues on the MBP surface rendering accessibility by PAD. The presence of lipid molecules around the arginine residues could make them inaccessible to PAD. The second factor is the overall conformation of the protein, which is altered by the presence of the lipid. In the extended conformation of MBP, more arginine residues are accessible or present on an accessible surface (Figure S4). The water-only simulations indicated that the arginine residues are solvent accessible (located on the surface) and could be easily accessed by PAD (Figure 6). The alternate case would be when these residues are not accessible to the solvent due to salt linkages or being buried inside the protein. In the LPC 18:1 simulations, the lipid molecules surround the arginine residues, possibly making it difficult for deimination by PAD to occur.

Figure 6. Change in MBP conformations and solvent accessibility of arginine residues. Representative structures are shown from simulations only in water, water with LPC, and water with PC. Helices are shown in red, β-strands in yellow, three N-terminal residues are colored green, and three C-terminal residues are colored cyan. Arginine residues are shown as green/blue sticks, while the lipid is shown as magenta/blue/orange sticks. Water was included in the simulations but omitted here for visualization purposes. In the case of water-only, the arginine residues are exposed on the surface; the presence of LPC protects a number of these residues, and in the presence of PC, MBP adopts an extended conformation, exposing several arginine residues to the solvent.

Figure 7. Hypothesis model of MBP–phospholipid complexation. (A) Working hypothesis for membrane disruption, incorporating key events associated with unstable myelin. The order of events has yet to be determined. (B) Model of phospholipid (PC 16:1/16:1, PI 18:0/20:4, PS 18:0/18:1, LPC 18:0, LPC 16:0, and LPC 14:0) competitive effects against PAD-mediated hyper-deimination and potential cascade of events. (C) Model of LPC 18:1 competitive effects against PAD-mediated hyper-deimination. Δ, “change in”; MBP, myelin basic protein; and PAD, peptidyl arginine deiminase. Open and C-shaped conformations are consistent with the previously published reports.

The behavior in the PC simulations is significantly different. First, in the presence of PC, there are several more arginine residues that appear to be solvent accessible. Second, there is a change in the structure of MBP. The averaged radius of gyration of MBP in the presence of PC is 19.1 Å, compared to 18.2 Å in the case of water-only simulations and 18.7 Å in the presence of LPC 18:1. The more extended conformations in the presence of PC allow more arginine residues to be accessible by other biomolecules, including PAD. This further supports that steric hindrance provided by LPC 18:1 competes against PAD-mediated hyper-deimination.

3. Conclusions

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Together, the data presented in this study demonstrate that LPC 18:1 undergoes complexation with MBP. This complexation prevents hyper-deimination when challenged with PAD. The severity of MS has shown strong and positive correlation with hyper-deimination of MBP. (6,10,23,24) Hyper-deiminated MBP is more easily unfolded (10) and becomes vulnerable to autolysis (56) and proteolysis, (22) resulting in the release of MBP peptides, which in turn triggers autoimmunogenicity. (10,28,57) Our findings reported here expands our understanding of the biochemistry associated with the previously recorded molecular events in MS pathogenicity (Figure 7).

4. Experimental Section

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4.1. Generation of Experimental Autoimmune Encephalomyelitis (EAE) Model

All animals were housed at the McKnight vivarium, University of Miami, FL, and animals utilized in this study were under the University of Miami Institutional Animal Care and Use Committee-approved protocols (IACUC protocol number: 16-235). Method was adapted from other studies. (58) Immunizing neuroantigen was prepared by the emulsion of 2 mg of myelin oligodendrocyte glycoprotein (MOG35-55) (Biosynthesis Inc., 12668, Lewisville, TX), incomplete Freund’s adjuvant (VWR, 90003-748, Radnor, PA), and 6 mg of Mycobacterium tuberculosis (H37RA, Fisher Scientific, DF3114-33-8, Hampton, NH). This stock solution was used to immunize six animals, in which each individual animal received 0.333 mg of myelin oligodendrocyte glycoprotein (MOG35-55) and 1 mg of M. tuberculosis. Pertussis toxin (Biological Laboratories, 181, Campbell, CA) was prepared at a concentration of 50 μg/mL in sterile phosphate-buffered saline (PBS), pH 7.4, with an intraperitoneal (i.p.) administration of 200 ng per injection. Female C57Bl6/J mice were injected i.p. with pertussis toxin the day before injection. They were then immunized subcutaneously (s.c.) at 2 months of age with 200 μL of neuroantigen (MOG35-55) followed by an i.p. injection of pertussis toxin 2 days post immunization. Three groups were created for this study, an immunized group with MOG35-55 (EAE, 15 animals), an immunized group without MOG35-55 (sham-injection control, 10 animals), and an injection control that received no injections (no-injection control, 10 animals). Animals were monitored for decline in body weight as well as manifestation of clinical signs indicative of encephalomyelitis following the same criteria as previous studies. (58)

4.2. Electroretinogram Recordings

Pattern electroretinogram (PERG) and flash electroretinogram (FERG) were recorded utilizing the Jörvec PERG system (Jörvec PERG Visual Stimulation Box, M014760L, Miami, FL). The experimental setup for FERG was as described in previously published protocols; however, there was a slight modification for the experimental setup for PERG. (59) The modification consisted of the subcutaneous insertion of the recording electrode between the two eyes, the reference electrode in the scalp and the ground electrode in the lower back adjacent to the tail. For both PERG and FERG, animals were anesthetized by the i.p. injection of 100 μL of ketamine and xylazine cocktail (1.5 mg/0.3 mg per 100 μL) per 20 g of body weight. A drop of the balanced saline solution was delivered to the eye to prevent dryness for the duration of the procedure. Animals were situated in a heating pad (Physitemp TCAT-2LV controller) with an anal reference thermometer set at 37 °C to stabilize the internal body temperature. PERG was recorded by three consecutive responses of 600 contrast reversals, and recording settings consisted of 10.0 K gain, 1.0 Hz high pass, 100.0 Hz low pass, and 360.0 μV rejection. FERG was recorded by three consecutive measurements of a flash, with a strength of 20.0 CDS/SM and a frequency of 1.0 Hz. Amplitude for PERG and FERG was measured as the difference in microvolts between the highest peak and the consecutive lowest trough. Latency for PERG and FERG was measured as the time (milliseconds) it took to reach the highest peak (from time zero to the time when the highest peak was recorded).

4.3. Optical Coherence Tomography (OCT)

Animals were anesthetized as described above, followed by the application of one drop of tropicamide ophthalmic solution (Akorn, 17478-102-12) to dilate the pupil and a drop of the balanced saline solution to prevent eye dryness. A 3.5 mm diameter mini contact lens (Ocuscience, ERGACC3) was placed for the visualization of retina. OCT images were taken with the Engineering Spectralis HRA + OCT (Franklin, MA) and analyzed with the Spectralis HRA + OCT software version 6.6.

4.4. Enzyme-Linked Immunosorbent Assay (ELISA)

Animals were anesthetized as described above, followed by exposure of the thoracic cavity. Using a 26G1/2 needle, about 1 mL of blood was aspirated from the left atrium. Animals were immediately decapitated to ensure euthanasia and prevent recovery from anesthesia. The blood was treated with 0.25 mL of anticoagulant citrate dextrose solution per 1 mL of blood. Blood was layered over 2 mL of Ficoll-Paque PLUS, a density of 1.077 g/mL (GE Healthcare, 17-1440-02), and centrifuged at 1800g for 35 min. Plasma was collected and store at −80 °C. Utilizing the AnaSpec Anti-MOG35-55 Quantitative ELISA kit (VWR, AS-54465) and following the manufacturer’s protocol, antibodies against MOG35-55 were detected and normalized to the total protein concentration in isolated blood plasma.

4.5. Immunohistochemistry

Dissected optic nerves were immersed fixed in 4% paraformaldehyde in PBS and incubated overnight at 4 °C. Tissue was washed using PBS, followed by cryoprotection using a gradient of 10% sucrose (overnight at 4 °C), 20% sucrose (overnight at 4 °C), and 30% sucrose (overnight at 4 °C). Tissue was then embedded in an optimum cutting temperature compound (VWR, 25608-930) and stored at −80 °C. Sections were collected at 10 μm thickness and stored at −80 °C. Immunohistochemistry standard protocol was used to detect MBP (Abcam, ab7349) and CD90 (CYMBUS Biotech, CBL1354), and the tissue was mounted using Vectashield with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, H-1200).

4.6. Tissue Preparation and Density Ultracentrifugation

Method was adapted from other studies. (60) Brains from 30 days postinjection mice (8–10 brains, ∼3.5 g total weight, when they manifested clinical signs indicative of encephalomyelitis) were rapidly dissected, flash-frozen by liquid nitrogen immersion followed by equilibration on dry ice, and stored at −80 °C. Tissue was homogenized at 4 °C utilizing 0.32 M sucrose solution with a protease inhibitor (5% w/v homogenate). Gradient I was established by layering the homogenate (18 mL) over 18 mL of 0.85 M sucrose at 4 °C and ultracentrifuged at 140 000g at 4 °C for 1 h (Beckman Rotor SW28). The low-density band was discarded, and the main band along with the dispersion band was collected. They were homogenized and diluted to 200 mL, utilizing a 10 mM ethylene glycol tetraacetic acid (EGTA) solution followed by centrifugation at 141 000g at 4 °C for 40 min (Beckman Rotor SW28). The supernatant was discarded, and the pellet was resuspended in 120 mL of 10 mM EGTA solution, homogenized, and ultracentrifuged at 35 000g at 4 °C for 15 min (Beckman Rotor SW28). This step was repeated once. The supernatant was discarded, and the pellet was resuspended in 18 mL with 0.85 M sucrose at 4 °C. Gradient II was established by layering from bottom to top over 2 mL of 1 M sucrose, 18 mL of 0.85 M sucrose-containing pellet, and 18 mL of 0.32 M sucrose at 4 °C followed by ultracentrifugation at 140 000g at 4 °C for 1.5 h (Beckman Rotor SW28). The low-density band was discarded, and the main band along with the dispersion band was collected followed by processing as described above; however, the pellet was finally resuspended in 13 mL with 0.85 M sucrose solution. Gradient III was established by layering from bottom to top over 12 mL of 0.85 M sucrose-containing pellet, 12 mL of 0.75 M sucrose, and 13 mL of 0.32 M sucrose at 4 °C followed by ultracentrifugation at 14 0000g at 4 °C for 16 h (Beckman Rotor SW28). Fractions were collected in separate tubes (1 mL each, 37 fractions total) along with the pellet. Fractions were washed with 2 mL of 2 mM EGTA solution followed by centrifugation at 18 000g 4 °C for 25 min (Beckman Rotor GH-3.8). The supernatant was discarded, and the pellets were resuspended in 1 mL of 50 mM Tris solution with a protease inhibitor and stored at −80 °C.

4.7. Gel Electrophoresis and Immunoblotting

Fractions from density centrifugation were heated to 85 °C for 3 min and diluted using Laemmli loading buffer (Amresco, M337). Equal volume of samples was loaded in 4–20% Tris–glycine gel (Bio-Rad, 5671095) to maximize the protein content in lower fractions and ran using Tris–glycine–sodium dodecyl sulfate (SDS) running buffer at 200 V for 33 min. Poly(vinylidene difluoride) (PVDF) membrane was activated with methanol and washed with ultrapure water and with transfer buffer (TGS-20% methanol) before transfer. Semidry transfer was carried out using the Trans-Blot Turbo Transfer System (Bio-Rad, 1704150). Membrane was blocked with a blocking buffer (Bio-Rad, 170-6404) and probed for either MBP using antibodies (Abcam, ab7349) at a 1:500 dilution or deiminated MBP (Cit-MBP, Millipore, MABS54887) at a 1:500 dilution. Membrane was washed using 25 mM Tris, 2.6 mM KCl, 0.14 M NaCl, 0.2% Tween-20, at pH 8, and chemiluminescence was detected using Western blotting substrate (Thermo, 32106). Dot immunoblotting was performed as described, with the exception that samples were not separated by gel electrophoresis. A modification to this protocol was made when detecting deiminated MBP by modification of the citrulline residues through a reaction with 2,3-butanedione monoxime and antipyrine in a strong acid solution (Millipore, 17-347B), which was performed immediately after transfer, and modification was incubated at 37 °C for 3 h.

4.8. Capillary Electrophoresis (CE)

After confirming the presence of MBP, fraction 32 was selected for further analysis. Fractions were separated using the Agilent Capillary Electrophoresis 7100 coupled with a bare fused silica capillary tube (Agilent, G1600-63311). (61) CE buffer consisted of 100 mM formic acid, 5 mM ammonium acetate, and 100 mM Tris at a pH of 7.3. The sample was prepared at a 1:10 ratio of density centrifugation fraction to CE buffer. Parameters for the CE system included injection at 50 mbar for 10 s, high voltage at 30 kV, 25 μA and 6 W, a cassette temperature of 25 °C, coupled with a high-pressure system of 10 mbar for the duration of the separation (70 min). Signal was detected using UV absorbance at 230 nm (bandwidth 4 nm) and a reference at 350 nm (bandwidth 50 nm). Fractions were collected in separate tubes in intervals of 10 min.

4.9. Lipid and Protein Extraction

Capillary electrophoresis fractions were centrifuged at 18 000g at 4 °C for 1 h (Beckman Microfuge 18). The supernatant was discarded, and the pellet (very small) was resuspended in methanol with 0.516 mg/mL butylated hydroxytoluene (BHT). The resuspended pellet was transferred to a glass tube to minimize contact with plastic, and methyl tert-butyl ether (MTBE) was added, maintaining a 10:3 ratio per volume of MTBE/methanol. Extraction was left shaking overnight (∼16 h) at 4 °C. Extractions were treated with 0.15 M ammonium acetate for phase separation while maintaining a 20:6:5 ratio per volume of MTBE/methanol/ammonium acetate. Extractions were centrifuged at 2000g at 4 °C for 10 min (Thermo Fisher Megafuge 8R), and the upper organic phase was collected in a separate glass tube. Extraction containers were washed with a 20:6:5 ratio per volume of MTBE/methanol/ammonium acetate, centrifuged, and the organic phase was added to the first collection. Organic phase samples were completely dried, utilizing speed vacuum at a temperature of 37 °C; the dried samples were stored at −80 °C and resuspended in 1:1 chloroform/methanol for mass spectrometry analysis. The aqueous phase was used for liposome flotation assay and protein–lipid overlay assay.

4.10. Sample Preparation for Protein Mass Spectrometry

Fifteen micrograms of total protein from the aqueous phase sample was added to four times the volume of acetone at 4 °C and incubated at room temperature for 15 min. Samples were centrifuged at 21 000g at 4 °C for 30 min (Thermo Fisher Megafuge 8R). The supernatant was discarded, and the pellet (very small) was air-dried for 10 min. The pellet was resuspended and reduced with 10 mM dithiothreitol in 100 mM ammonium bicarbonate for 45 min at room temperature. Following reduction, the resuspended pellet was alkylated with 55 mM iodoacetamide in 100 mM ammonium bicarbonate for 30 min while maintained in darkness. Two times the volume of acetone at room temperature was added and centrifuged at 21 000g at room temperature for 15 min (Thermo Fisher Megafuge 8R). The supernatant was discarded, and the pellet (very small) was air-dried for 10 min. The pellet was resuspended in 0.1 μg/μL of chymotrypsin (Promega, V106A) in 15 mM 4-ethylmorpholine and incubated overnight (∼16 h) at 37 °C. Samples were centrifuged at 21 000g at room temperature for 5 min (Thermo Fisher Megafuge 8R) and dried, utilizing speed vacuum at a temperature of 40 °C, leaving about 50 μL of sample. Samples were stored at −80 °C.

4.11. High-Performance Liquid Chromatography (HPLC)–Mass Spectrometry

Mass spectrometry lipidomic chromatography and identification were carried out as described in detail in our previous publication. (62) In brief, samples were run through an Acclaim C30 column (particle size 3.0 μm, 150 × 2.1 mm² ID; Thermo Fisher Scientific, Waltham, MA). An HPLC Accela instrument (equipped with an autosampler and a 600 pump) was coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific). Heated electrospray ionization (HESI) was used as the method of ionization by coupling a HESI probe to the Q-exactive instrument. The instrument was set at full scan with a collision energy of 30 and 19 eV in positive mode. Lipids were identified using LipidSearch software version 4.1, developed by Ryo Taguchi and Mitsui Knowledge Industry Co. (Tokyo, Japan). Mass spectrometry proteomics was performed on a Q-Exactive instrument after fractionation on a coupled Easy nLC 1000 nano-liquid chromatography system (Thermo Fisher Scientific), as described in our other published reports. (63) Peaks were generated using a Thermo Scientific Xcalibur (version 4.1.31.9, released 2017); proteins were identified using Proteome Discoverer 2.2 (version 2.2.0.388, released 2017). UniProt sequence database was used for the identification of proteins (downloaded July 2019). Proteome Discoverer search parameters for chymotrypsin-digested enzymes (max missed cleavage sites: 2, min peptide length: 6, max peptide length: 144), post-translational modification: deimination (+0.984 Da (R), max modification per peptide: 3), precursor mass tolerance: 10 ppm, fragment mass tolerance: 0.02 Da, signal/noise threshold for spectra: 1.5, false discovery rate (FDR): strict for PSMs 0.01, and strict for peptides 0.01. In brief, false discovery rates are calculated as follows: first, the software ascertains whether there are q-values and PEPs available for PSMs. If so, the software uses them and assigns the PSM confidences based on target FDRs for PSMs. Next, the software calculates q-values and PEPs for peptides engaging the quality algorithm. Peptide confidences are then assigned based on target FDRs for peptides. If there are no q-values and PEPs available for PSMs, the PSM confidences are set based on our defined target FDRs for PSM employing the respective search engine scores.

4.12. Liposome Extrusion and Liposome Flotation Assay

Candidate lipids were purchased at Avanti Polar Lipids Inc. (PC 16:0/22:6 cat no. 850461, PC 16:1/16:1 cat no. 850358, PI 18:0/20:4 cat no. 850144, PS 18:0/18:1 cat no. 840039, LPC 18:1 cat no. 855773, LPC 18:0 cat no. 855774, LPC 16:0 cat no. 855675, LPC 14:0 cat no. 855575, PC 16:0/20:4 cat no. 850459, PC 18:1/18:1 cat no. 999989). Lipids were resuspended in chloroform, and 30 μg of lipid was aliquoted in glass vials, desiccated in speed vacuum, and resuspended in 100 μL of PBS using sonication. Lipids were extruded using the NanoSizer MINI extruder kit (T&T Scientific, TT-030-0001) through a 100 nm NanoSizer (T&T Scientific, TT-002-0010). Extruded liposomes (100 μL) were incubated with 2.5 μg (1 μL) of isolated ex vivo mouse protein from the lipid–protein extraction of CE fractions (no-injection control, sham-injection control, and EAE groups; see Section 4.9) for 30 min at room temperature. Liposomes/micelles and protein mixture were then resuspended in 50% sucrose. Sucrose gradient was prepared by layering from bottom to top over 1 mL of liposome/micelle–protein mixture in 50% sucrose, 2 mL of 25% sucrose in PBS, and 1 mL of PBS. The gradient was ultracentrifuged at 114 000g at 4 °C for 3.5 h. Fractions were collected using a mechanical pulp to aspirate 1 mL of the bottom fraction followed by 2 mL of the middle fraction and 1 mL of the top fraction. The collected fractions were washed using PBS followed by centrifugation at 18 000g 4 °C for 25 min (Beckman Rotor GH-3.8). The supernatant was discarded, and the pellet was processed for dot immunoblot analysis, as described above.

4.13. Protein–Lipid Overlay Assay

Candidate lipids listed above were dotted on a PVDF membrane at 30 μg per dot and allowed to completely dry. The membrane was incubated for 1 h at 4 °C with 2.5 μg of isolated ex vivo mouse protein from the lipid–protein extraction of CE fractions (no-injection control, sham-injection control, and EAE groups; see Section 4.9) in PBS, followed by cross-linking at 1200 × 100 μJ for 50 s (Stratagene, La Jolla, CA, Stratalinker UV crosslinker, model 1800). The membrane was blocked, washed, and probed using primary and secondary antibodies, as described in Section 4.7.

4.14. MBP–Lipid Deimination Assay

Candidate lipids (30 μg per lipid) listed above were incubated in the presence of 10 μg of a vendor-purified MBP protein (Millipore, 13-104, Burlington, MA) in PBS and allowed to interact for 30 min at room temperature. Sequence alignment of bovine, mouse, and human MBP demonstrated high conservation between species (Figure S5). After incubation, each reaction was adjusted to a final concentration of 1.66 mM CaCl₂ and incubated overnight at 37 °C in the presence of a purified peptidyl arginine deiminase (PAD, Sigma-Aldrich, P1584). Proteins were then precipitated using standard acetone precipitation protocols and analyzed as described in Section 4.7. Densitometry analysis was done using ImageJ program (National Institute of Health, Bethesda, MD). Deiminated MBP (MBP–PAD) densitometry signal was normalized to the MBP signal, and fold change was adjusted to MBP.

4.15. Circular Dichroism

Candidate lipids (PI and LPC, 180 μg per lipid) and 60 μg of a vendor-purified MBP (Millipore, 13-104, Burlington, MA) were separately resuspended in CD buffer (10 mM potassium phosphate, 50 mM ammonium sulfate). Circular dichroism (Jasco, Jasco J-815, Easton, MD) settings included measured range 250–190 nm, data pitch 1 nm, bandwidth 3 nm, scanning speed 20 nm/min, data integration time (D.I.T.) 2 s, and variable temperatures 20 and 105 °C. Baseline measurements were recorded for the CD buffer, and candidate lipids were resuspended in CD buffer before binding assays were performed. Following these measurements, a vendor-purified MBP was added to the corresponding candidate lipids or CD buffer sample and allowed to interact for 30 min at room temperature. CD spectra were recorded for MBP–lipid samples at room temperature and MBP without the presence of any lipids at two temperatures (20 and 105 °C). Baseline CD buffer and candidate lipid spectra were subtracted from their corresponding sample. Samples were then incubated overnight at 37 °C in the presence of purified PAD and 1 mM calcium phosphate. CD spectra were recoded, and baseline spectra were subtracted for each sample. CD analysis was done on the CD analysis and plotting tool (CAPITO) software (mean with SEM). (64)

4.16. Computational Modeling

There is no atomic structure available for the full-length MBP. Previous NMR studies had indicated that a part of the sequence adopts a helical structure, while additional parts are unstructured. (65) Previous studies have suggested that MBP may be an intrinsically disordered protein (IDP). (43,54,55) A check with the PrDOS disordered structure prediction server (66) indicated that MBP sequence matches the criteria for disordered proteins. Robetta server (http://new.robetta.org/) was used to obtain structure prediction (based on the Rosetta method) for MBP. (67) We used five best predictions provided by the server as the starting point and performed MD simulations, allowing MBP to explore the landscape in the presence of water, as well as in the presence of PC and LPC.

4.17. Model Preparation

AMBER’s LEaP module was used for model preparation. Starting with the five structural predictions obtained from the Robetta server (secondary structure depicted in the Supporting Information; Figure S4), five separate systems were prepared. Each structure was immersed in a rectangular box of explicit solvent (extended simple point charge (SPC/E)) (68) such that the protein coordinates are at least 10 Å from the edge of the box, and counterions were added to make the system charge neutral. AMBER’s ff14SB (69) was used for all simulations.

For PC and LPC systems, the coordinates for the protein were surrounded with 25 molecules of LPC or PC, using a model development procedure used and validated in our lab for other protein systems in nonaqueous solvents. (70) These calculations were performed by PackMol (71) such that there was one molecule of MBP for every 25 molecules of lipid, in a cube of 80 Å × 80 Å × 80 Å. The size of this box was selected after trial and error, such that the lipid molecules could easily fit but the cube is smaller in size, possibly to keep the number of atoms in the system at a minimum. Then, the system was immersed in a box of water similar to the case of water-only simulations. The charges for PC and LPC lipids were obtained using the protocol described in AMBER, and ff14SB parameters were used.

4.18. MD Simulations

Each of the 15 systems (five in water, five in PC + water, and five in LPC + water) was equilibrated separately using a protocol developed in our laboratory. (72) Equilibration removes vacuum bubbles and equilibrates the density closer to the density of water and was found to be in the range of 1.05–1.08 g/mL for the 15 model systems. The final temperature was 300 K. Following the equilibration, each of these systems was slowly heated to 305, 310, 315, and 320 K to generate conformational diversity in the MD simulation of 1 ns. These four points were separately cooled to 300 K. Therefore, for each of the 15 systems, there were five starting conformations (the equilibrated conformations at 300 K and four additional ones obtained by heating and cooling) to represent the possible conformational diversity.

For each of these 15 systems with five different starting conformations, production MD simulations were performed for a duration 100 ns under constant energy conditions (NVE ensemble). A total of 2.5 μs aggregated MD sampling was obtained for each system (with water, with water + LPC, and water + PC). NVE was used as this ensemble offers better computational stability and performance for longer MD simulations. (73) The production simulations were performed at a temperature of 300 K and a time step of 2 fs (with SHAKE applied to bonds and angles involving hydrogens). As NVE ensemble was used for production runs, these values correspond to the initial temperature at the start of simulations. A temperature adjusting thermostat was not used in simulations; over the course of 200 ns simulations, the temperature fluctuated around 300 K, with the fluctuations between 2 and 4 K, which is typical for well-equilibrated systems. A total of 100 conformational snapshots collected from each MD run was used for analysis.

4.19. Computational Modeling Analysis

The conformational snapshots from five runs with different starting conformations were collected and analyzed. Therefore, a total of 2500 conformations were analyzed for MBP in water, MBP in PC and water, and MBP in LPC and water. The goal here was to capture as much conformational diversity that MBP samples in the disordered state. The radius of gyration was calculated for the three systems separately, for each of the five structures. Secondary structure was analyzed using AMBER’s PTRAJ program. (74) Previously, we have successfully used the radius of gyration as a measure of conformational flexibility of proteins. (75)

4.20. Statistics

Data are expressed as mean ± standard error (S.E.) of three separate replicates. Statistical significance was assessed using paired (for PERG data) and unpaired (remaining data) t-tests with Prism software version 8 (GraphPad). p < 0.05 was considered statistically significant. Lipid abundance ratios were normalized to the protein concentration found in each samples used for lipidomic analysis. Deimination levels were the measurement of raw densitometry for Cit-MBP normalized to MBP densitometry after membrane stripping. Deimination fold changes reported were all compared and normalized to the endogenous levels of deimination in the MBP sample.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c01590.

Confirmation of the EAE model, variation of hydrocarbon chain, computational modeling of MBP, and five model structures of MBP and MBP sequence alignment (Figures S1–S5); mass spectrometry results for arginine residues and confirmation of MBP presence in gel-excised band (Table S1); and computer simulations of MBP (Movies S1–S3) (ZIP)

ao0c01590_si_001.zip (22.62 MB)

Accession Codes

MBP (UniProt accession: P02686-5 and P02687). Mass spectrometry data (PRIDE archive accession: PXD015494).

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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Corresponding Author
- Sanjoy K. Bhattacharya - Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miami, Florida 33136, United States; Neuroscience Graduate Program, University of Miami, Miami, Florida 33136, United States; http://orcid.org/0000-0003-3759-647X; Email: [email protected]
Authors
- Anddre Osmar Valdivia - Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miami, Florida 33136, United States; Neuroscience Graduate Program, University of Miami, Miami, Florida 33136, United States; Present Address: Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami, 1638 NW 10th Avenue, Room 706A, Miami Florida 33136, United States; http://orcid.org/0000-0002-8425-9102
- Pratul K. Agarwal - Department of Biochemistry & Cell and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996, United States; Department of Physiological Sciences and High Performance Computing Center and, Oklahoma State University, Stillwater, 106 Math Sciences, Stillwater, Oklahoma 74078-1010, United States; http://orcid.org/0000-0002-3848-9492
Author Contributions
A.O.V. and S.K.B., conceptualization; A.O.V and P.K.A., data acquisition and curation; A.O.V., formal analysis; A.O.V. and S.K.B., supervision; S.K.B., funding acquisition; A.O.V., validation; S.K.B., principal investigator; A.O.V. and S.K.B., methodology; A.O.V., writing-original draft; A.O.V. and S.K.B., project administration; and A.O.V. and S.K.B., writing: review and editing.
Funding
This work was supported by an unrestricted grant to the University of Miami from Research to Prevent Blindness (RPB), Department of Defense Grant WHX81-16-0715 and NIH Grant EY027257, EY14801, and NIH Grant GM105978 (to P.K.A.). This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1548562. XSEDE computing allocation were awarded to P.K.A. (MCB-180199 and MCB-190044).
Notes
The authors declare no competing financial interest.

Acknowledgments

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The authors would like to thank Dr. Jae Lee for his valuable insight into the preparation of the manuscript, Drs. Roberta Brambilla and Haritha Desu for their guidance in the generation of the EAE mouse model, and Dr. David Broyles for his guidance during circular dichroism experiments, EY14801and NIH Grant GM105978 (to PKA). This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1548562. XSEDE computing allocation were awarded to P.K.A. (MCB-180199 and MCB-190044).

Abbreviations

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MS	multiple sclerosis
MBP	myelin basic protein
PE	phosphatidylethanolamines
PC	phosphatidylcholines
PI	phosphatidylinositols
PS	phosphatidylserines
EAE	experimental autoimmune encephalomyelitis
MOG	myelin oligodendrocyte glycoprotein
PERG	pattern electroretinogram
FERG	flash electroretinogram
BHT	butylated hydroxytoluene
MTBE	methyl tert-butyl ether
OCT	optical coherence tomography
CE	capillary electrophoresis
PAD	peptidyl arginine deiminase
LFA	liposome floatation assay
PLOA	protein–lipid overlay assay
CD	circular dichroism
MD	molecular dynamic
IDP	intrinsic-disordered protein
CNS	central nervous system

References

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This article references 75 other publications.

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Nylander, A.; Hafler, D. A. Multiple sclerosis. J. Clin. Invest. 2012, 122, 1180, DOI: 10.1172/JCI58649
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Multiple sclerosis
Nylander, Alyssa; Hafler, David A.
Journal of Clinical Investigation (2012), 122 (4), 1180-1188CODEN: JCINAO; ISSN:0021-9738. (American Society for Clinical Investigation)
A review. Multiple sclerosis (MS) is a multifocal demyelinating disease with progressive neurodegeneration caused by an autoimmune response to self-antigens in a genetically susceptible individual. While the formation and persistence of meningeal lymphoid follicles suggest persistence of antigens to drive the continuing inflammatory and humoral response, the identity of an antigen or infectious agent leading to the oligoclonal expansion of B and T cells is unknown. In this review we examine new paradigms for understanding the immunopathol. of MS, present recent data defining the common genetic variants underlying disease susceptibility, and explore how improved understanding of immune pathway disruption can inform MS prognosis and treatment decisions.
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Lassmann, H. What drives disease in multiple sclerosis: Inflammation or neurodegeneration?. Clin. Exp. Neuroimmunol. 2010, 1, 2– 11, DOI: 10.1111/j.1759-1961.2009.00003.x
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What drives disease in multiple sclerosis: inflammation or neurodegeneration?
Lassmann, Hans
Clinical & Experimental Neuroimmunology (2010), 1 (1), 2-11CODEN: CENLCG; ISSN:1759-1961. (John Wiley & Sons Ltd.)
A review. Multiple sclerosis (MS) is defined as a chronic inflammatory disease of the central nervous system, which leads to focal inflammatory demyelinated lesions with secondary neurodegeneration. However, this concept has recently been challenged by several observations suggesting that in this disease neurodegeneration might occur independently of inflammation. Here, these new findings are critically discussed and evidence that active neurodegeneration in MS is invariably assocd. with inflammation is provided. The present review shows, however, that the inflammatory reaction is much more complex, as thought before, and that in the progressive stage of the disease it might become trapped in the central nervous system behind a repaired blood-brain barrier. Future therapeutic options for this disease are discussed on the basis of recent knowledge of the mechanisms of inflammation and neurodegeneration.
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Bielekova, B.; Sung, M.-H.; Kadom, N.; Simon, R.; McFarland, H.; Martin, R. Expansion and Functional Relevance of High-Avidity Myelin-Specific CD4+ T Cells in Multiple Sclerosis. J. Immunol. 2004, 172, 3893– 3904, DOI: 10.4049/jimmunol.172.6.3893
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Expansion and Functional Relevance of High-Avidity Myelin-Specific CD4+ T Cells in Multiple Sclerosis
Bielekova, Bibiana; Sung, Myong-Hee; Kadom, Nadja; Simon, Richard; McFarland, Henry; Martin, Roland
Journal of Immunology (2004), 172 (6), 3893-3904CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
Multiple sclerosis (MS) is an autoimmune disease in which myelin-specific T cells are believed to play a crucial pathogenic role. Nevertheless, so far it has been extremely difficult to demonstrate differences in T cell reactivity to myelin Ag between MS patients and controls. The authors believe that by using unphysiol. high Ag concns. previous studies have missed a highly relevant aspect of autoimmune responses, i.e., T cells recognizing Ag with high functional avidity. Therefore, the authors focused on the characterization of high-avidity myelin-specific CD4+ T cells in a large cohort of MS patients and controls that was matched demog. and with respect to expression of MHC class II alleles. The authors demonstrated that their frequency is significantly higher in MS patients while the nos. of control T cells specific for influenza hemagglutinin are virtually identical between the two cohorts; that high-avidity T cells are enriched for previously in vivo-activated cells and are significantly skewed toward a proinflammatory phenotype. Moreover, the immunodominant epitopes that were most discriminatory between MS patients and controls differed from those described previously and were clearly biased toward epitopes with lower predicted binding affinities to HLA-DR mols., pointing at the importance of thymic selection for the generation of the autoimmune T cell repertoire. Correlations between selected immunol. parameters and magnetic resonance imaging markers indicate that the specificity and function of these cells influences phenotypic disease expression. These data have important implications for autoimmunity research and should be considered in the development of Ag-specific therapies in MS.
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Moscarello, M. A.; Wood, D. D.; Boulias, C.; Ackerley, C. Myelin in multiple sclerosis is developmentally immature. J. Clin. Invest. 1994, 94, 146– 154, DOI: 10.1172/JCI117300
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Myelin in multiple sclerosis is developmentally immature
Moscarello, Mario A.; Wood, D. Denise; Ackerley, Cameron; Boulias, Christos
Journal of Clinical Investigation (1994), 94 (1), 146-54CODEN: JCINAO; ISSN:0021-9738.
The etiol. of multiple sclerosis (MS) is considered to involve genetic, environmental, infective, and immunol. factors which affect the integrity of a normally assembled myelin sheath, either directly or indirectly resulting in demyelination. In a correlative study involving protein chem., mass spectrometric, and electron microscopic techniques the authors have detd. that myelin obtained from victims of MS is arrested at the level of the first growth spurt (within the first 6 yr of life) and is therefore developmentally immature. The data supporting this conclusion include (a) the pattern of microheterogeneity of myelin basic protein (MBP); (b) the NH2-terminal acylation of the least cationic component of MBP ("C-8"); (c) the phase transition temp. (Tc) of myelin isolated from victims of MS correlated with the increased proportion of the least cationic component of MBP; and (d) immunogold electron microscopy using an antibody specific for "C-8" showed that the distribution of gold particles in a 2-yr old infant was similar to the distribution found in a victim of MS. The authors postulate that this developmentally immature myelin is more susceptible to degrdn. by one or a combination of factors mentioned above, providing the initial antigenic material to the immune system.
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Kursula, P. Structural properties of proteins specific to the myelin sheath. Amino Acids 2008, 34, 175– 185, DOI: 10.1007/s00726-006-0479-7
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Structural properties of proteins specific to the myelin sheath
Kursula, P.
Amino Acids (2008), 34 (2), 175-185CODEN: AACIE6; ISSN:0939-4451. (Springer Wien)
A review. The myelin sheath is an insulating membrane layer surrounding myelinated axons in vertebrates, which is formed when the plasma membrane of an oligodendrocyte or a Schwann cell wraps itself around the axon. A large fraction of the total protein in this membrane layer is comprised of only a small no. of individual proteins, which have certain intriguing structural properties. The myelin proteins are implicated in a no. of neurol. diseases, including, for example, autoimmune diseases and peripheral neuropathies. In this review, the structural properties of a no. of myelin-specific proteins are described.
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Hartline, D. K.; Colman, D. R. Rapid conduction and the evolution of giant axons and myelinated fibers. Curr. Biol. 2007, 17, R29– R35, DOI: 10.1016/j.cub.2006.11.042
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Rapid Conduction and the Evolution of Giant Axons and Myelinated Fibers
Hartline, D. K.; Colman, D. R.
Current Biology (2007), 17 (1), R29-R35CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
Nervous systems have evolved two basic mechanisms for increasing the conduction speed of the elec. impulse. The first is through axon gigantism: using axons several times larger in diam. than the norm for other large axons, as for example in the well-known case of the squid giant axon. The second is through encasing axons in helical or concentrically wrapped multilamellar sheets of insulating plasma membrane - the myelin sheath. Each mechanism, alone or in combination, is employed in nervous systems of many taxa, both vertebrate and invertebrate. Myelin is a unique way to increase conduction speeds along axons of relatively small caliber. It seems to have arisen independently in evolution several times in vertebrates, annelids and crustacea. Myelinated nerves, regardless of their source, have in common a multilamellar membrane wrapping, and long myelinated segments interspersed with 'nodal' loci where the myelin terminates and the nerve impulse propagates along the axon by 'saltatory' conduction. For all of the differences in detail among the morphologies and biochemistries of the sheath in the different myelinated animal classes, the function is remarkably universal.
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Valdivia, A. O.; Farr, V.; Bhattacharya, S. K. A novel myelin basic protein transcript variant in the murine central nervous system. Mol. Biol. Rep. 2019, 46, 2547– 2553, DOI: 10.1007/s11033-019-04635-8
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A novel myelin basic protein transcript variant in the murine central nervous system
Valdivia, Anddre Osmar; Farr, Valentina; Bhattacharya, Sanjoy K.
Molecular Biology Reports (2019), 46 (2), 2547-2553CODEN: MLBRBU; ISSN:0301-4851. (Springer)
Myelin basic protein is a multifunctional protein whose primary role is to adhere membranes of the myelin sheath. There are various isoforms that have been identified, 6 distinct isoforms in human and 13 distinct isoforms in mice. These distinct isoforms are the product of alternative splicing of a single gene. The present study sought out to identify the different isoforms found in the murine central nervous system. Neuronal tissue (brain) from five different C57BL6/J mice at 2 mo of age was harvested and used for mRNA extn. MRNA was reversed transcribed to cDNA and transcripts were detected through PCR amplification and DNA agarose gel sepn. Primers for exon 1, exon 5b and exon 11 of the myelin basic protein gene were used to capture all the possible transcripts that are naturally found in the murine central nervous system. Unknown transcript was sequenced at Genewiz facilities (South Plainfield, NJ) and mass spectrometry protein sequence anal. demonstrated the presence of a novel myelin basic protein transcript variant. We identified a novel transcript variant of myelin basic protein. This novel transcript variant corresponds to a myelin basic protein of 32.5 kDa which has not been previously reported. This novel transcript variant presents relevant clin. significance to various demyelinating diseases due to its contribution to the understanding of the natural state of the murine central nervous system.
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Musse, A. A.; Boggs, J. M.; Harauz, G. Deimination of membrane-bound myelin basic protein in multiple sclerosis exposes an immunodominant epitope. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4422– 4427, DOI: 10.1073/pnas.0509158103
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Deimination of membrane-bound myelin basic protein in multiple sclerosis exposes an immunodominant epitope
Musse, Abdiwahab A.; Boggs, Joan M.; Harauz, George
Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (12), 4422-4427CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
The degrdn. of myelin in the CNS is the hallmark of multiple sclerosis. Redn. in the net pos. charge of myelin basic protein (MBP), through deimination, correlates strongly with disease severity and may mediate myelin instability and loss of compaction. Using Cys scanning, spin labeling, EPR spectroscopy, and site-specific proteolysis, we show that in the membrane-bound state the primary immunodominant epitope, V83-T92, of the less cationic recombinant murine MBP C8 mimic (rmC8) forms a more highly surface-exposed and shorter amphipathic α-helix than in the unmodified form, recombinant murine MBP C1 mimic (rmC1), analogous to the most cationic and abundant isomer of MBP in normal myelin. Moreover, cathepsin D digested lipid-assocd. rmC8 3-fold faster than rmC1, and cleavage at F86-F87 occurred more readily in rmC8 than rmC1. These findings suggest a mechanism for initial loss of myelin stability and the autoimmune pathogenesis of multiple sclerosis.
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Kidd, B. A.; Ho, P. P.; Sharpe, O.; Zhao, X.; Tomooka, B. H.; Kanter, J. L.; Steinman, L.; Robinson, W. H. Epitope spreading to citrullinated antigens in mouse models of autoimmune arthritis and demyelination. Arthritis Res. Ther. 2008, 10, R119 DOI: 10.1186/ar2523
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Epitope spreading to citrullinated antigens in mouse models of autoimmune arthritis and demyelination
Kidd Brian A; Ho Peggy P; Sharpe Orr; Zhao Xiaoyan; Tomooka Beren H; Kanter Jennifer L; Steinman Lawrence; Robinson William H
Arthritis research & therapy (2008), 10 (5), R119 ISSN:.
INTRODUCTION: Anti-citrullinated protein antibodies have a diagnostic role in rheumatoid arthritis (RA); however, little is known about their origins and contribution to pathogenesis. Citrullination is the post-translational conversion of arginine to citrulline by peptidyl arginine deiminase, and increased citrullination of proteins is observed in the joint tissue in RA and in brain tissue in multiple sclerosis (MS). METHODS: We applied synovial and myelin protein arrays to examine epitope spreading of B cell responses to citrullinated epitopes in both the collagen-induced arthritis (CIA) model for RA and the experimental autoimmune encephalomyelitis (EAE) model for MS. Synovial and myelin protein arrays contain a spectrum of proteins and peptides, including native and citrullinated forms, representing candidate autoantigens in RA and MS, respectively. We applied these arrays to characterise the specificity of autoantibodies in serial serum samples derived from mice with acute and chronic stages of CIA and EAE. RESULTS: In samples from pre-disease CIA and acute-disease EAE, we observed autoantibody targeting of the immunising antigen and responses to a limited set of citrullinated epitopes. Over the course of diseases, the autoantibody responses expanded to target multiple citrullinated epitopes in both CIA and EAE. Using immunoblotting and mass spectrometry analysis, we identified citrullination of multiple polypeptides in CIA joint and EAE brain tissue that have not previously been described as citrullinated. CONCLUSIONS: Our results suggest that anti-citrulline antibody responses develop in the early stages of CIA and EAE, and that autoimmune inflammation results in citrullination of joint proteins in CIA and brain proteins in EAE, thereby creating neoantigens that become additional targets in epitope spreading of autoimmune responses.
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Cuzner, M. L.; Davison, A. N. Changes in cerebral lysosomal enzyme activity and lipids in multiple sclerosis. J. Neurol. Sci. 1973, 19, 29– 36, DOI: 10.1016/0022-510X(73)90053-1
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Changes in cerebral lysosomal enzyme activity and lipids in multiple sclerosis
Cuzner, M. Louise; Davison, A. N.
Journal of the Neurological Sciences (1973), 19 (1), 29-36CODEN: JNSCAG; ISSN:0022-510X.
In most cases of multiple sclerosis no change was seen in the lipid compn. of apparently normal white matter. Where there was depletion of lipid, chem. and enzymatic evidence of demyelination was found. Raised lysosomal enzyme activity was normally restricted to plaques but in acute cases of multiple sclerosis changes were more extensive. Even in apparently normal areas of white matter cholesterol esters were found and lysosomal acid proteinase, β-glucuronidase and arylsulfatase activities were significantly increased. The significance of these observations to the pathology of multiple sclerosis is discussed.
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Einstein, E. R.; Csejtey, J.; Dalal, K. B.; Adams, C. W.; Bayliss, O. B.; Hallpike, J. F. Proteolytic activity and basic protein loss in and around multiple sclerosis plaques: combined biochemical and histochemical observations. J. Neurochem. 1972, 19, 653– 662, DOI: 10.1111/j.1471-4159.1972.tb01382.x
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Proteolytic activity and basic protein loss in and around multiple sclerosis plaques. Combined biochemical and histochemical observations
Einstein, Elizabeth R.; Csejtey, Judit; Dalal, K. B.; Adams, C. W. M.; Bayliss, O. B.; Hallpike, J. F.
Journal of Neurochemistry (1972), 19 (3), 653-62CODEN: JONRA9; ISSN:0022-3042.
This combined histochem. and biochem. study has shown that acid proteinase activity (pH 3.5) is increased around histol. defined active plaques of multiple sclerosis (MS). Biochem. estn. showed that the enzyme is more active in most samples of normal white matter in MS than in controls. A gradient of enzyme activity was obsd.: control white matter-white matter distant from plaque-close white matter-edge-plaque. Both electrophoretic and histochem. techniques revealed a redn. or absence of basic (encephalitogenic) protein in the plaques. Electrophoresis showed a diminution of encephalitogenic protein outside some plaques. Phospholipids that remain on the base line of thin layer chromatoplates were predominantly phosphoinositides combined with encephalitogenic protein.
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Richards, P. T.; Cuzner, M. L. Proteolytic activity in CSF. Adv. Exp. Med. Biol. 1978, 100, 521– 527, DOI: 10.1007/978-1-4684-2514-7_38
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Proteolytic activity in CSF
Richards, P. T.; Cuzner, M. Louise
Advances in Experimental Medicine and Biology (1978), 100 (Myelination Demyelination), 521-7CODEN: AEMBAP; ISSN:0065-2598.
Cerebrospinal fluid cellular neutral proteinase and supernatant acid proteinase were increased in acute multiple sclerosis and in central nervous system infections.
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Dreyton, C. J.; Knuckley, B.; Jones, J. E.; Lewallen, D. M.; Thompson, P. R. Mechanistic studies of protein arginine deiminase 2: evidence for a substrate-assisted mechanism. Biochemistry 2014, 53, 4426, DOI: 10.1021/bi500554b
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Mechanistic studies of protein arginine deiminase 2: Evidence for a substrate-assisted mechanism
Dreyton, Christina J.; Knuckley, Bryan; Jones, Justin E.; Lewallen, Daniel M.; Thompson, Paul R.
Biochemistry (2014), 53 (27), 4426-4433CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
Citrullination, which is catalyzed by protein arginine deiminases (PADs 1-4 and 6), is a post-translational modification (PTM) that effectively neutralizes the pos. charge of a guanidinium group by its replacement with a neutral urea. Given the sequence similarity of PAD2 across mammalian species and the genomic organization of the PAD2 gene, PAD2 is predicted to be the ancestral homolog of the PADs. Although PAD2 has long been known to play a role in myelination, it has only recently been linked to other cellular processes, including gene transcription and macrophage extracellular trap formation. For example, PAD2 deiminates histone H3 at Arg-26, and this PTM leads to the increased transcription of >200 genes under the control of the estrogen receptor. Given that the understanding of PAD2 biol. remains incomplete, the authors initiated mechanistic studies on this enzyme to aid the development of PAD2-specific inhibitors. Here, the authors report that the substrate specificity and Ca2+ dependence of PAD2 were similar to those of PADs 1, 3, and 4. However, unlike those isoenzymes, PAD2 appeared to use a substrate-assisted mechanism of catalysis in which the pos. charged substrate, guanidinium, depressed the pKa of the nucleophilic Cys residue. By contrast, PADs 1, 3, and 4 use a reverse-protonation mechanism. These mechanistic differences will aid the development of isoenzyme-specific inhibitors.
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Vossenaar, E. R.; Zendman, A. J.; van Venrooij, W. J.; Pruijn, G. J. PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. BioEssays 2003, 25, 1106– 1118, DOI: 10.1002/bies.10357
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PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease
Vossenaar, Erik R.; Zendman, Albert J. W.; van Venrooij, Walther J.; Pruijn, Ger J. M.
BioEssays (2003), 25 (11), 1106-1118CODEN: BIOEEJ; ISSN:0265-9247. (John Wiley & Sons, Inc.)
A review. Peptidylarginine deiminase (PAD, EC 3.5.3.15) enzymes catalyze the conversion of protein-bound Arg to citrulline. This post-translational modification may have a big impact on the structure and function of the target protein. In this review, the authors will discuss the effects of citrullination and its involvement in several human diseases, including rheumatoid arthritis and multiple sclerosis. So far, 4 isotypes of PAD were described in mammals. We describe the existence of PAD in non-mammalian vertebrates and the existence of a fifth mammalian PAD. In addn., tissue-specific expression, genomic organization and evolutionary conservation of the different PAD isotypes will be discussed in detail. This article contains supplementary material which may be viewed at the BioEssays website at http://www.interscience.wiley.com/jpages/0265-9247/suppmat/2003/25/v25.1106.html.
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Tarcsa, E.; Marekov, L. N.; Mei, G.; Melino, G.; Lee, S. C.; Steinert, P. M. Protein unfolding by peptidylarginine deiminase. Substrate specificity and structural relationships of the natural substrates trichohyalin and filaggrin. J. Biol. Chem. 1996, 271, 30709, DOI: 10.1074/jbc.271.48.30709
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Protein unfolding by peptidylarginine deiminase. Substrate specificity and structural relationships of the natural substrates trichohyalin and filaggrin
Tarcsa, Edit; Marekov, Lyuben N.; Mei, Giampiero; Melino, Gerry; Lee, Seung-Chul; Steinert, Peter M.
Journal of Biological Chemistry (1996), 271 (48), 30709-30716CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
Peptidylarginine deiminases, which are commonly found in mammalian cells, catalyze the deimination of protein-bound arginine residues to citrullines. However, very little is known about their substrate requirements and the significance or consequences of this post-synthetic modification. We have explored this reaction in vitro with two known substrates filaggrin and trichohyalin. First, the degree and rate of modification of arginines to citrullines directly correlates with the structural order of the substrate. In filaggrin, which has little structural order, the reaction proceeded rapidly to >95% completion. However, in the highly α-helical protein trichohyalin, the reaction proceeded slowly to about 25% and could be forced to a max. of about 65%. Second, the rate and degree of modification depends on the sequence location of the target arginines. Third, we show by gel electrophoresis, CD, and fluorescence spectroscopy that the reaction interferes with organized protein structure: the net formation of ≥10% citrulline results in protein denaturation. Cyanate modification of the lysines in model α-helix-rich proteins to homocitrullines also results in loss of organized structure. These data suggest that the ureido group on the citrulline formed by the peptidylarginine deiminase enzyme modification functions to unfold proteins due to decrease in net charge, loss of potential ionic bonds, and interference with H bonds.
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Takahara, H. A History of Deimination Research in Japan: The Founding Fathers. In Protein Deimination in Human Health and Disease; Nicholas, A. P.; Bhattacharya, S. K.; Thompson, P. R., Eds.; Springer International Publishing: Cham, 2017; pp 1– 10.
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Cao, L.; Goodin, R.; Wood, D.; Moscarello, M. A.; Whitaker, J. N. Rapid release and unusual stability of immunodominant peptide 45-89 from citrullinated myelin basic protein. Biochemistry 1999, 38, 6157– 6163, DOI: 10.1021/bi982960s
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Rapid Release and Unusual Stability of Immunodominant Peptide 45-89 from Citrullinated Myelin Basic Protein
Cao, Ligong; Goodin, Richard; Wood, Denise; Moscarello, Mario A.; Whitaker, John N.
Biochemistry (1999), 38 (19), 6157-6163CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
Myelin basic protein (MBP) exists in a population of isoforms and isomers. The 18.5 kDa MBP-C1, the main human adult isoform, has 170 residues and is relatively unmodified, whereas the same isoform can be citrullinated on six arginine residues to create the MBP-C8 (MBP Cit6) isomer. MBP Cit6 dominates in MS brain, accounting for 45% rather than 25% of the population of MBP isomers. In the fulminant form of MS, known as Marburg's Disease, 18 of the 19 arginines in MBP are citrullinated (MBP Cit18). Citrullination of MBP could lead to instability of myelin or limited remyelination. In this investigation, the susceptibilities to degrdn. by cathepsin D of MBP Cit6 and MBP-C1, both from normal and MS brain tissue, and Marburg MBP Cit18 were compared. The pattern of digestion was similar, and no differences of corresponding isomers in normal and MS brain were noted. However, normal MBP Cit6 was degraded 10-fold more rapidly than MBP-C1, and MBP Cit18 was degraded even more rapidly. MBP peptide 45-89 was preserved regardless of isomer type or source. Its generation was directly related to the citrulline content of the MBP substrate being 4 times faster in normal MBP Cit6 and 35 times faster in Marburg MBP Cit18 than in normal MBP-C1. Peptide 45-89 from a citrullinated MBP exhibited more deamidation, and, regardless of source, showed an α-helix structure in a lipid mimetic environment. We postulate that the generation of MBP peptides, including those that are dominant and encephalitogenic, is directly related to deimination of arginine to citrulline in MBP.
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Whitaker, J. N. Myelin encephalitogenic protein fragments in cerebrospinal fluid of persons with multiple sclerosis. Neurology 1977, 27, 911– 920, DOI: 10.1212/WNL.27.10.911
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Myelin encephalitogenic protein fragments in cerebrospinal fluid of persons with multiple sclerosis
Whitaker, John N.
Neurology (1977), 27 (10), 911-20CODEN: NEURAI; ISSN:0028-3878.
With a double-antibody radioimmunoassay performed on unconcd. cerebrospinal fluid, 8 of 14 patients in an acute phase of multiple sclerosis had levels of 3.4 to 15.4 ng/mL of the P1 fragment (residues 43-88) of myelin encephalitogenic protein. Encephalitogenic protein-P1 was found only in the acute phase and was present in 6 of 7 persons in the first week of an exacerbation and absent in 29 multiple sclerosis patients who were stable or had a gradually progressive course. Six of 117 controls had detectable cerebrospinal fluid encephalitogenic protein-P1. Only in 2 of these, one with a recent cerebral infarction and one with diabetic nephropathy who was in coma, were the levels in the range encountered in patients in the acute phase of multiple sclerosis. Although not entirely specific for multiple sclerosis, the presence of material in the cerebrospinal fluid of multiple sclerosis patients cross-reacting with encephalitogenic protein-P1 appears to be a characteristic of acute exacerbations.
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Whitaker, J. R.; Granum, P. E. An absolute method for protein determination based on difference in absorbance at 235 and 280 nm. Anal. Biochem. 1980, 109, 156– 159, DOI: 10.1016/0003-2697(80)90024-X
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An absolute method for protein determination based on difference in absorbance at 235 and 280 nm
Whitaker, John R.; Granum, Per Einar
Analytical Biochemistry (1980), 109 (1), 156-9CODEN: ANBCA2; ISSN:0003-2697.
Protein (in mg/mL) is detd. spectrometrically by subtracting the absorbance at 280 nm from the absorbance at 235 nm, then dividing by 2.51 (which is the difference between the av. absorptivities at the 2 wavelengths). This method does not require a std. curve, is not subject to interference by nucleic acids, and is independent of amino acid compn. The sensitivity of this method is 45% that of the Lowry method.
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Pritzker, L. B.; Joshi, S.; Gowan, J. J.; Moscarello, M. A.; Harauz, G. Deimination of myelin basic protein. 1. Effect of deimination of arginyl residues of myelin basic protein on its structure and susceptibility to digestion by cathepsin D. Biochemistry 2000, 39, 5374– 5381, DOI: 10.1021/bi9925569
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Deimination of Myelin Basic Protein. 1. Effect of Deimination of Arginyl Residues of Myelin Basic Protein on Its Structure and Susceptibility to Digestion by Cathepsin D
Pritzker, Laura B.; Joshi, Shashikant; Gowan, Jessica J.; Harauz, George; Moscarello, Mario A.
Biochemistry (2000), 39 (18), 5374-5381CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
The effect of deimination of arginyl residues in bovine myelin basic protein (MBP) on its susceptibility to digestion by cathepsin D has been studied. Using bovine component 1 (C-1) of MBP, the most unmodified of the components with all 18 arginyl residues intact, we have generated a no. of citrullinated forms by treatment of the protein with purified peptidylarginine deiminase (PAD) in vitro. We obtained species contg. 0-9.9 mol of citrulline/mol of MBP. These various species were digested with cathepsin D, a metalloproteinase which cleaves proteins at Phe-Phe linkages. The rate of digestion compared to component 1 was only slightly affected when 2.7 or 3.8 mol of citrulline/mol of MBP was present. With 7.0 mol of citrulline/mol of MBP, a large increase in the rate of digestion occurred. No further increase was obsd. with 9.9 mol of citrulline/mol of MBP. The immunodominant peptide 43-88 (bovine sequence) was released slowly when 2.7 and 3.8 mol of citrulline/mol of MBP was present, but it was released rapidly when 7.0 mol of citrulline/mol of MBP was present. The dramatic change in digestion with 7.0 mol of citrulline/mol of MBP or more could be explained by a change in three-dimensional structure. Mol. dynamics simulation showed that increasing the no. of citrullinyl residues above 7 mol/mol of MBP generated a more open structure, consistent with exptl. observations in the literature. We conclude that PAD, which deiminates arginyl residues in proteins, decreases both the charge and compact structure of MBP. This structural change allows better access of the Phe-Phe linkages to cathepsin D. This scheme represents an effective way of generating the immunodominant peptide which sensitizes T-cells for the autoimmune response in demyelinating disease.
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Wood, D. D.; Bilbao, J. M.; O’Connors, P.; Moscarello, M. A. Acute multiple sclerosis (Marburg type) is associated with developmentally immature myelin basic protein. Ann. Neurol. 1996, 40, 18– 24, DOI: 10.1002/ana.410400106
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Acute multiple sclerosis (Marburg type) is associated with developmentally immature myelin basic protein
Wood D D; Bilbao J M; O'Connors P; Moscarello M A
Annals of neurology (1996), 40 (1), 18-24 ISSN:0364-5134.
We have studied a case of acute, fulminating multiple sclerosis (MS) (Marburg type) at the pathological and biochemical levels. Postmortem examination of the brain revealed extensive areas of gross rarefaction in the hemispheric white matter. Histologically, well-demarcated areas of demyelination with a large influx of macrophages and a subtle perivascular infiltration of lymphocytes were seen with relative preservation of the axis cylinders. Myelin basic protein (MBP) was isolated and purified [correction of purifed] from noninvolved white matter. It was slightly larger in molecular weight than MBP from normal brain or from chronic MS brain. The increase in mass was accounted for, in part, by the deimination of 18 of 19 arginyl residues to citrulline, making the patient's MBP much less cationic than MBP from normal white matter. When expressed as the ratio of least cationic form of MBP to the most cationic (C-8/C-1), the normal ratio was 0.82, chronic MS 2.5, and the patient in this study 6.7. Because the ratio of 6.7 was similar to 7.5 found for a 15-month-old infant, MBP was considered to be of the immature form. The data are consistent with a genetic factor influencing the charge microheterogeneity of MBP. The resulting less cationic MBP cannot carry out its normal function of compacting multilayers.
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Mastronardi, F. G.; Wood, D. D.; Mei, J.; Raijmakers, R.; Tseveleki, V.; Dosch, H. M.; Probert, L.; Casaccia-Bonnefil, P.; Moscarello, M. A. Increased citrullination of histone H3 in multiple sclerosis brain and animal models of demyelination: a role for tumor necrosis factor-induced peptidylarginine deiminase 4 translocation. J. Neurosci. 2006, 26, 11387– 11396, DOI: 10.1523/JNEUROSCI.3349-06.2006
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Increased citrullination of histone H3 in multiple sclerosis brain and animal models of demyelination: a role for tumor necrosis factor-induced peptidylarginine deiminase 4 translocation
Mastronardi, Fabrizio G.; Wood, D. Denise; Mei, Jiang; Raijmakers, Reinout; Tseveleki, Vivian; Dosch, Hans-Michael; Probert, Lesley; Casaccia-Bonnefil, Patrizia; Moscarello, Mario A.
Journal of Neuroscience (2006), 26 (44), 11387-11396CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
Modification of arginine residues by citrullination is catalyzed by peptidylarginine deiminases (PADs), of which five are known, generating irreversible protein structural modifications. We have shown previously that enhanced citrullination of myelin basic protein contributed to destabilization of the myelin membrane in the CNS of multiple sclerosis (MS) patients. We now report increased citrullination of nucleosomal histones by PAD4 in normal-appearing white matter (NAWM) of MS patients and in animal models of demyelination. Histone citrullination was attributable to increased levels and activity of nuclear PAD4. PAD4 translocation into the nucleus was attributable to elevated tumor necrosis factor-α (TNF-α) protein. The elevated TNF-α in MS NAWM was not assocd. with CD3+ or CD8+ lymphocytes, nor was it assocd. with CD68+ microglia/macrophages. GFAP, a measure of astrocytosis, was the only cytol. marker that was consistently elevated in the MS NAWM, suggesting that TNF-α may have been derived from astrocytes. In cell cultures of mouse and human oligodendroglial cell lines, PAD4 was predominantly cytosolic but TNF-α treatment induced its nuclear translocation. To address the involvement of TNF-α in targeting PAD4 to the nucleus, we found that transgenic mice overexpressing TNF-α also had increased levels of citrullinated histones and elevated nuclear PAD4 before demyelination. In conclusion, high citrullination of histones consequent to PAD4 nuclear translocation is part of the process that leads to irreversible changes in oligodendrocytes and may contribute to apoptosis of oligodendrocytes in MS.
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Nicholas, A. P.; Whitaker, J. N. Preparation of a monoclonal antibody to citrullinated epitopes: its characterization and some applications to immunohistochemistry in human brain. Glia 2002, 37, 328– 336, DOI: 10.1002/glia.10039
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Preparation of a monoclonal antibody to citrullinated epitopes: its characterization and some applications to immunohistochemistry in human brain
Nicholas Anthony P; Whitaker John N
Glia (2002), 37 (4), 328-36 ISSN:0894-1491.
Using hybridoma technology, an IgM monoclonal antibody (mAb), designated as F95, was developed against a deca-citrullinated peptide (DCP) consisting of 10 citrulline residues and a carboxyl Gly-Gly-Cys through which DCP was covalently linked to an activated carrier protein, keyhole limpet hemocyanin (KLH). Clones were selected on the basis of not reacting with human unmodified and noncitrullinated myelin basic protein (MBP), MBP-C1, but reacting well with human citrullinated MBP (MBP-C8). When tested by ELISA, this mAb demonstrated minimal reactivity with human MBP-C1, varying reactivity with the C2-C5 isomers of human MBP, moderate binding with guinea pig MBP-C8, and strong reactivity with human MBP-C8. By ELISA, mAb F95 was directed predominantly against citrulline, not MBP, as revealed by its binding to DCP linked with activated KLH, bovine serum albumin (BSA), or ovalbumin (OA), but not with KLH, BSA, or OA alone. Immunohistochemistry of normal human brain demonstrated that F95 stained central nervous system myelin and a subset of astrocytes. Given the citrulline-directed features of mAb F95, this immunohistochemical pattern suggests that certain astroglial filaments expressing glial fibrillary acidic protein also contain citrulline-bearing components. These potentially implicate citrullinated proteins, notably in astroglial filaments, in a variety of normal and pathological neurobiological processes.
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Raijmakers, R.; Vogelzangs, J.; Croxford, J. L.; Wesseling, P.; van Venrooij, W. J.; Pruijn, G. J. Citrullination of central nervous system proteins during the development of experimental autoimmune encephalomyelitis. J. Comp. Neurol. 2005, 486, 243– 253, DOI: 10.1002/cne.20529
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Citrullination of central nervous system proteins during the development of experimental autoimmune encephalomyelitis
Raijmakers Reinout; Vogelzangs Judith; Croxford J Ludovic; Wesseling Pieter; van Venrooij Walther J; Pruijn Ger J M
The Journal of comparative neurology (2005), 486 (3), 243-53 ISSN:0021-9967.
Immunization of mammals with central nervous system (CNS)-derived proteins or peptides induces experimental autoimmune encephalomyelitis (EAE), a disease resembling the human autoimmune disease multiple sclerosis (MS). Both diseases are accompanied by destruction of a part of the of the myelin sheaths, which surround neurites in the CNS. Previous studies in MS have described alterations in the citrullination of myelin basic protein, one of the main protein constituents of the myelin sheath. Here, we show that, also during the development of EAE in mice, hypercitrullination occurs in the areas of the spinal cord that show the highest degree of inflammation and that myelin basic protein and glial fibrillary acidic protein are among the hypercitrullinated proteins. We conclude that hypercitrullination of myelin proteins in the CNS is a common phenomenon in demyelinating disease. Hypercitrullination may cause conformational changes in proteins, so the affected proteins may be involved in the pathogenesis of CNS autoimmune disease by acting as autoreactive T-cell epitopes. This is the first report in which hypercitrullination of CNS proteins in EAE is described and in which proteins other than myelin basic protein are reported to be citrullinated during autoimmune-mediated CNS inflammation.
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Cao, L.; Sun, D.; Whitaker, J. N. Citrullinated myelin basic protein induces experimental autoimmune encephalomyelitis in Lewis rats through a diverse T cell repertoire. J. Neuroimmunol. 1998, 88, 21– 29, DOI: 10.1016/S0165-5728(98)00063-0
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Citrullinated myelin basic protein induces experimental autoimmune encephalomyelitis in Lewis rats through a diverse T cell repertoire
Cao, Ligong; Sun, Deming; Whitaker, John N.
Journal of Neuroimmunology (1998), 88 (1,2), 21-29CODEN: JNRIDW; ISSN:0165-5728. (Elsevier Science B.V.)
An increased proportion of citrullinated MBP (MBP-C8) occurs in the brains of multiple sclerosis (MS) patients. In this study, MBP-C8 from guinea pig (GP) brains was isolated and found encephalitogenic in Lewis rats upon immunization. An encephalitogenic T cell line selected with MBP-C8 preferentially reacted with MBP-C8 over unmodified MBP. This T cell line responded weakly to the dominant encephalitogenic epitope, GP-MBP peptide 70-88, and did not display restricted TCR β-chain usage (such as Vβ8.2). The distinctive features of MBP-C8 were also demonstrated by its ability to reinduce active EAE in 70% of rats which had recovered from unmodified MBP induced EAE. These findings raise the possibility that citrullinated MBP may elicit a different pathogenic T cell repertoire for the recurrent phases of inflammatory demyelination.
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Carrillo-Vico, A.; Leech, M. D.; Anderton, S. M. Contribution of myelin autoantigen citrullination to T cell autoaggression in the central nervous system. J. Immunol. 2010, 184, 2839– 2846, DOI: 10.4049/jimmunol.0903639
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Contribution of Myelin Autoantigen Citrullination to T Cell Autoaggression in the Central Nervous System
Carrillo-Vico, Antonio; Leech, Melanie D.; Anderton, Stephen M.
Journal of Immunology (2010), 184 (6), 2839-2846CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
Breakdown in immunol. self tolerance, leading to autoimmune diseases such as multiple sclerosis, might arise from immune recognition of self proteins that have undergone heightened posttranslational modification under pathophysiol. conditions. A posttranslational modification of particular interest is the deimination of Arg to citrulline, catalyzed by peptidylarginyl deiminase (PAD) enzymes. As a CD4+ T cell-driven model of multiple sclerosis, the authors used exptl. autoimmune encephalomyelitis (EAE) induced with the immunodominant 35-55 peptide of myelin oligodendrocyte glycoprotein (pMOG) in C57BL/6 mice to test whether citrullination of a T cell epitope can contribute to disease etiopathol. Immunization with an altered peptide ligand (APL) of pMOG with an Arg citrulline conversion at a TCR contact (residue 41) led to the activation of two populations of APL-responsive T cells that either did, or did not cross-react with the native pMOG peptide. This APL could induce EAE. However, this reflected the activation of T cells that cross-reacted with the native pMOG epitope, because prior tolerization of these T cells using pMOG prevented APL-induced EAE. Using a passive transfer model, the authors found that T cells that responded specifically to the citrullinated form of pMOG were neither necessary, nor sufficient to initiate the EAE lesion. Nevertheless, these cells could provoke exacerbation of pathol. if transferred into mice with ongoing EAE. The PAD2 and PAD4 enzymes were markedly upregulated in the inflamed CNS. Therefore, once inflammation is established, citrullination of target autoantigens can allow an expanded repertoire of T cells to contribute to CNS pathol.
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Quinn, T. A.; Dutt, M.; Shindler, K. S. Optic neuritis and retinal ganglion cell loss in a chronic murine model of multiple sclerosis. Front. Neurol. 2011, 2, 50 DOI: 10.3389/fneur.2011.00050
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Optic neuritis and retinal ganglion cell loss in a chronic murine model of multiple sclerosis
Quinn Thomas A; Dutt Mahasweta; Shindler Kenneth S
Frontiers in neurology (2011), 2 (), 50 ISSN:.
Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are neurodegenerative diseases with characteristic inflammatory demyelination in the central nervous system, including the optic nerve. Neuronal and axonal damage is considered to be the main cause of long-term disability in patients with MS. Neuronal loss, including retinal ganglion cell (RGC) apoptosis in eyes with optic neuritis (ON), also occurs in EAE. However, there is significant variability in the clinical course and level of neuronal damage in MS and EAE. The current studies examine the mechanisms and kinetics of RGC loss in C57/BL6 mice immunized with myelin oligodendrocyte glycoprotein to induce a chronic EAE disease. Clinical progression of EAE was scored daily and vision was assessed by optokinetic responses. At various time points, RGCs were counted and optic nerves were examined for inflammatory cell infiltration. Almost all EAE mice develop ON by day 15 post-immunization; however, RGC loss is delayed in these mice. No RGC loss is detected 25 days post-immunization, whereas RGC numbers in EAE mice significantly and progressively decrease compared to controls from 35 to 50 days post-immunization. The delayed time course of RGC loss is in stark contrast to that reported in relapsing EAE, as well as in rats with chronic EAE. Results suggest that different clinical disease courses of optic nerve inflammation may trigger distinct mechanisms of neuronal damage, or RGCs in different rodent strains may have variable resistance to neuronal degeneration.
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Beniac, D. R.; Luckevich, M. D.; Czarnota, G. J.; Tompkins, T. A.; Ridsdale, R. A.; Ottensmeyer, F. P.; Moscarello, M. A.; Harauz, G. Three-dimensional structure of myelin basic protein. I. Reconstruction via angular reconstitution of randomly oriented single particles. J. Biol. Chem. 1997, 272, 4261– 4268, DOI: 10.1074/jbc.272.7.4261
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Three-dimensional structure of myelin basic protein. I. Reconstruction via angular reconstitution of randomly oriented single particles
Beniac, Daniel R.; Luckevich, Maria D.; Czarnota, Gregory J.; Tompkins, Thomas A.; Ridsdale, Ross A.; Ottensmeyer, F. Peter; Moscarello, Mario A.; Harauz, George
Journal of Biological Chemistry (1997), 272 (7), 4261-4268CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
Myelin basic protein (MBP) plays an integral role in the structure and function of the myelin sheath. In humans and cattle, an 18.5-kDa isoform of MBP predominates and exists as a multitude of charge isomers resulting from extensive and varied post-translational modifications. We have purified the least modified isomer (named C1) of the 18.5-kDa isoform of MBP from fresh bovine brain and imaged this protein as neg. stained single particle adsorbed to a lipid monolayer. Under these conditions, MBP/C1 presented diverse projections whose relative orientations were detd. using an iterative quaternion-assisted angular reconstitution scheme. In different buffers, one with a low salt and the other with a high salt concn., the conformation of the protein was slightly different. In low salt buffer, the three-dimensional reconstruction, solved to a resoln. of 4 nm, had an overall "C" shape of outer radius 5.5 nm, inner radius 3 nm, overall circumference 15 nm, and height 4.7 nm. The three-dimensional reconstruction of the protein in high salt buffer, solved to a resoln. of 2.8 nm, was essentially the same in terms of overall dimensions but had a somewhat more compact architecture. These results are the first structures achieved directly for this unusual macromol., which plays a key role in the development of multiple sclerosis.
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Ridsdale, R. A.; Beniac, D. R.; Tompkins, T. A.; Moscarello, M. A.; Harauz, G. Three-dimensional structure of myelin basic protein. II. Molecular modeling and considerations of predicted structures in multiple sclerosis. J. Biol. Chem. 1997, 272, 4269– 4275, DOI: 10.1074/jbc.272.7.4269
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Three-dimensional structure of myelin basic protein. II. Molecular modeling and considerations of predicted structures in multiple sclerosis
Ridsdale, Ross A.; Beniac, Daniel R.; Tompkins, Thomas A.; Moscarello, Mario A.; Harauz, George
Journal of Biological Chemistry (1997), 272 (7), 4269-4275CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
A computational model of myelin basic protein (MBP) has been constructed based on the premise of a phylogenetically conserved β-sheet backbone and on electron microscopical three-dimensional reconstructions. Many residues subject to post-translational modification (phosphorylation, methylation, or conversion of arginines to citrullines) were located in loop regions and thus accessible to modifying enzymes. The triproline segment (residues 99-101) is fully exposed on the back surface of the protein in a long crossover connection between two parallel β-strands. The proximity of this region to the underlying β-sheet suggests that post-translational modifications here might have potential synergistic effects on the entire structure. Post-translational modifications that lead to reduced surface charge could result first in a weakened attachment to the myelin membrane rather than in a gross conformational change of the protein itself. Such mechanisms could be operative in demyelinating diseases such as multiple sclerosis.
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Haas, H.; Oliveira, C. L.; Torriani, I. L.; Polverini, E.; Fasano, A.; Carlone, G.; Cavatorta, P.; Riccio, P. Small angle x-ray scattering from lipid-bound myelin basic protein in solution. Biophys. J. 2004, 86, 455– 460, DOI: 10.1016/S0006-3495(04)74122-3
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Small angle X-ray scattering from lipid-bound myelin basic protein in solution
Haas, H.; Oliveira, C. L. P.; Torriani, I. L.; Polverini, E.; Fasano, A.; Carlone, G.; Cavatorta, P.; Riccio, P.
Biophysical Journal (2004), 86 (1, Pt. 1), 455-460CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
The structure of myelin basic protein (MBP), purified from the myelin sheath in both lipid-free (LF-MBP) and lipid-bound (LB-MBP) forms, was investigated in soln. by small angle x-ray scattering. The water-sol. LF-MBP, extd. at pH < 3.0 from defatted brain, is the classical prepn. of MBP, commonly regarded as an intrinsically unfolded protein. LB-MBP is a lipoprotein-detergent complex extd. from myelin with its native lipidic environment at pH > 7.0. Under all conditions, the scattering from the two protein forms was different, indicating different mol. shapes. For the LB-MBP, well-defined scattering curves were obtained, suggesting that the protein had a unique, compact (but not globular) structure. Furthermore, these data were compatible with earlier results from mol. modeling calcns. on the MBP structure which have been refined by us. In contrast, the LF-MBP data were in accordance with the expected open-coil conformation. The results represent the first direct structural information from x-ray scattering measurements on MBP in its native lipidic environment in soln.
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Jahn, O.; Tenzer, S.; Werner, H. Myelin Proteomics: Molecular Anatomy of an Insulating Sheath. Mol. Neurobiol. 2009, 40, 55– 72, DOI: 10.1007/s12035-009-8071-2
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Myelin Proteomics: Molecular Anatomy of an Insulating Sheath
Jahn, Olaf; Tenzer, Stefan; Werner, Hauke B.
Molecular Neurobiology (2009), 40 (1), 55-72CODEN: MONBEW; ISSN:0893-7648. (Humana Press Inc.)
A review. Fast-transmitting vertebrate axons are elec. insulated with multiple layers of nonconductive plasma membrane of glial cell origin, termed myelin. The myelin membrane is dominated by lipids, and its protein compn. has historically been viewed to be of very low complexity. In this review, we discuss an updated ref. compendium of 342 proteins assocd. with central nervous system myelin that represents a valuable resource for analyzing myelin biogenesis and white matter homeostasis. Cataloging the myelin proteome has been made possible by tech. advances in the sepn. and mass spectrometric detection of proteins, also referred to as proteomics. This led to the identification of a large no. of novel myelin-assocd. proteins, many of which represent low abundance components involved in catalytic activities, the cytoskeleton, vesicular trafficking, or cell adhesion. By mass spectrometry-based quantification, proteolipid protein and myelin basic protein constitute 17% and 8% of total myelin protein, resp., suggesting that their abundance was previously overestimated. As the biochem. profile of myelin-assocd. proteins is highly reproducible, differential proteome analyses can be applied to material isolated from patients or animal models of myelin-related diseases such as multiple sclerosis and leukodystrophies.
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Fewster, M. E.; Hirono, H.; Mead, J. F. Lipid composition of myelin in multiple sclerosis. J. Neurol. 1976, 213, 119– 131, DOI: 10.1007/BF00313273
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Lipid composition of myelin in multiple sclerosis
Fewster, Mona E.; Hirono, H.; Mead, J. F.
Journal of Neurology (1976), 213 (2), 119-31CODEN: JNRYA9; ISSN:0340-5354.
Myelin was isolated from histol. normal white matter and plaques from multiple sclerosis (MS) patients and from white matter of neurol. normal controls. No difference was found in the total lipid content. There were no detectable deficits in MS myelin of phosphoglycerides, plasmalogens or sphingolipids. Gangliosides and lysolecithin were not detected. Anal. of the fatty aldehyde compn. of the phosphoglycerides and the fatty acid compn. of the cholesteryl esters, phosphoglycerides and sphingolipids did not show any differences between the normal and MS myelin.
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Wilson, R.; Tocher, D. R. Lipid and fatty acid composition is altered in plaque tissue from multiple sclerosis brain compared with normal brain white matter. Lipids 1991, 26, 9– 15, DOI: 10.1007/BF02544017
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Lipid and fatty acid composition is altered in plaque tissue from multiple sclerosis brain compared with normal brain white matter
Wilson, Robert; Tocher, Douglas R.
Lipids (1991), 26 (1), 9-15CODEN: LPDSAP; ISSN:0024-4201.
Plaques and white matter from brains of multiple sclerosis (MS) patients were analyzed for lipid content, class compn., and fatty acid compn. of total lipid, together with the fatty acid compn. of plaque glycerophospholipids, and the results were compared with white matter from normal brain. Plaques contained less than 30% of the lipid present in normal white matter. Plaque lipid was characterized by significantly increased proportions of glycerophospholipids and decreased cerebrosides and sulfatides. In addn., a subacute plaque contained approx. 10 times the proportion of steryl esters obsd. in chronic plaques or normal white matter. Total lipid from all the MS plaques showed significantly increased percentages of satd. fatty acids, n-6, n-3 and total polyunsatd. fatty acids and decreased percentages of monoenes and alk-1-enyl ethers in comparison with normal brains. These results were consistent with increased cellularity and astrogliosis assocd. with MS plaques. However, anal. of plaque glycerophospholipids showed that the fatty acid changes obsd. in total lipid were not simply due to the increased myelin lipids, but that the fatty acid compn. of the individual glycerophospholipids was different.
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Alling, C.; Vanier, M. T.; Svennerholm, L. Lipid alterations in apparently normal white matter in multiple sclerosis. Brain Res. 1971, 35, 325– 336, DOI: 10.1016/0006-8993(71)90478-1
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Lipid alterations in apparently normal white matter in multiple sclerosis
Alling, Christer; Vanier, Marie T.; Svennerholm, Lars
Brain Research (1971), 35 (2), 325-36CODEN: BRREAP; ISSN:0006-8993.
Apparently normal white matter from 6 subjects with multiple sclerosis (MS) of 5-41 years duration was compared with roughly age-matched normal tissue. Cholesterol, phospholipids, and galactolipids were slightly lower in MS brains, but significantly lower values were found only for serine phosphoglycerides and sulfatides. The fatty acid patterns of MS brains were strikingly similar to those of normal brains. Nevertheless, the concns. of some of the fatty acids characteristic of white matter were slightly lower in MS brains. There was no evidence for a primary lipid defect of myelin. The abnormalities in the lipid and the fatty acid compn. were explained by the presence of small lesions overlooked at isolation of apparently normal white matter.
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Woelk, H.; Borri, P. Lipid and fatty acid composition of myelin purified from normal and MS brains. Eur. Neurol. 1973, 10, 250– 260, DOI: 10.1159/000114281
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Lipid and fatty acid composition of myelin purified from normal and MS [multiple sclerosis] brains
Woelk, Helmut; Borri, Piero
European Neurology (1973), 10 (4), 250-60CODEN: EUNEAP; ISSN:0014-3022.
The concns. of phospholipids and galactolipids and their fatty acid compns. were detd. in purified myelin from apparently normal white matter of 11 MS and 11 control brains. Myelin from MS brains showed a significant decrease in serine phosphatide and a slight decrease in the ethanolamine phosphatide fraction when compared with normal myelin; increases were found in the lysocholine and lysoethanolamine phosphoglycerides. In the galactolipids, lower figures were obtained for sulfatides, resulting in a higher cerebroside/sulfatide ratio for MS myelin. Anal. of the fatty acid pattern of phospholipids in MS brains revealed lower values for 18:1 and, except for the lyso compds., slightly higher amts. for 20:4 and 22:6. Lower levels for the sum of 18:3 and 20:1 fatty acids were found for the ethanolamine and inositol phosphoglyceride fractions.
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Gerstl, B.; Eng, L. F.; Tavaststjerna, M.; Smith, J. K.; Kruse, S. L. Lipids and proteins in multiple sclerosis white matter. J. Neurochem. 1970, 17, 677– 689, DOI: 10.1111/j.1471-4159.1970.tb00547.x
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Lipids and proteins in multiple sclerosis white matter
Gerstl, Bruno; Eng, Lawrence F.; Tavaststjerna, M.; Smith, James Knox; Kruse, S. L.
Journal of Neurochemistry (1970), 17 (5), 677-89CODEN: JONRA9; ISSN:0022-3042.
Quant. anals. of white matter from four brains of patients with multiple sclerosis (MS) and four control brains were carried out for total and sol. proteins, individual lipid fractions, and their corresponding fatty acids. In three specimens from two of the MS brains there were redns. of cerebrosides and of the C20:1 acid in the ethanolamine glycerophosphatide (EGP) fraction and a slight increase of tetraenes and trienes, while all other components were present in concns. similar to those in the controls. In three other samples from two of the MS brains, galactolipids were deficient to a greater extent than cholesterol, EGP, or CGP (choline glycerophosphatide), while proteins were within the control range. In samples where thinning of myelin was observed in Luxol-blue stained sections, there were proportional decreases of all components. The percentage of C20:1 acid in the EGP fraction was reduced in two of three myelin prepns. from corresponding samples of MS white matter, and that of C24:1 acid in the cerebroside fraction was reduced in all three MS myelin prepns. when compared with the two controls. The data suggest that inadequacy of the fatty acid elongation process together with deficits of cerebrosides represent one of the early biochem. lesions in the white matter of the MS brain.
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Arnetoli, G.; Pazzagli, A.; Amaducci, L. Fatty acid and aldehyde changes in choline- and ethanolamine-containing phospholipids in the white matter of multiple sclerosis brains. J. Neurochem. 1969, 16, 461– 463, DOI: 10.1111/j.1471-4159.1969.tb10387.x
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Fatty acid and aldehyde changes in choline- and ethanolamine-containing phospholipids in the white matter of multiple sclerosis brains
Arnetoli, G.; Pazzagli, Adolfo; Amaducci, Luigi
Journal of Neurochemistry (1969), 16 (3), 461-3CODEN: JONRA9; ISSN:0022-3042.
As detd. by gas chromatog. of the fatty acid methyl esters, the fatty acid compn. of the choline-contg. phospholipids from central white matter of 5 multiple sclerosis brains was not different from that in 4 control brains. In multiple sclerosis brains the percentage of monounsatd. C18 fatty acids in ethanolamine-contg. phospholipids from central white matter was decreased and the percentage of satd. C18 fatty acids and (or) monounsatd. C18 fatty aldehydes was increased compared to controls. Thus, the ratio of satd. C18 acid and (or) monounsatd. C18-aldehyde to monounsatd. C18-fatty acid in ethanolamine-contg. phospholipids was 1.51 in multiple sclerosis brains compared to 0.82 in controls. Thus ethanolamine-contg. phospholipids may be affected earlier or to a greater extent than choline-contg. phospholipids in this type of disease.
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Göpfert, E.; Pytlik, S.; Debuch, H. 2′,3′-Cyclic nucleotide 3′-phosphohydrolase and lipids of myelin from multiple sclerosis and normal brains. J. Neurochem. 1980, 34, 732– 739, DOI: 10.1111/j.1471-4159.1980.tb11205.x
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2',3'-Cyclic nucleotide 3'-phosphohydrolase and lipids of myelin from multiple sclerosis and normal brains
Gopfert E; Pytlik S; Debuch H
Journal of neurochemistry (1980), 34 (3), 732-9 ISSN:0022-3042.
There is no expanded citation for this reference.
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Neu, I.; Woelk, H. Investigations of the lipid metabolism of the white matter in multiple sclerosis Changes in glycero-phosphatides and lipid-splitting enzymes. Neurochem. Res. 1982, 7, 727– 735, DOI: 10.1007/BF00965525
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Investigations of the lipid metabolism of the white matter in multiple sclerosis: changes in glycero-phosphatides and lipid-splitting enzymes
Neu I; Woelk H
Neurochemical research (1982), 7 (6), 727-35 ISSN:0364-3190.
Phospho- and galacto- lipids and lipidhydrolyzing enzymes have been determined in the white matter of a young patient with a subacute course of multiple sclerosis (MS). Significant changes were observed for the concentration of glycerophosphatides and the fatty acid pattern of the normal appearing with matter surrounding MS-plaques. Among the individual glycerophosphatides a significant decrease of phosphatidylserine and phosphatidylinositol was found, whereas the ethanolamine containing phosphatides showed lower figures (non significant). The fatty acid pattern of the ethanolamine-phosphatide-fraction of the diseased tissue a decrease of the 18:1 and the sum of 20:1 and 18:3 fatty acids as compared to the normal control, whereas the highly unsaturated, long-chained fatty acids 20:4 (arachidonic acid) and 22:6 (docosahexaenic acid) were elevated. The measurement of lipidhydrolyzing enzymes resulted in an increased phospholipase A1 activity in the diseased tissue. The experimental data point to a decreased activity of the fatty acid elongation system in the course of MS. The decrease of the acidic glycerophosphatides might be due to the increased phospholipase A1 activity.
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Del Boccio, P.; Pieragostino, D.; Di Ioia, M.; Petrucci, F.; Lugaresi, A.; De Luca, G.; Gambi, D.; Onofrj, M.; Di Ilio, C.; Sacchetta, P.; Urbani, A. Lipidomic investigations for the characterization of circulating serum lipids in multiple sclerosis. J. Proteomics 2011, 74, 2826– 2836, DOI: 10.1016/j.jprot.2011.06.023
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Lipidomic investigations for the characterization of circulating serum lipids in multiple sclerosis
Del Boccio, Piero; Pieragostino, Damiana; Di Ioia, Maria; Petrucci, Francesca; Lugaresi, Alessandra; De Luca, Giovanna; Gambi, Domenico; Onofrj, Marco; Di Ilio, Carmine; Sacchetta, Paolo; Urbani, Andrea
Journal of Proteomics (2011), 74 (12), 2826-2836CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)
Multiple Sclerosis (MS) is a neurodegenerative autoimmune demyelinating disease affecting young adults. The etiol. still remains a mystery and diagnosis is impaired by the lack of defined mol. markers. Autoimmune response remains the main topic under investigation and recent studies suggest addnl. non-proteic mediators of brain inflammation such as lipids. We carried out an LC-MS based lipidomics approach to highlight serum lipids profiling in MS. Method was optimized and applied in a preliminary clin. cross-sectional investigation of MS patients vs Healthy Controls (HC) and patients with Other Neurol. Diseases (OND). Ten significant metabolites were highlighted and tentatively identified by accurate mass and MS/MS expts. Our most relevant data show altered level of lyso-glycerophosphatidylcholine (lysoPC) and glycerophosphatidylcholine (PC) species. Total lysoPC/PC ratio showed significant decrease in pathol. groups (MS, OND) and, in addn., MS subjects had a relevant decrease of this ratio also in respect to OND. These findings suggest that there may be an altered phospholipid metab. in MS that can be evaluated in serum. Some of these features are distinctive and may be considered specific for MS. Our lipidomics data show, for the first time, evidence in serum of a relationship between LysoPC/PC ratio and MS.
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Boggs, J. M. Myelin Basic Protein; Nova Science Publishers, Incorporated: New York, 2008.
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44
Procaccini, C.; De Rosa, V.; Pucino, V.; Formisano, L.; Matarese, G. Animal models of Multiple Sclerosis. Eur. J. Pharmacol. 2015, 759, 182– 191, DOI: 10.1016/j.ejphar.2015.03.042
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Animal models of Multiple Sclerosis
Procaccini, Claudio; De Rosa, Veronica; Pucino, Valentina; Formisano, Luigi; Matarese, Giuseppe
European Journal of Pharmacology (2015), 759 (), 182-191CODEN: EJPHAZ; ISSN:0014-2999. (Elsevier B.V.)
Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) which involves a complex interaction between immune system and neural cells. Animal modeling has been crit. for addressing MS pathogenesis. The three most characterized animal models of MS are (1) the exptl. autoimmune/allergic encephalomyelitis (EAE); (2) the virally-induced chronic demyelinating disease, known as Theiler's murine encephalomyelitis virus (TMEV) infection and (3) the toxin-induced demyelination. All these models, in a complementary way, have allowed to reach a good knowledge of the pathogenesis of MS. Specifically, EAE is the model which better reflects the autoimmune pathogenesis of MS and is extremely useful to study potential exptl. treatments. Furthermore, both TMEV and toxin-induced demyelination models are suitable for characterizing the role of the axonal injury/repair and the remyelination process in MS. In conclusion, animal models, despite their limitations, remain the most useful instrument for implementing the study of MS.
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Niroomand, H.; Pamu, R.; Mukherjee, D.; Khomami, B. Microenvironment alterations enhance photocurrents from photosystem I confined in supported lipid bilayers. J. Mater. Chem. A 2018, 6, 12281– 12290, DOI: 10.1039/C8TA00898A
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Microenvironment alterations enhance photocurrents from photosystem I confined in supported lipid bilayers
Niroomand, Hanieh; Pamu, Ravi; Mukherjee, Dibyendu; Khomami, Bamin
Journal of Materials Chemistry A: Materials for Energy and Sustainability (2018), 6 (26), 12281-12290CODEN: JMCAET; ISSN:2050-7496. (Royal Society of Chemistry)
Transmembrane photosynthetic proteins, photosystem I (PSI), are nano-scale biol. photodiodes that enable light-activated unidirectional electron flow. The robust photochem. properties of PSI make it a promising candidate for harnessing solar energy. However, the role of natural membrane confinements of PSI in orchestrating this photoactivated charge sepn. with near unity quantum efficiency, which is central to the rational design of PSI-based energy conversion systems, is still ill-understood. Motivated by this lack of fundamental understanding, herein the authors investigate the photoactivity of biomimetic constructs of cyanobacterial PSI encapsulated within solid-supported lipid bilayers (SLB) assembled on electrodes. PSI confined in SLBs is assembled from PSI-proteoliposomes that are synthesized from the recently developed facile routes for engineering neg. charged phospholipid (DPhPG) bilayer membranes. Specifically, detailed chronoamperometry measurements have been used to investigate photocurrent variations arising from the SLBs supported on self-assembled monolayer (SAM) substrates. These measurements, in conjunction with cryo-TEM, at. force microscopy imaging and force spectroscopy, allow for direct visualization and detection of the SLBs of PSI-proteoliposomes on the substrates. The results indicate the crit. role of microenvironment alterations, heretofore not considered, in achieving ∼4-5-fold enhancements in photocurrents generated from PSI complexes under SLB confinements as compared to those from a dense monolayer of equiv. concns. of PSI on SAM substrates.
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Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. Theory of self-assembly of lipid bilayers and vesicles. Biochim. Biophys. Acta, Biomembr. 1977, 470, 185– 201, DOI: 10.1016/0005-2736(77)90099-2
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Theory of self-assembly of lipid bilayers and vesicles
Israelachvili, Jacob N.; Mitchell, D. John; Ninham, Barry W.
Biochimica et Biophysica Acta, Biomembranes (1977), 470 (2), 185-201CODEN: BBBMBS; ISSN:0005-2736.
A simple theory is developed that explains the formation of bilayers and vesicles and accounts quant. for many of their phys. properties. Properties including vesicle size distributions and bilayer elasticity, emerge from a unified theory that links thermodn., interaction free energy, and mol. geometry. The theory may be applied to the anal. of more complicated membrane structures and mechanisms.
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Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press, 2015.
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48
Wood, D. D.; Moscarello, M. A. The isolation, characterization, and lipid-aggregating properties of a citrulline containing myelin basic protein. J. Biol. Chem. 1989, 264, 5121– 5127
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The isolation, characterization, and lipid-aggregating properties of a citrulline containing myelin basic protein
Wood, D. D.; Moscarello, M. A.
Journal of Biological Chemistry (1989), 264 (9), 5121-7CODEN: JBCHA3; ISSN:0021-9258.
Human myelin basic protein was fractionated into its various charge isomers by CM52 cation exchange chromatog. Of the total charge applied to the column, ∼25-30% appeared in the void vol. This material, termed C-8, was further purified by reversed phase HPLC. Amino acid analyses of C-8 revealed low arginine (Arg) (7 residue % in C-8 compared to 11-12 residue % in C-1) and increased glutamic acid or glutamine residues. The low Arg was accounted for by a corresponding amt. of citrulline. Sequence anal. after chem. fragmentation (CNBr and BNPS-skatole) and enzymic (cathepsin D and carboxypeptidase S-1) digestion localized the citrulline at residues 25, 31, 122, 130, 159, and 170 of the amino acid sequence. The effect of this loss of pos. charge on the ability of the protein to aggregate lipid vesicles was demonstrated with vesicles composed of phosphatidylcholine (92.2 mol %) and phosphatidylserine (7.8 mol %). C-1 was the most effective charge isomer, and C-8 was the least effective. The ability of these charge isomers to aggregate vesicles correlated with the net pos. charge on each. Vesicles composed of phosphatidylcholine alone were not aggregated by lipophilin or any of the charge isomers. However, when lipophilin was incorporated into phosphatidylcholine vesicles (50%), small, optically clear suspensions of vesicles were formed. None of C-1, C-2, or C-3 aggregated these vesicles, but C-8 produced rapid vesicle aggregation. Since the substitution of citrulline for Arg would generate several relatively long apolar sequences, these would enhance the ability of C-8 to interact with the hydrophobic lipophilin mol., promoting vesicle aggregation by hydrophobic interactions.
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Harauz, G.; Musse, A. A Tale of Two Citrullines—Structural and Functional Aspects of Myelin Basic Protein Deimination in Health and Disease. Neurochem. Res. 2007, 32, 137– 158, DOI: 10.1007/s11064-006-9108-9
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A Tale of Two Citrullines-Structural and Functional Aspects of Myelin Basic Protein Deimination in Health and Disease
Harauz, George; Musse, Abdiwahab A.
Neurochemical Research (2007), 32 (2), 137-158CODEN: NEREDZ; ISSN:0364-3190. (Springer)
A review. Myelin basic protein (MBP) binds to neg. charged lipids on the cytosolic surface of oligodendrocyte membranes and is responsible for adhesion of these surfaces in the multilayered myelin sheath. The pattern of extensive post-translational modifications of MBP is dynamic during normal central nervous system (CNS) development and during myelin degeneration in multiple sclerosis (MS), affecting its interactions with the myelin membranes and with other mols. In particular, the degree of deimination (or citrullination) of MBP is correlated with the severity of MS, and may represent a primary defect that precedes neurodegeneration due to autoimmune attack. That the degree of MBP deimination is also high in early CNS development indicates that this modification plays major physiol. roles in myelin assembly. In this review, we describe the structural and functional consequences of MBP deimination in healthy and diseased myelin.
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Matsumoto, T.; Kobayashi, T.; Kamata, K. Role of lysophosphatidylcholine (LPC) in atherosclerosis. Curr. Med. Chem. 2007, 14, 3209– 3220, DOI: 10.2174/092986707782793899
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Role of lysophosphatidylcholine (LPC) in atherosclerosis
Matsumoto, Takayuki; Kobayashi, Tsuneo; Kamata, Katsuo
Current Medicinal Chemistry (2007), 14 (30), 3209-3220CODEN: CMCHE7; ISSN:0929-8673. (Bentham Science Publishers Ltd.)
A review. Lysophosphatidylcholine (LPC) is a bioactive proinflammatory lipid generated by pathol. activities. LPC is also a major phospholipid component of oxidized low-d. lipoprotein (Ox-LDL) and is implicated as a crit. factor in the atherogenic activity of Ox-LDL. LPC is believed to play an important role in atherosclerosis and inflammatory diseases by altering various functions in a no. of cell-types, including endothelial cells, smooth muscle cells, monocytes, macrophages, and T-cells. LPC activates several second messengers - including protein kinase C, extracellular-signal-regulated kinases, protein tyrosine kinases, and Ca2+ -- implicating the engagement of transduction mechanisms in its obsd. actions. Moreover, recent evidence suggests that in several cell-types, cloned orphan G-protein-coupled receptors may serve as the specific receptors via which LPC modulates second messenger pathways (although LPC may not be a direct ligand of such receptors). In addn., current evidence suggests that LPC impairs the endothelium-dependent relaxations mediated by endothelium-derived relaxing factors and directly modulates contractile responses in vascular smooth muscle. However, despite all this, and although elevated levels of LPC have been linked to the cardiovascular complications assocd. with atherosclerosis, ischemia, and diabetes, the precise pathophysiol. roles played by LPC in several states remain to be established. In this review, we focus in some detail on the entirety of the signal-transduction system for LPC, its pathophysiol. implications, and the vascular abnormalities assocd. with it.
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Muramatsu, R.; Kuroda, M.; Matoba, K.; Lin, H.; Takahashi, C.; Koyama, Y.; Yamashita, T. Prostacyclin prevents pericyte loss and demyelination induced by lysophosphatidylcholine in the central nervous system. J. Biol. Chem. 2015, 290, 11515– 11525, DOI: 10.1074/jbc.M114.587253
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Prostacyclin Prevents Pericyte Loss and Demyelination Induced by Lysophosphatidylcholine in the Central Nervous System
Muramatsu, Rieko; Kuroda, Mariko; Matoba, Ken; Lin, Hsiaoyun; Takahashi, Chisato; Koyama, Yoshihisa; Yamashita, Toshihide
Journal of Biological Chemistry (2015), 290 (18), 11515-11525CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
Pericytes play pivotal roles in physiol. and pathophysiol. conditions in the central nervous system. As pericytes prevent vascular leakage, they can halt neuronal damage stemming from a compromised blood-brain barrier. Therefore, pericytes may be a good target for the treatment of neurodegenerative disorders, although evidence is lacking. In this study, we show that prostacyclin attenuates lysophosphatidylcholine (LPC)-mediated vascular dysfunction through pericyte protection in the adult mouse spinal cord. LPC decreased the no. of pericytes in an in vitro blood-brain barrier model, and this decrease was prevented by iloprost treatment, a prostacyclin analog. Intrathecal administration of iloprost attenuated vascular barrier disruption after LPC injection in the mouse spinal cord. Furthermore, iloprost treatment diminished demyelination and motor function deficits in mice injected with LPC. These results support the notion that prostacyclin acts on pericytes to maintain vascular barrier integrity.
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Plemel, J. R.; Michaels, N. J.; Weishaupt, N.; Caprariello, A. V.; Keough, M. B.; Rogers, J. A.; Yukseloglu, A.; Lim, J.; Patel, V. V.; Rawji, K. S.; Jensen, S. K.; Teo, W.; Heyne, B.; Whitehead, S. N.; Stys, P. K.; Yong, V. W. Mechanisms of lysophosphatidylcholine-induced demyelination: A primary lipid disrupting myelinopathy. Glia 2018, 66, 327– 347, DOI: 10.1002/glia.23245
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Mechanisms of lysophosphatidylcholine-induced demyelination: A primary lipid disrupting myelinopathy
Plemel Jason R; Michaels Nathan J; Caprariello Andrew V; Keough Michael B; Rogers James A; Yukseloglu Aran; Lim Jaehyun; Rawji Khalil S; Jensen Samuel K; Teo Wulin; Stys Peter K; Yong V Wee; Weishaupt Nina; Patel Vikas V; Whitehead Shawn N; Heyne Belinda
Glia (2018), 66 (2), 327-347 ISSN:.
For decades lysophosphatidylcholine (LPC, lysolecithin) has been used to induce demyelination, without a clear understanding of its mechanisms. LPC is an endogenous lysophospholipid so it may cause demyelination in certain diseases. We investigated whether known receptor systems, inflammation or nonspecific lipid disruption mediates LPC-demyelination in mice. We found that LPC nonspecifically disrupted myelin lipids. LPC integrated into cellular membranes and rapidly induced cell membrane permeability; in mice, LPC injury was phenocopied by other lipid disrupting agents. Interestingly, following its injection into white matter, LPC was cleared within 24 hr but by five days there was an elevation of endogenous LPC that was not associated with damage. This elevation of LPC in the absence of injury raises the possibility that the brain has mechanisms to buffer LPC. In support, LPC injury in culture was significantly ameliorated by albumin buffering. These results shed light on the mechanisms of LPC injury and homeostasis.
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Birgbauer, E.; Rao, T. S.; Webb, M. Lysolecithin induces demyelination in vitro in a cerebellar slice culture system. J. Neurosci. Res. 2004, 78, 157– 166, DOI: 10.1002/jnr.20248
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Lysolecithin induces demyelination in vitro in a cerebellar slice culture system
Birgbauer, Eric; Rao, Tadimeti S.; Webb, Michael
Journal of Neuroscience Research (2004), 78 (2), 157-166CODEN: JNREDK; ISSN:0360-4012. (Wiley-Liss, Inc.)
Demyelination is a hallmark of several human diseases, including multiple sclerosis. To understand better the process of demyelination and remyelination, we explored the use of an in vitro organotypic cerebellar slice culture system. Para-sagittal slices of postnatal Day 10 (P10) rat cerebellar cultured in vitro demonstrated significant myelination after 1 wk in culture. Treatment of the cultures at 7 days in vitro (DIV) with the bioactive lipid lysolecithin (lysophosphatidylcholine) for 15-17 h in vitro produced marked demyelination. This demyelination was obsd. by immunostaining for the myelin components myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). After a transient demyelinating insult with lysolecithin in vitro, the cultures recovered with oligodendrocyte differentiation recapitulating a normal time course; there was initially re-expression of CNPase and MBP during this recovery, and this was followed by MOG. In addn., there seemed to be some limited remyelination during the recovery phase. Lysolecithin thus induces demyelination in an in vitro organotypic cerebellar slice culture system, providing a model system for studying myelination, demyelination, and remyelination in vitro.
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Boggs, J. M. Myelin basic protein: a multifunctional protein. Cell. Mol. Life Sci. 2006, 63, 1945– 1961, DOI: 10.1007/s00018-006-6094-7
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Myelin basic protein: a multifunctional protein
Boggs, J. M.
Cellular and Molecular Life Sciences (2006), 63 (17), 1945-1961CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
A review. Myelin basic protein (MBP), the 2nd most abundant protein in central nervous system myelin, is responsible for adhesion of the cytosolic surfaces of multilayered compact myelin. A member of the 'intrinsically disordered' or conformationally adaptable protein family, it also appears to have several other functions. It can interact with a no. of polyanionic proteins including actin, tubulin, Ca2+-calmodulin, and clathrin, and neg. charged lipids, and acquires structure on binding to them. It may act as a membrane actin-binding protein, which might allow it to participate in transmission of extracellular signals to the cytoskeleton in oligodendrocytes and tight junctions in myelin. Some size isoforms of MBP are transported into the nucleus and thus they may also bind polynucleotides. Extracellular signals received by myelin or cultured oligodendrocytes cause changes in the phosphorylation of MBP, suggesting that MBP is also involved in signaling. Further study of this very abundant protein will reveal how it is utilized by the oligodendrocyte and myelin for different purposes.
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Harauz, G.; Ishiyama, N.; Hill, C. M.; Bates, I. R.; Libich, D. S.; Fares, C. Myelin basic protein-diverse conformational states of an intrinsically unstructured protein and its roles in myelin assembly and multiple sclerosis. Micron 2004, 35, 503– 542, DOI: 10.1016/j.micron.2004.04.005
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Myelin basic protein - diverse conformational states of an intrinsically unstructured protein and its roles in myelin assembly and multiple sclerosis
Harauz, George; Ishiyama, Noboru; Hill, Christopher M. D.; Bates, Ian R.; Libich, David S.; Fares, Christophe
Micron (2004), 35 (7), 503-542CODEN: MCONEN; ISSN:0968-4328. (Elsevier Science B.V.)
A review. The 18.5-kDa isoform of myelin basic protein (MBP) is a major component of the myelin sheath in the central nervous system of higher vertebrates, and a member of a larger family of proteins with a multiplicity of forms and post-translational modifications (PTMs). The 18.5-kDa protein is the exemplar of the family, being most abundant in adult myelin, and thus the most-studied. It is peripherally membrane-assocd., but has generally been investigated in isolated form. MBP is an 'intrinsically unstructured' protein with a high proportion (∼75%) of random coil, but postulated to have core elements of β-sheet and α-helix. Here, the authors review the properties of the MBP family, esp. of the 18.5-kDa isoform, and discuss how its 3-dimensional (3D) structure may be resolved by direct techniques available to them, viz., x-ray and electron crystallog., and soln. and solid-state NMR spectrometry. In particular, it is emphasized that creating an appropriate environment in which the protein can adopt a physiol. relevant fold is crucial to such endeavors. By solving the 3D structure of 18.5-kDa MBP and the effects of PTMs, a better understanding of myelin architecture, and of the mol. mechanisms that transpire in demyelinating diseases such as multiple sclerosis, will be attained.
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D’Souza, C. A.; Wood, D. D.; She, Y.-M.; Moscarello, M. A. Autocatalytic cleavage of myelin basic protein: An alternative to molecular mimicry. Biochemistry 2005, 44, 12905– 12913, DOI: 10.1021/bi051152f
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Autocatalytic Cleavage of Myelin Basic Protein: An Alternative to Molecular Mimicry
D'Souza, Cheryl A.; Wood, D. Denise; She, Yi-Min; Moscarello, Mario A.
Biochemistry (2005), 44 (38), 12905-12913CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
Although multiple sclerosis (MS) is thought to be an autoimmune disease, the mechanisms by which immunodominant epitopes are generated and lymphocytes are activated are not known. Here, myelin basic protein-component 1 (MBP-C1) from MS tissue was shown to undergo autocatalytic cleavage at slightly alk. pH. Importantly, one of the major peptides released contained the immunodominant epitope 84-89. Interestingly, MBP isolated from MS patients showed a faster time course of cleavage and a more robust release of epitope 84-89 than MBP isolated from normal individuals. The cleavage reaction was not inhibited by protease inhibitors, except for phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor. Since PMSF inhibition suggested a role for a serine residue in the cleavage, we labeled myelin basic protein with diisopropyl fluorophosphate (DFP), known to bind active site serine residues. Mass spectrometry was used to identify the labeled peptide, which consisted of residues 140-152. Since this peptide contained a single serine residue, we concluded it to be the active serine. The importance of this cleavage mechanism is that it provides for a ready source of the immunodominant peptide for sensitization of T-cells. It is not necessary to invoke other mechanisms such as mol. mimicry.
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Zhou, S. R.; Moscarello, M. A.; Whitaker, J. N. The effects of citrullination or variable amino-terminus acylation on the encephalitogenicity of human myelin basic protein in the PL/J mouse. J. Neuroimmunol. 1995, 62, 147– 152, DOI: 10.1016/0165-5728(95)00112-3
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The effects of citrullination or variable amino-terminus acylation on the encephalitogenicity of human myelin basic protein in the PL/J mouse
Zhou, Shan-Ren; Moscarello, Mario; Whitaker, John N.
Journal of Neuroimmunology (1995), 62 (2), 147-152CODEN: JNRIDW; ISSN:0165-5728. (Elsevier)
The post-translational modifications of myelin basic protein (MBP) in the form of citrullination and varying length of amino-terminus acylation may modify the biol. functions and immunol. features of MBP. Both modifications influence the reaction of antibodies and specific T cells recognizing MBP. The present study was undertaken to compare the encephalitogenicity of the citrullinated isomer of MBP (MBP-C8) with the unmodified isomer of MBP (MBP-C1) and to det. if the length of amino-terminal acylation of MBP peptide 1-21 altered an encephalitogenic epitope. MBP-C8, whether from patients with or without multiple sclerosis (MS), and MBP-C1 could induce active exptl. allergic encephalomyelitis (EAE) in PL/J mice. A trend of reduced severity of EAE was obsd. in MBP-C8-injected animals. An increase in the length of amino-terminus fatty acid decreased the encephalitogenicity of MBP peptide 1-21 for both active and adoptive EAE in PL/J mice. Only lymph node cells sensitive to MBP peptide acetyl 1-21 and Bu 1-21 could transfer clin. EAE. In adoptive EAE, MBP peptides hexyl and octyl 1-21 induced moderate histopathol. but no clin. change, whereas MBP peptide decyl 1-21 caused neither. A broadening in the antibody response could be detected in the sera of mice with active EAE induced by MBP-acylated peptides 1-21. Our findings demonstrate that encephalitogenicity is retained in the presence of citrullination but that the length of amino-terminus acylation diminishes the encephalitogenicity of MBP in the PL/J mouse. These findings may be relevant to MS where central nervous system myelin shows differences from normal in both MBP-C8 content and MBP amino-terminus acylation.
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Madsen, P. M.; Motti, D.; Karmally, S.; Szymkowski, D. E.; Lambertsen, K. L.; Bethea, J. R.; Brambilla, R. Oligodendroglial TNFR2 Mediates Membrane TNF-Dependent Repair in Experimental Autoimmune Encephalomyelitis by Promoting Oligodendrocyte Differentiation and Remyelination. J. Neurosci. 2016, 36, 5128– 5143, DOI: 10.1523/JNEUROSCI.0211-16.2016
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Oligodendroglial TNFR2 Mediates Membrane TNF-Dependent Repair in Experimental Autoimmune Encephalomyelitis by Promoting Oligodendrocyte Differentiation and Remyelination
Madsen Pernille M; Motti Dario; Karmally Shaffiat; Brambilla Roberta; Szymkowski David E; Lambertsen Kate Lykke; Bethea John R
The Journal of neuroscience : the official journal of the Society for Neuroscience (2016), 36 (18), 5128-43 ISSN:.
UNLABELLED: Tumor necrosis factor (TNF) is associated with the pathophysiology of various neurological disorders, including multiple sclerosis. It exists as a transmembrane form tmTNF, signaling via TNF receptor 2 (TNFR2) and TNFR1, and a soluble form, solTNF, signaling via TNFR1. Multiple sclerosis is associated with the detrimental effects of solTNF acting through TNFR1, while tmTNF promotes repair and remyelination. Here we demonstrate that oligodendroglial TNFR2 is a key mediator of tmTNF-dependent protection in experimental autoimmune encephalomyelitis (EAE). CNP-cre:TNFR2(fl/fl) mice with TNFR2 ablation in oligodendrocytes show exacerbation of the disease with increased axon and myelin pathology, reduced remyelination, and increased loss of oligodendrocyte precursor cells and mature oligodendrocytes. The clinical course of EAE is not improved by the solTNF inhibitor XPro1595 in CNP-cre:TNFR2(fl/fl) mice, indicating that for tmTNF to promote recovery TNFR2 in oligodendrocytes is required. We show that TNFR2 drives differentiation of oligodendrocyte precursor cells, but not proliferation or survival. TNFR2 ablation leads to dysregulated expression of microRNAs, among which are regulators of oligodendrocyte differentiation and inflammation, including miR-7a. Our data provide the first direct in vivo evidence that TNFR2 in oligodendrocytes is important for oligodendrocyte differentiation, thereby sustaining tmTNF-dependent repair in neuroimmune disease. Our studies identify TNFR2 in the CNS as a molecular target for the development of remyelinating agents, addressing the most pressing need in multiple sclerosis therapy nowadays. SIGNIFICANCE STATEMENT: Our study, using novel TNF receptor 2 (TNFR2) conditional KO mice with selective TNFR2 ablation in oligodendrocytes, provides the first direct evidence that TNFR2 is an important signal for oligodendrocyte differentiation. Following activation by transmembrane TNF, TNFR2 initiates pathways that drive oligodendrocytes into a reparative mode contributing to remyelination following disease. This identifies TNFR2 as a new molecular target for the development of therapeutic agents in multiple sclerosis.
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Porciatti, V.; Saleh, M.; Nagaraju, M. The pattern electroretinogram as a tool to monitor progressive retinal ganglion cell dysfunction in the DBA/2J mouse model of glaucoma. Invest. Ophthalmol. Visual Sci. 2007, 48, 745– 751, DOI: 10.1167/iovs.06-0733
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The pattern electroretinogram as a tool to monitor progressive retinal ganglion cell dysfunction in the DBA/2J mouse model of glaucoma
Porciatti Vittorio; Saleh Maher; Nagaraju Mahesh
Investigative ophthalmology & visual science (2007), 48 (2), 745-51 ISSN:0146-0404.
PURPOSE: To determine the baseline characteristics, reliability, and dynamic range of the pattern electroretinogram (PERG) as a tool to monitor progressive RGC dysfunction in the DBA/2J mouse model of glaucoma with spontaneously elevated intraocular pressure (IOP). METHODS: PERGs were recorded from 56 undilated eyes of 28 anesthetized (ketamine-xylazine-acepromazine) DBA/2J mice of different ages (2-4 months, n = 44 eyes; 12-14 months, n = 12 eyes) in response to contrast reversal of gratings that maximize PERG amplitude (95% contrast, 1-Hz reversal, 0.05 cyc/deg spatial frequency, 50 degrees x 56 degrees field size). Robust averaging (1800 sweeps) was used to isolate PERG from background noise. Cone-driven ERGs in response to diffuse light flashes superimposed on a rod-adapting background (FERG) were also recorded. RESULTS: PERGs had consistent waveforms and were reproducible across batches of mice and operators. In 2- to 4-month-old mice (prehypertensive stage), the PERG amplitude (mean, 8.15 +/- 0.4 microV [SEM]) was considerably larger than the noise (mean 1.18 +/- 0.1 microV). The test-retest variability (two different sessions 1 week apart) and interocular asymmetry of PERG amplitude was approximately 30%, and that of PERG latency was approximately 17%. In 12- to 14-month-old mice (advanced hypertensive stage) the PERG amplitude (mean, 1.29 +/- 0.12 microV) was close to that of noise. In 12- to 14-month-old mice the FERG was reduced to a lesser extent compared with the PERG. CONCLUSIONS: The PERG has an adequate signal-to-noise ratio, reproducibility, and dynamic range to monitor the progression of functional changes in the inner retina in DBA/2J mice.
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Menon, K.; Rasband, M. N.; Taylor, C. M.; Brophy, P.; Bansal, R.; Pfeiffer, S. E. The myelin–axolemmal complex: biochemical dissection and the role of galactosphingolipids. J. Neurochem. 2003, 87, 995– 1009, DOI: 10.1046/j.1471-4159.2003.02075.x
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The myelin-axolemmal complex: Biochemical dissection and the role of galactosphingolipids
Menon, Krishna; Rasband, Matthew N.; Taylor, Christopher M.; Brophy, Peter; Bansal, Rashmi; Pfeiffer, Steven E.
Journal of Neurochemistry (2003), 87 (4), 995-1009CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)
Myelin-axolemmal interactions regulate many cellular and mol. events, including gene expression, oligodendrocyte survival and ion channel clustering. Here we report the biochem. fractionation and enrichment of distinct subcellular domains from myelinated nerve fibers. Using antibodies against proteins found in compact myelin, non-compact myelin and axolemma, we show that a rigorous procedure designed to purify myelin also results in the isolation of the myelin-axolemmal complex, a high-affinity protein complex consisting of axonal and oligodendroglial components. Further, the isolation of distinct subcellular domains from galactolipid-deficient mice with disrupted axoglial junctions is altered in a manner consistent with the delocalization of axolemmal proteins obsd. in these animals. These results suggest a paradigm for identification of proteins involved in neuroglial signaling.
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Valdivia, A. O.; Myer, C.; Suarez, M. F.; Bhattacharya, S. K. Protein–Lipid Complex Separation Utilizing a Capillary Electrophoresis System. In Metabolomics: Methods and Protocols; Bhattacharya, S. K., Ed.; Springer: New York, 1996; pp 95– 100.
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Chauhan, M. Z.; Valencia, A. K.; Piqueras, M. C.; Enriquez-Algeciras, M.; Bhattacharya, S. K. Optic Nerve Lipidomics Reveal Impaired Glucosylsphingosine Lipids Pathway in Glaucoma. Invest. Ophthalmol. Visual Sci. 2019, 60, 1789– 1798, DOI: 10.1167/iovs.18-25802
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Optic nerve lipidomics reveal impaired glucosylsphingosine lipids pathway in glaucoma
Chauhan, Muhammad Zain; Valencia, Ann-Katrin; Piqueras, Maria Carmen; Enriquez-Algeciras, Mabel; Bhattacharya, Sanjoy K.
Investigative Ophthalmology & Visual Science (2019), 60 (5), 1789-1798CODEN: IOVSDA; ISSN:1552-5783. (Association for Research in Vision and Ophthalmology)
To det. major differences in lipid profile between human control and glaucomatous optic nerve. To assess major enzymes in lipid pathway if aberration is revealed for a lipid class by profiling. Optic nerve (ON) samples were obtained from human cadaveric donors [control (n = 11) and primary open-angle glaucoma (POAG; n = 12)]; the lipids were extd. using Bligh and Dyer methods. Control and glaucoma donors were all Caucasians age 72.3 ± 5.9 and 70.3 ± 10.5 (inclusive of both sexes), resp. Lipids were extd. after weighing the tissue; the protein amts. in the corresponding aq. phase of org. solvent extn. were recorded. High-resoln. mass spectrometry was performed using a Qexactive mass spectrometer coupled with an EASY-nLC 1000 liq. chromatograph instrument. Bioinformatics and statistical anal. were performed using LipidSearch v.4.1 and MetaboAnalyst 4.0/STATA 14.2. Protein amts. were detd. using Bradford's method. Western blot, ELISA, and immunohistochem. utilized established protocols and were performed for protein quantification and localization, resp. Addnl. donor tissues were utilized for Western blot, ELISA, and immunohistochem. Principal component anal. (PCA) placed control and glaucomatous ONs in two distinct groups based on anal. of lipid profiles. Total lipid, total phospholipids, total ceramide, and total sphingolipids were similar (without significant difference) between control and glaucoma. However, we found a significant increase in glucosylsphingosine in glaucoma compared to control samples. We found similar levels of glucocerebrosidase (GBA), ceramide glucosyltransferase (UGCG), decreased nonlysosomal glucocerebrosidase (GBA2), and increased lysosomal and nonlysosomal acylsphingosine amidohydrolase (ASAH1 and ASAH2) levels in glaucomatous ON compared to control. We found significant differences in glucosylsphingosine lipids, consistent with decreased GBA and GBA2 and increased ASAH1 and ASAH2 immunoreactivity in glaucoma, suggesting the potential impairment of sphingolipid enzymic pathways in lysosomal and nonlysosomal cellular compartments.
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Travers, T. S.; Harlow, L.; Rosas, I. O.; Gochuico, B. R.; Mikuls, T. R.; Bhattacharya, S. K.; Camacho, C. J.; Ascherman, D. P. Extensive Citrullination Promotes Immunogenicity of HSP90 through Protein Unfolding and Exposure of Cryptic Epitopes. J. Immunol. 2016, 197, 1926– 1936, DOI: 10.4049/jimmunol.1600162
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Extensive Citrullination Promotes Immunogenicity of HSP90 through Protein Unfolding and Exposure of Cryptic Epitopes
Travers, Timothy S.; Harlow, Lisa; Rosas, Ivan O.; Gochuico, Bernadette R.; Mikuls, Ted R.; Bhattacharya, Sanjoy K.; Camacho, Carlos J.; Ascherman, Dana P.
Journal of Immunology (2016), 197 (5), 1926-1936CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
Post-translational protein modifications such as citrullination have been linked to the breach of immune tolerance and clin. autoimmunity. Previous studies from our lab. support this concept, demonstrating that autoantibodies targeting citrullinated isoforms of heat shock protein 90 (HSP90) are assocd. with rheumatoid arthritis complicated by interstitial lung disease. To further explore the relationship between citrullination and structural determinants of HSP90 immunogenicity, we employed a combination of ELISA-based epitope profiling, computational modeling, and mass-spectrometric sequencing of peptidylarginine deiminase (PAD)-modified protein. Remarkably, ELISAs involving selected citrullinated HSP90β/α peptides identified a key epitope corresponding to an internal Arg residue (R502 [HSP90β]/R510 [HSP90α]) that is normally buried within the crystal structure of native/unmodified HSP90. In vitro time/dose-response expts. reveal an ordered pattern of PAD-mediated deimination events culminating in citrullination of R502/R510. Conventional as well as scaled mol. dynamics simulations further demonstrate that citrullination of selected Arg residues leads to progressive disruption of HSP90 tertiary structure, promoting exposure of R502/R510 to PAD modification and subsequent autoantibody binding. Consistent with this process, ELISAs incorporating variably deiminated HSP90 as substrate Ag indicate a direct relationship between the degree of citrullination and the level of ex vivo Ab recognition. Overall, these data support a novel structural paradigm whereby citrullination-induced shifts in protein structure generate cryptic epitopes capable of bypassing B cell tolerance in the appropriate genetic context.
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Wiedemann, C.; Bellstedt, P.; Gorlach, M. CAPITO--a web server-based analysis and plotting tool for circular dichroism data. Bioinformatics 2013, 29, 1750– 1757, DOI: 10.1093/bioinformatics/btt278
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CAPITO-a web server-based analysis and plotting tool for circular dichroism data
Wiedemann, Christoph; Bellstedt, Peter; Goerlach, Matthias
Bioinformatics (2013), 29 (14), 1750-1757CODEN: BOINFP; ISSN:1367-4803. (Oxford University Press)
Motivation: CD (CD) spectroscopy is one of the most versatile tools to study protein folding and to validate the proper fold of purified proteins. Here, we aim to provide a readily accessible, user-friendly and platform-independent tool capable of analyzing multiple CD datasets of virtually any format and returning results as high-quality graphical output to the user. Results: CAPITO (CD Anal. and Plotting Tool) is a novel web server-based tool for analyzing and plotting CD data. It allows reliable estn. of secondary structure content utilizing different approaches. CAPITO accepts multiple CD datasets and, hence, is well suited for a wide application range such as the anal. of temp. or pH-dependent (un)folding and the comparison of mutants.
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Ahmed, M. A.; De Avila, M.; Polverini, E.; Bessonov, K.; Bamm, V. V.; Harauz, G. Solution nuclear magnetic resonance structure and molecular dynamics simulations of a murine 18.5 kDa myelin basic protein segment (S72-S107) in association with dodecylphosphocholine micelles. Biochemistry 2012, 51, 7475– 7487, DOI: 10.1021/bi300998x
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Solution Nuclear Magnetic Resonance Structure and Molecular Dynamics Simulations of a Murine 18.5 kDa Myelin Basic Protein Segment (S72-S107) in Association with Dodecylphosphocholine Micelles
Ahmed, Mumdooh A. M.; De Avila, Miguel; Polverini, Eugenia; Bessonov, Kyrylo; Bamm, Vladimir V.; Harauz, George
Biochemistry (2012), 51 (38), 7475-7487CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
The 18.5 kDa myelin basic protein (MBP), the most abundant splice isoform in adult mammalian myelin, is a multifunctional, intrinsically disordered protein involved in the development and compaction of the myelin sheath in the central nervous system. A highly conserved central segment comprises a membrane-anchoring amphipathic α-helix followed by a proline-rich segment that represents a ligand for SH3 domain-contg. proteins. Here, we have detd. using soln. NMR spectroscopy the structure of a 36-residue peptide fragment of MBP (murine 18.5 kDa residues S72-S107, denoted the α2-peptide) comprising these two structural motifs, in assocn. with dodecylphosphocholine (DPC) micelles. The structure was calcd. using CS-ROSETTA (version 1.01) because the nuclear Overhauser effect restraints were insufficient for this protein. The exptl. studies were complemented by mol. dynamics (MD) simulations of a corresponding 24-residue peptide fragment (murine 18.5 kDa residues E80-G103, denoted the MD-peptide), also in assocn. with a DPC micelle in silico. The exptl. and theor. results agreed well with one another, despite the independence of the starting structures and analyses, both showing membrane assocn. via the amphipathic α-helix, and a sharp bend in the vicinity of the Pro93 residue (murine 18.5 kDa sequence numbering). Overall, the conformations elucidated here show how the SH3 ligand is presented to the cytoplasm for interaction with SH3 domain-contg. proteins such as Fyn and contribute to our understanding of myelin architecture at the mol. level.
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Ishida, T.; Kinoshita, K. PrDOS: prediction of disordered protein regions from amino acid sequence. Nucleic Acids Res. 2007, 35, W460– W464, DOI: 10.1093/nar/gkm363
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Song, Y.; DiMaio, F.; Wang, R. Y.; Kim, D.; Miles, C.; Brunette, T.; Thompson, J.; Baker, D. High-resolution comparative modeling with RosettaCM. Structure 2013, 21, 1735– 1742, DOI: 10.1016/j.str.2013.08.005
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High-Resolution Comparative Modeling with RosettaCM
Song, Yifan; DiMaio, Frank; Wang, Ray Yu-Ruei; Kim, David; Miles, Chris; Brunette, T. J.; Thompson, James; Baker, David
Structure (Oxford, United Kingdom) (2013), 21 (10), 1735-1742CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
We describe an improved method for comparative modeling, RosettaCM, which optimizes a phys. realistic all-atom energy function over the conformational space defined by homologous structures. Given a set of sequence alignments, RosettaCM assembles topologies by recombining aligned segments in Cartesian space and building unaligned regions de novo in torsion space. The junctions between segments are regularized using a loop closure method combining fragment superposition with gradient-based minimization. The energies of the resulting models are optimized by all-atom refinement, and the most representative low-energy model is selected. The CASP10 expt. suggests that RosettaCM yields models with more accurate side-chain and backbone conformations than other methods when the sequence identity to the templates is greater than ∼15%.
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Berendsen, H. J. C.; Grigera, J. R.; Straatsma, T. P. The missing term in effective pair potentials. J. Phys. Chem. A 1987, 91, 6269– 6271, DOI: 10.1021/j100308a038
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The missing term in effective pair potentials
Berendsen, H. J. C.; Grigera, J. R.; Straatsma, T. P.
Journal of Physical Chemistry (1987), 91 (24), 6269-71CODEN: JPCHAX; ISSN:0022-3654.
Effective pair potentials used for simulations of polar liqs. include the av. effects of polarization. Such potentials are generally adjusted to produce the exptl. heat of vaporization. It has not been recognized before that the self-energy term inherent in any polarizable model should be included in effective pair potentials as well. Inclusion of the self-energy correction with a consequent reparametrization of the simple point charge model of water yields an improvement of the effective pair potential for water, as exemplified by d., radial distribution functions, and diffusion const.
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ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB
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Journal of Chemical Theory and Computation (2015), 11 (8), 3696-3713CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
Mol. mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Av. errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a phys. motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reprodn. of NMR χ1 scalar coupling measurements for proteins in soln. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.
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Duff, M. R., Jr.; Borreguero, J. M.; Cuneo, M. J.; Ramanathan, A.; He, J.; Kamath, G.; Chennubhotla, S. C.; Meilleur, F.; Howell, E. E.; Herwig, K. W.; Myles, D. A. A.; Agarwal, P. K. Modulating Enzyme Activity by Altering Protein Dynamics with Solvent. Biochemistry 2018, 57, 4263– 4275, DOI: 10.1021/acs.biochem.8b00424
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Modulating Enzyme Activity by Altering Protein Dynamics with Solvent
Duff, Michael R.; Borreguero, Jose M.; Cuneo, Matthew J.; Ramanathan, Arvind; He, Junhong; Kamath, Ganesh; Chennubhotla, S. Chakra; Meilleur, Flora; Howell, Elizabeth E.; Herwig, Kenneth W.; Myles, Dean A. A.; Agarwal, Pratul K.
Biochemistry (2018), 57 (29), 4263-4275CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
Optimal enzyme activity depends on a no. of factors, including structure and dynamics. The role of enzyme structure is well recognized, however, the linkage between protein dynamics and enzyme activity has given rise to a contentious debate. We have developed an approach that uses an aq. mixt. of org. solvent to control the functionally relevant enzyme dynamics (without changing the structure), which in turn modulates the enzyme activity. Using this approach, we predicted that the hydride transfer reaction catalyzed by the enzyme dihydrofolate reductase (DHFR) from Escherichia coli in aq. mixts. of isopropanol (IPA) with water will decrease by ∼3 fold at 20% (vol./vol.) IPA concn. Stop flow kinetic measurements find that the pH-independent khydride rate decreases by 2.2 fold. X-ray crystallog. enzyme structures shows no noticeable differences, while computational studies indicate that the transition-state and electrostatic effects were identical for water and mixed solvent conditions; and quasi-elastic neutron scattering studies show that the dynamical enzyme motions are suppressed. Our approach provides a unique avenue to modulating enzyme activity through changes in enzyme dynamics. Further it provides vital insights that show the altered motions of DHFR cause significant changes in the enzyme's ability to access its functionally relevant conformational sub-states, explaining the decreased khydride rate. This approach has important implications for obtaining fundamental insights into the role of rate-limiting dynamics in catalysis and as well as for enzyme engineering.
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Martínez, L.; Andrade, R.; Birgin, E. G.; Martinez, J. M. PACKMOL: a package for building initial configurations for molecular dynamics simulations. J. Comput. Chem. 2009, 30, 2157– 2164, DOI: 10.1002/jcc.21224
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PACKMOL: A package for building initial configurations for molecular dynamics simulations
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Journal of Computational Chemistry (2009), 30 (13), 2157-2164CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
Adequate initial configurations for mol. dynamics simulations consist of arrangements of mols. distributed in space in such a way to approx. represent the system's overall structure. In order that the simulations are not disrupted by large van der Waals repulsive interactions, atoms from different mols. must keep safe pairwise distances. Obtaining such a mol. arrangement can be considered a packing problem: Each type mol. must satisfy spatial constraints related to the geometry of the system, and the distance between atoms of different mols. must be greater than some specified tolerance. We have developed a code able to pack millions of atoms, grouped in arbitrarily complex mols., inside a variety of three-dimensional regions. The regions may be intersections of spheres, ellipses, cylinders, planes, or boxes. The user must provide only the structure of one mol. of each type and the geometrical constraints that each type of mol. must satisfy. Building complex mixts., interfaces, solvating biomols. in water, other solvents, or mixts. of solvents, is straightforward. In addn., different atoms belonging to the same mol. may also be restricted to different spatial regions, in such a way that more ordered mol. arrangements can be built, as micelles, lipid double-layers, etc. The packing time for state-of-the-art mol. dynamics systems varies from a few seconds to a few minutes in a personal computer. The input files are simple and currently compatible with PDB, Tinker, Molden, or Moldy coordinate files. The package is distributed as free software and can be downloaded from . © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009.
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Agarwal, P. K. Cis/trans isomerization in HIV-1 capsid protein catalyzed by cyclophilin A: insights from computational and theoretical studies. Proteins 2004, 56, 449– 463, DOI: 10.1002/prot.20135
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Cis/trans isomerization in HIV-1 capsid protein catalyzed by cyclophilin A: Insights from computational and theoretical studies
Agarwal, Pratul K.
Proteins: Structure, Function, and Bioinformatics (2004), 56 (3), 449-463CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
A network of protein vibrations has recently been identified in the enzyme cyclophilin A (CypA) that is assocd. with its peptidylprolyl cis/trans isomerization activity of small peptide substrates. It has been suggested that this network may have a role in promoting the catalytic step during the isomerization reaction. This work presents the results from the characterization of this network during the isomerization of the Gly89-Pro90 peptide bond in the N-terminal domain of the capsid protein (CAN) from human immunodeficiency virus type 1 (HIV-1), which is a naturally occurring, biol. relevant protein substrate for CypA. A variety of computational and theor. studies are utilized to investigate the protein dynamics of the CypA-CAN complex, at multiple time scales, during the isomerization step. The results provide insights into the detailed mechanism of isomerization and confirm the presence of previously reported network of protein vibrations coupled to the reaction. Conserved CypA residues at the complex interface and at positions distal to the interface form parts of this network. There is HIV-1 related medical interest in CypA; incorporation of CypA, complexed with the capsid protein, into the virion is required for the infectious activity of HIV-1. Interaction energy and dynamical cross-correlation calcns. are used for a detailed investigation of the protein-protein interactions in the CypA-CAN complex. The results show that CAN residues His87-Ala-Gly-Pro-Ile-Ala92 form the majority of the interactions with CypA residues. New protein-protein interactions distal to the active site (CypA Arg148-CAN Gln95 and CypA Arg148-CAN Asn121) are also identified.
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Methods for molecular dynamics simulations of protein folding/unfolding in solution
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Methods (San Diego, CA, United States) (2004), 34 (1), 112-120CODEN: MTHDE9; ISSN:1046-2023. (Elsevier)
All atom mol. dynamics simulations have become a std. method for mapping equil. protein dynamics and nonequil. events like folding and unfolding. Here, we present detailed methods for performing such simulations. Generic protocols for minimization, solvation, simulation, and anal. derived from previous studies are also presented. As a measure of validation, our water model is compared with expt. An example of current applications of these methods, simulations of the ultrafast folding protein Engrailed Homeodomain are presented including the exptl. evidence used to verify their results. Ultrafast folders are an invaluable tool for studying protein behavior as folding and unfolding events measured by expt. occur on timescales accessible with the high-resoln. mol. dynamics methods we describe. Finally, to demonstrate the prospect of these methods for folding proteins, a temp. quench simulation of a thermal unfolding intermediate of the Engrailed Homeodomain is described.
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Roe, D. R.; Cheatham, T. E., 3rd PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. J. Chem. Theory Comput. 2013, 9, 3084– 3095, DOI: 10.1021/ct400341p
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PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data
Roe, Daniel R.; Cheatham, Thomas E.
Journal of Chemical Theory and Computation (2013), 9 (7), 3084-3095CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
We describe PTRAJ and its successor CPPTRAJ, two complementary, portable, and freely available computer programs for the anal. and processing of time series of three-dimensional at. positions (i.e., coordinate trajectories) and the data therein derived. Common tools include the ability to manipulate the data to convert among trajectory formats, process groups of trajectories generated with ensemble methods (e.g., replica exchange mol. dynamics), image with periodic boundary conditions, create av. structures, strip subsets of the system, and perform calcns. such as RMS fitting, measuring distances, B-factors, radii of gyration, radial distribution functions, and time correlations, among other actions and analyses. Both the PTRAJ and CPPTRAJ programs and source code are freely available under the GNU General Public License version 3 and are currently distributed within the AmberTools 12 suite of support programs that make up part of the Amber package of computer programs (see http://ambermd.org). This overview describes the general design, features, and history of these two programs, as well as algorithmic improvements and new features available in CPPTRAJ.
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Shukla, S.; Bafna, K.; Gullett, C.; Myles, D. A. A.; Agarwal, P. K.; Cuneo, M. J. Differential Substrate Recognition by Maltose Binding Proteins Influenced by Structure and Dynamics. Biochemistry 2018, 57, 5864– 5876, DOI: 10.1021/acs.biochem.8b00783
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Differential Substrate Recognition by Maltose Binding Proteins Influenced by Structure and Dynamics
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Biochemistry (2018), 57 (40), 5864-5876CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
The genome of the hyperthermophile Thermotoga maritima contains three isoforms of maltose binding protein (MBP) that are high-affinity receptors for di-, tri-, and tetrasaccharides. Two of these proteins (tmMBP1 and tmMBP2) share significant sequence identity, approx. 90%, while the third (tmMBP3) shares less than 40% identity. MBP from Escherichia coli (ecMBP) shares 35% sequence identity with the tmMBPs. This subset of MBP isoforms offers an interesting opportunity to investigate the mechanisms underlying the evolution of substrate specificity and affinity profiles in a genome where redundant MBP genes are present. In this study, the X-ray crystal structures of tmMBP1, tmMBP2, and tmMBP3 are reported in the absence and presence of oligosaccharides. tmMBP1 and tmMBP2 have binding pockets that are larger than that of tmMBP3, enabling them to bind to larger substrates, while tmMBP1 and tmMBP2 also undergo substrate-induced hinge bending motions (∼52°) that are larger than that of tmMBP3 (∼35°). Small-angle X-ray scattering was used to compare protein behavior in soln., and computer simulations provided insights into dynamics of these proteins. Comparing quant. protein-substrate interactions and dynamical properties of tmMBPs with those of the promiscuous ecMBP and disaccharide selective Thermococcus litoralis MBP provides insights into the features that enable selective binding. Collectively, the results provide insights into how the structure and dynamics of tmMBP homologues enable them to differentiate between a myriad of chem. entities while maintaining their common fold.
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Abstract
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Figure 1
Figure 1. Myelin basic protein in EAE is deiminated. (A) Representative sucrose density ultracentrifugation fractions of mouse brains probing for myelin basic protein (Abcam-ab7349). *Sucrose fraction 32 (SF 32) from each group was used for further analysis (coomassie, Western blot, mass spectrometry, and capillary electrophoresis (CE)). EAE, n = 15; sham-injection control, n = 10; and no-injection control, n = 10. Each immunoblot was repeated three times. (B) Coomassie blue stain and Western blot for sucrose fraction 32. Bands were excised from coomassie gel and analyzed by mass spectrometry. Western blot probed for levels of deimination (citrulline, Millipore-MABS54887). (C) Mass spectrometry analysis of excised bands from coomassie blue stain gel. PSM = peptide-spectrum match.
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Figure 2
Figure 2. Myelin basic protein isolated from EAE has different phospholipid compositions than controls. (A) Representative capillary electrophoresis (CE) chromatogram of sucrose fraction 32 (SF 32). Arrow points toward the peak collected for protein and phospholipid analysis. (B) Dot immunoblot for MBP in CE fractions, 10 min before the peak, peak (as noted by the arrow), and 10 min after the peak. MBP was probed with an anti-MBP antibody (Abcam, ab7349). (C) Phospholipid analysis of CE peak fractions, three replicates, and bars correspond to standard error mean (SEM) (*p < 0.05).
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Figure 3
Figure 3. Confirmation of MBP–phospholipid complexation. (A) Structure of candidate lipids: PC 16:0/22:6, 1-((4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoyl)-2-hexadecanoyl-sn-glycero-3-phosphocholine, LMGP01011116; PC 16:1/16:1, 1,2-di-((9Z)-hexadecanoyl)-sn-glycero-3-phosphocholine, LMGP01010684; PI 18:0/20:4, 1-octadecanoyl-2-((5Z,8Z,11Z,14Z)-eicosatetraenoyl)-sn-glycero-3-phospho-(1′-myo-inositol), LMGP06010010; PS 18:0/18:1, 1-octadecanoyl-2-((9Z)-octadecanoyl)-sn-glycero-3-phosphoserine, LMGP03010025; LPC 18:1, 2-((9Z)-octadecanoyl)-sn-glycero-3-phosphocholine, LMGP01050082. (B) Liposome flotation assay (LFA) of candidate phospholipids demonstrating MBP complexation with phospholipids. B, bottom fraction; M, middle fraction; and T, top fraction (MBP, Abcam, ab7349). (C) Protein–lipid overlay assay (PLOA) of candidate phospholipids demonstrating MBP complexation with phospholipids (MBP, Abcam, ab7349).
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Figure 4
Figure 4. Complexation of LPC 18:1 with MBP is competitive for MBP hyper-deimination. (A) Dot blot analysis of deimination reaction for MBP (MBP, Abcam-ab7349) and deiminated MBP (citrulline, Millipore-MABS54887) in the presence of candidate phospholipids. PAD, peptidyl arginine deiminase. (B) Deimination fold change analysis of Western blot, three replicates, and densitometry using ImageJ was normalized to MBP (***p < 0.0005, comparison to MBP–PAD). (C) Representative circular dichroism (CD) spectra of deimination reaction for MBP in the presence of candidate phospholipids, three replicates per spectra. (D) CD spectra analysis of secondary structure probability, utilizing CD analysis and plotting tool (CAPITO) software (mean with SEM).
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Figure 5
Figure 5. LPC 18:1 competes with hyper-deimination of MBP arginine residues and has differential complexation with MBP. (A) Mass spectrometry analysis of MBP arginine residues that are being deiminated in in vitro deimination assay (Millipore, 13-104, Burlington, MA). The deiminated Myelin basic protein (UniProt accession: P02687) peptides were identified by mass spectrometry (see Table 3). Gray highlight, deiminated arginine; red bold square, arginine not deiminated in LPC 18:1 reaction (residues 63, 112, and 129). (B) Protein–lipid overlay assay of LPC 18:1, LPC 14:0, LPC 16:0, and LPC 18:0 MBP was probed with an anti-MBP antibody (Abcam, ab7349).
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Figure 6
Figure 6. Change in MBP conformations and solvent accessibility of arginine residues. Representative structures are shown from simulations only in water, water with LPC, and water with PC. Helices are shown in red, β-strands in yellow, three N-terminal residues are colored green, and three C-terminal residues are colored cyan. Arginine residues are shown as green/blue sticks, while the lipid is shown as magenta/blue/orange sticks. Water was included in the simulations but omitted here for visualization purposes. In the case of water-only, the arginine residues are exposed on the surface; the presence of LPC protects a number of these residues, and in the presence of PC, MBP adopts an extended conformation, exposing several arginine residues to the solvent.
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Figure 7
Figure 7. Hypothesis model of MBP–phospholipid complexation. (A) Working hypothesis for membrane disruption, incorporating key events associated with unstable myelin. The order of events has yet to be determined. (B) Model of phospholipid (PC 16:1/16:1, PI 18:0/20:4, PS 18:0/18:1, LPC 18:0, LPC 16:0, and LPC 14:0) competitive effects against PAD-mediated hyper-deimination and potential cascade of events. (C) Model of LPC 18:1 competitive effects against PAD-mediated hyper-deimination. Δ, “change in”; MBP, myelin basic protein; and PAD, peptidyl arginine deiminase. Open and C-shaped conformations are consistent with the previously published reports.
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  Lassmann, Hans
  Clinical & Experimental Neuroimmunology (2010), 1 (1), 2-11CODEN: CENLCG; ISSN:1759-1961. (John Wiley & Sons Ltd.)
  A review. Multiple sclerosis (MS) is defined as a chronic inflammatory disease of the central nervous system, which leads to focal inflammatory demyelinated lesions with secondary neurodegeneration. However, this concept has recently been challenged by several observations suggesting that in this disease neurodegeneration might occur independently of inflammation. Here, these new findings are critically discussed and evidence that active neurodegeneration in MS is invariably assocd. with inflammation is provided. The present review shows, however, that the inflammatory reaction is much more complex, as thought before, and that in the progressive stage of the disease it might become trapped in the central nervous system behind a repaired blood-brain barrier. Future therapeutic options for this disease are discussed on the basis of recent knowledge of the mechanisms of inflammation and neurodegeneration.
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5. 5
  Bielekova, B.; Sung, M.-H.; Kadom, N.; Simon, R.; McFarland, H.; Martin, R. Expansion and Functional Relevance of High-Avidity Myelin-Specific CD4+ T Cells in Multiple Sclerosis. J. Immunol. 2004, 172, 3893– 3904, DOI: 10.4049/jimmunol.172.6.3893
  5
  Expansion and Functional Relevance of High-Avidity Myelin-Specific CD4+ T Cells in Multiple Sclerosis
  Bielekova, Bibiana; Sung, Myong-Hee; Kadom, Nadja; Simon, Richard; McFarland, Henry; Martin, Roland
  Journal of Immunology (2004), 172 (6), 3893-3904CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
  Multiple sclerosis (MS) is an autoimmune disease in which myelin-specific T cells are believed to play a crucial pathogenic role. Nevertheless, so far it has been extremely difficult to demonstrate differences in T cell reactivity to myelin Ag between MS patients and controls. The authors believe that by using unphysiol. high Ag concns. previous studies have missed a highly relevant aspect of autoimmune responses, i.e., T cells recognizing Ag with high functional avidity. Therefore, the authors focused on the characterization of high-avidity myelin-specific CD4+ T cells in a large cohort of MS patients and controls that was matched demog. and with respect to expression of MHC class II alleles. The authors demonstrated that their frequency is significantly higher in MS patients while the nos. of control T cells specific for influenza hemagglutinin are virtually identical between the two cohorts; that high-avidity T cells are enriched for previously in vivo-activated cells and are significantly skewed toward a proinflammatory phenotype. Moreover, the immunodominant epitopes that were most discriminatory between MS patients and controls differed from those described previously and were clearly biased toward epitopes with lower predicted binding affinities to HLA-DR mols., pointing at the importance of thymic selection for the generation of the autoimmune T cell repertoire. Correlations between selected immunol. parameters and magnetic resonance imaging markers indicate that the specificity and function of these cells influences phenotypic disease expression. These data have important implications for autoimmunity research and should be considered in the development of Ag-specific therapies in MS.
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6. 6
  Moscarello, M. A.; Wood, D. D.; Boulias, C.; Ackerley, C. Myelin in multiple sclerosis is developmentally immature. J. Clin. Invest. 1994, 94, 146– 154, DOI: 10.1172/JCI117300
  6
  Myelin in multiple sclerosis is developmentally immature
  Moscarello, Mario A.; Wood, D. Denise; Ackerley, Cameron; Boulias, Christos
  Journal of Clinical Investigation (1994), 94 (1), 146-54CODEN: JCINAO; ISSN:0021-9738.
  The etiol. of multiple sclerosis (MS) is considered to involve genetic, environmental, infective, and immunol. factors which affect the integrity of a normally assembled myelin sheath, either directly or indirectly resulting in demyelination. In a correlative study involving protein chem., mass spectrometric, and electron microscopic techniques the authors have detd. that myelin obtained from victims of MS is arrested at the level of the first growth spurt (within the first 6 yr of life) and is therefore developmentally immature. The data supporting this conclusion include (a) the pattern of microheterogeneity of myelin basic protein (MBP); (b) the NH2-terminal acylation of the least cationic component of MBP ("C-8"); (c) the phase transition temp. (Tc) of myelin isolated from victims of MS correlated with the increased proportion of the least cationic component of MBP; and (d) immunogold electron microscopy using an antibody specific for "C-8" showed that the distribution of gold particles in a 2-yr old infant was similar to the distribution found in a victim of MS. The authors postulate that this developmentally immature myelin is more susceptible to degrdn. by one or a combination of factors mentioned above, providing the initial antigenic material to the immune system.
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7. 7
  Kursula, P. Structural properties of proteins specific to the myelin sheath. Amino Acids 2008, 34, 175– 185, DOI: 10.1007/s00726-006-0479-7
  7
  Structural properties of proteins specific to the myelin sheath
  Kursula, P.
  Amino Acids (2008), 34 (2), 175-185CODEN: AACIE6; ISSN:0939-4451. (Springer Wien)
  A review. The myelin sheath is an insulating membrane layer surrounding myelinated axons in vertebrates, which is formed when the plasma membrane of an oligodendrocyte or a Schwann cell wraps itself around the axon. A large fraction of the total protein in this membrane layer is comprised of only a small no. of individual proteins, which have certain intriguing structural properties. The myelin proteins are implicated in a no. of neurol. diseases, including, for example, autoimmune diseases and peripheral neuropathies. In this review, the structural properties of a no. of myelin-specific proteins are described.
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8. 8
  Hartline, D. K.; Colman, D. R. Rapid conduction and the evolution of giant axons and myelinated fibers. Curr. Biol. 2007, 17, R29– R35, DOI: 10.1016/j.cub.2006.11.042
  8
  Rapid Conduction and the Evolution of Giant Axons and Myelinated Fibers
  Hartline, D. K.; Colman, D. R.
  Current Biology (2007), 17 (1), R29-R35CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
  Nervous systems have evolved two basic mechanisms for increasing the conduction speed of the elec. impulse. The first is through axon gigantism: using axons several times larger in diam. than the norm for other large axons, as for example in the well-known case of the squid giant axon. The second is through encasing axons in helical or concentrically wrapped multilamellar sheets of insulating plasma membrane - the myelin sheath. Each mechanism, alone or in combination, is employed in nervous systems of many taxa, both vertebrate and invertebrate. Myelin is a unique way to increase conduction speeds along axons of relatively small caliber. It seems to have arisen independently in evolution several times in vertebrates, annelids and crustacea. Myelinated nerves, regardless of their source, have in common a multilamellar membrane wrapping, and long myelinated segments interspersed with 'nodal' loci where the myelin terminates and the nerve impulse propagates along the axon by 'saltatory' conduction. For all of the differences in detail among the morphologies and biochemistries of the sheath in the different myelinated animal classes, the function is remarkably universal.
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9. 9
  Valdivia, A. O.; Farr, V.; Bhattacharya, S. K. A novel myelin basic protein transcript variant in the murine central nervous system. Mol. Biol. Rep. 2019, 46, 2547– 2553, DOI: 10.1007/s11033-019-04635-8
  9
  A novel myelin basic protein transcript variant in the murine central nervous system
  Valdivia, Anddre Osmar; Farr, Valentina; Bhattacharya, Sanjoy K.
  Molecular Biology Reports (2019), 46 (2), 2547-2553CODEN: MLBRBU; ISSN:0301-4851. (Springer)
  Myelin basic protein is a multifunctional protein whose primary role is to adhere membranes of the myelin sheath. There are various isoforms that have been identified, 6 distinct isoforms in human and 13 distinct isoforms in mice. These distinct isoforms are the product of alternative splicing of a single gene. The present study sought out to identify the different isoforms found in the murine central nervous system. Neuronal tissue (brain) from five different C57BL6/J mice at 2 mo of age was harvested and used for mRNA extn. MRNA was reversed transcribed to cDNA and transcripts were detected through PCR amplification and DNA agarose gel sepn. Primers for exon 1, exon 5b and exon 11 of the myelin basic protein gene were used to capture all the possible transcripts that are naturally found in the murine central nervous system. Unknown transcript was sequenced at Genewiz facilities (South Plainfield, NJ) and mass spectrometry protein sequence anal. demonstrated the presence of a novel myelin basic protein transcript variant. We identified a novel transcript variant of myelin basic protein. This novel transcript variant corresponds to a myelin basic protein of 32.5 kDa which has not been previously reported. This novel transcript variant presents relevant clin. significance to various demyelinating diseases due to its contribution to the understanding of the natural state of the murine central nervous system.
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10. 10
  Musse, A. A.; Boggs, J. M.; Harauz, G. Deimination of membrane-bound myelin basic protein in multiple sclerosis exposes an immunodominant epitope. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4422– 4427, DOI: 10.1073/pnas.0509158103
  10
  Deimination of membrane-bound myelin basic protein in multiple sclerosis exposes an immunodominant epitope
  Musse, Abdiwahab A.; Boggs, Joan M.; Harauz, George
  Proceedings of the National Academy of Sciences of the United States of America (2006), 103 (12), 4422-4427CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  The degrdn. of myelin in the CNS is the hallmark of multiple sclerosis. Redn. in the net pos. charge of myelin basic protein (MBP), through deimination, correlates strongly with disease severity and may mediate myelin instability and loss of compaction. Using Cys scanning, spin labeling, EPR spectroscopy, and site-specific proteolysis, we show that in the membrane-bound state the primary immunodominant epitope, V83-T92, of the less cationic recombinant murine MBP C8 mimic (rmC8) forms a more highly surface-exposed and shorter amphipathic α-helix than in the unmodified form, recombinant murine MBP C1 mimic (rmC1), analogous to the most cationic and abundant isomer of MBP in normal myelin. Moreover, cathepsin D digested lipid-assocd. rmC8 3-fold faster than rmC1, and cleavage at F86-F87 occurred more readily in rmC8 than rmC1. These findings suggest a mechanism for initial loss of myelin stability and the autoimmune pathogenesis of multiple sclerosis.
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11. 11
  Kidd, B. A.; Ho, P. P.; Sharpe, O.; Zhao, X.; Tomooka, B. H.; Kanter, J. L.; Steinman, L.; Robinson, W. H. Epitope spreading to citrullinated antigens in mouse models of autoimmune arthritis and demyelination. Arthritis Res. Ther. 2008, 10, R119 DOI: 10.1186/ar2523
  11
  Epitope spreading to citrullinated antigens in mouse models of autoimmune arthritis and demyelination
  Kidd Brian A; Ho Peggy P; Sharpe Orr; Zhao Xiaoyan; Tomooka Beren H; Kanter Jennifer L; Steinman Lawrence; Robinson William H
  Arthritis research & therapy (2008), 10 (5), R119 ISSN:.
  INTRODUCTION: Anti-citrullinated protein antibodies have a diagnostic role in rheumatoid arthritis (RA); however, little is known about their origins and contribution to pathogenesis. Citrullination is the post-translational conversion of arginine to citrulline by peptidyl arginine deiminase, and increased citrullination of proteins is observed in the joint tissue in RA and in brain tissue in multiple sclerosis (MS). METHODS: We applied synovial and myelin protein arrays to examine epitope spreading of B cell responses to citrullinated epitopes in both the collagen-induced arthritis (CIA) model for RA and the experimental autoimmune encephalomyelitis (EAE) model for MS. Synovial and myelin protein arrays contain a spectrum of proteins and peptides, including native and citrullinated forms, representing candidate autoantigens in RA and MS, respectively. We applied these arrays to characterise the specificity of autoantibodies in serial serum samples derived from mice with acute and chronic stages of CIA and EAE. RESULTS: In samples from pre-disease CIA and acute-disease EAE, we observed autoantibody targeting of the immunising antigen and responses to a limited set of citrullinated epitopes. Over the course of diseases, the autoantibody responses expanded to target multiple citrullinated epitopes in both CIA and EAE. Using immunoblotting and mass spectrometry analysis, we identified citrullination of multiple polypeptides in CIA joint and EAE brain tissue that have not previously been described as citrullinated. CONCLUSIONS: Our results suggest that anti-citrulline antibody responses develop in the early stages of CIA and EAE, and that autoimmune inflammation results in citrullination of joint proteins in CIA and brain proteins in EAE, thereby creating neoantigens that become additional targets in epitope spreading of autoimmune responses.
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12. 12
  Cuzner, M. L.; Davison, A. N. Changes in cerebral lysosomal enzyme activity and lipids in multiple sclerosis. J. Neurol. Sci. 1973, 19, 29– 36, DOI: 10.1016/0022-510X(73)90053-1
  12
  Changes in cerebral lysosomal enzyme activity and lipids in multiple sclerosis
  Cuzner, M. Louise; Davison, A. N.
  Journal of the Neurological Sciences (1973), 19 (1), 29-36CODEN: JNSCAG; ISSN:0022-510X.
  In most cases of multiple sclerosis no change was seen in the lipid compn. of apparently normal white matter. Where there was depletion of lipid, chem. and enzymatic evidence of demyelination was found. Raised lysosomal enzyme activity was normally restricted to plaques but in acute cases of multiple sclerosis changes were more extensive. Even in apparently normal areas of white matter cholesterol esters were found and lysosomal acid proteinase, β-glucuronidase and arylsulfatase activities were significantly increased. The significance of these observations to the pathology of multiple sclerosis is discussed.
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13. 13
  Einstein, E. R.; Csejtey, J.; Dalal, K. B.; Adams, C. W.; Bayliss, O. B.; Hallpike, J. F. Proteolytic activity and basic protein loss in and around multiple sclerosis plaques: combined biochemical and histochemical observations. J. Neurochem. 1972, 19, 653– 662, DOI: 10.1111/j.1471-4159.1972.tb01382.x
  13
  Proteolytic activity and basic protein loss in and around multiple sclerosis plaques. Combined biochemical and histochemical observations
  Einstein, Elizabeth R.; Csejtey, Judit; Dalal, K. B.; Adams, C. W. M.; Bayliss, O. B.; Hallpike, J. F.
  Journal of Neurochemistry (1972), 19 (3), 653-62CODEN: JONRA9; ISSN:0022-3042.
  This combined histochem. and biochem. study has shown that acid proteinase activity (pH 3.5) is increased around histol. defined active plaques of multiple sclerosis (MS). Biochem. estn. showed that the enzyme is more active in most samples of normal white matter in MS than in controls. A gradient of enzyme activity was obsd.: control white matter-white matter distant from plaque-close white matter-edge-plaque. Both electrophoretic and histochem. techniques revealed a redn. or absence of basic (encephalitogenic) protein in the plaques. Electrophoresis showed a diminution of encephalitogenic protein outside some plaques. Phospholipids that remain on the base line of thin layer chromatoplates were predominantly phosphoinositides combined with encephalitogenic protein.
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14. 14
  Richards, P. T.; Cuzner, M. L. Proteolytic activity in CSF. Adv. Exp. Med. Biol. 1978, 100, 521– 527, DOI: 10.1007/978-1-4684-2514-7_38
  14
  Proteolytic activity in CSF
  Richards, P. T.; Cuzner, M. Louise
  Advances in Experimental Medicine and Biology (1978), 100 (Myelination Demyelination), 521-7CODEN: AEMBAP; ISSN:0065-2598.
  Cerebrospinal fluid cellular neutral proteinase and supernatant acid proteinase were increased in acute multiple sclerosis and in central nervous system infections.
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15. 15
  Dreyton, C. J.; Knuckley, B.; Jones, J. E.; Lewallen, D. M.; Thompson, P. R. Mechanistic studies of protein arginine deiminase 2: evidence for a substrate-assisted mechanism. Biochemistry 2014, 53, 4426, DOI: 10.1021/bi500554b
  15
  Mechanistic studies of protein arginine deiminase 2: Evidence for a substrate-assisted mechanism
  Dreyton, Christina J.; Knuckley, Bryan; Jones, Justin E.; Lewallen, Daniel M.; Thompson, Paul R.
  Biochemistry (2014), 53 (27), 4426-4433CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  Citrullination, which is catalyzed by protein arginine deiminases (PADs 1-4 and 6), is a post-translational modification (PTM) that effectively neutralizes the pos. charge of a guanidinium group by its replacement with a neutral urea. Given the sequence similarity of PAD2 across mammalian species and the genomic organization of the PAD2 gene, PAD2 is predicted to be the ancestral homolog of the PADs. Although PAD2 has long been known to play a role in myelination, it has only recently been linked to other cellular processes, including gene transcription and macrophage extracellular trap formation. For example, PAD2 deiminates histone H3 at Arg-26, and this PTM leads to the increased transcription of >200 genes under the control of the estrogen receptor. Given that the understanding of PAD2 biol. remains incomplete, the authors initiated mechanistic studies on this enzyme to aid the development of PAD2-specific inhibitors. Here, the authors report that the substrate specificity and Ca2+ dependence of PAD2 were similar to those of PADs 1, 3, and 4. However, unlike those isoenzymes, PAD2 appeared to use a substrate-assisted mechanism of catalysis in which the pos. charged substrate, guanidinium, depressed the pKa of the nucleophilic Cys residue. By contrast, PADs 1, 3, and 4 use a reverse-protonation mechanism. These mechanistic differences will aid the development of isoenzyme-specific inhibitors.
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16. 16
  Vossenaar, E. R.; Zendman, A. J.; van Venrooij, W. J.; Pruijn, G. J. PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. BioEssays 2003, 25, 1106– 1118, DOI: 10.1002/bies.10357
  16
  PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease
  Vossenaar, Erik R.; Zendman, Albert J. W.; van Venrooij, Walther J.; Pruijn, Ger J. M.
  BioEssays (2003), 25 (11), 1106-1118CODEN: BIOEEJ; ISSN:0265-9247. (John Wiley & Sons, Inc.)
  A review. Peptidylarginine deiminase (PAD, EC 3.5.3.15) enzymes catalyze the conversion of protein-bound Arg to citrulline. This post-translational modification may have a big impact on the structure and function of the target protein. In this review, the authors will discuss the effects of citrullination and its involvement in several human diseases, including rheumatoid arthritis and multiple sclerosis. So far, 4 isotypes of PAD were described in mammals. We describe the existence of PAD in non-mammalian vertebrates and the existence of a fifth mammalian PAD. In addn., tissue-specific expression, genomic organization and evolutionary conservation of the different PAD isotypes will be discussed in detail. This article contains supplementary material which may be viewed at the BioEssays website at http://www.interscience.wiley.com/jpages/0265-9247/suppmat/2003/25/v25.1106.html.
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17. 17
  Tarcsa, E.; Marekov, L. N.; Mei, G.; Melino, G.; Lee, S. C.; Steinert, P. M. Protein unfolding by peptidylarginine deiminase. Substrate specificity and structural relationships of the natural substrates trichohyalin and filaggrin. J. Biol. Chem. 1996, 271, 30709, DOI: 10.1074/jbc.271.48.30709
  17
  Protein unfolding by peptidylarginine deiminase. Substrate specificity and structural relationships of the natural substrates trichohyalin and filaggrin
  Tarcsa, Edit; Marekov, Lyuben N.; Mei, Giampiero; Melino, Gerry; Lee, Seung-Chul; Steinert, Peter M.
  Journal of Biological Chemistry (1996), 271 (48), 30709-30716CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  Peptidylarginine deiminases, which are commonly found in mammalian cells, catalyze the deimination of protein-bound arginine residues to citrullines. However, very little is known about their substrate requirements and the significance or consequences of this post-synthetic modification. We have explored this reaction in vitro with two known substrates filaggrin and trichohyalin. First, the degree and rate of modification of arginines to citrullines directly correlates with the structural order of the substrate. In filaggrin, which has little structural order, the reaction proceeded rapidly to >95% completion. However, in the highly α-helical protein trichohyalin, the reaction proceeded slowly to about 25% and could be forced to a max. of about 65%. Second, the rate and degree of modification depends on the sequence location of the target arginines. Third, we show by gel electrophoresis, CD, and fluorescence spectroscopy that the reaction interferes with organized protein structure: the net formation of ≥10% citrulline results in protein denaturation. Cyanate modification of the lysines in model α-helix-rich proteins to homocitrullines also results in loss of organized structure. These data suggest that the ureido group on the citrulline formed by the peptidylarginine deiminase enzyme modification functions to unfold proteins due to decrease in net charge, loss of potential ionic bonds, and interference with H bonds.
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18. 18
  Takahara, H. A History of Deimination Research in Japan: The Founding Fathers. In Protein Deimination in Human Health and Disease; Nicholas, A. P.; Bhattacharya, S. K.; Thompson, P. R., Eds.; Springer International Publishing: Cham, 2017; pp 1– 10.
  There is no corresponding record for this reference.
19. 19
  Cao, L.; Goodin, R.; Wood, D.; Moscarello, M. A.; Whitaker, J. N. Rapid release and unusual stability of immunodominant peptide 45-89 from citrullinated myelin basic protein. Biochemistry 1999, 38, 6157– 6163, DOI: 10.1021/bi982960s
  19
  Rapid Release and Unusual Stability of Immunodominant Peptide 45-89 from Citrullinated Myelin Basic Protein
  Cao, Ligong; Goodin, Richard; Wood, Denise; Moscarello, Mario A.; Whitaker, John N.
  Biochemistry (1999), 38 (19), 6157-6163CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  Myelin basic protein (MBP) exists in a population of isoforms and isomers. The 18.5 kDa MBP-C1, the main human adult isoform, has 170 residues and is relatively unmodified, whereas the same isoform can be citrullinated on six arginine residues to create the MBP-C8 (MBP Cit6) isomer. MBP Cit6 dominates in MS brain, accounting for 45% rather than 25% of the population of MBP isomers. In the fulminant form of MS, known as Marburg's Disease, 18 of the 19 arginines in MBP are citrullinated (MBP Cit18). Citrullination of MBP could lead to instability of myelin or limited remyelination. In this investigation, the susceptibilities to degrdn. by cathepsin D of MBP Cit6 and MBP-C1, both from normal and MS brain tissue, and Marburg MBP Cit18 were compared. The pattern of digestion was similar, and no differences of corresponding isomers in normal and MS brain were noted. However, normal MBP Cit6 was degraded 10-fold more rapidly than MBP-C1, and MBP Cit18 was degraded even more rapidly. MBP peptide 45-89 was preserved regardless of isomer type or source. Its generation was directly related to the citrulline content of the MBP substrate being 4 times faster in normal MBP Cit6 and 35 times faster in Marburg MBP Cit18 than in normal MBP-C1. Peptide 45-89 from a citrullinated MBP exhibited more deamidation, and, regardless of source, showed an α-helix structure in a lipid mimetic environment. We postulate that the generation of MBP peptides, including those that are dominant and encephalitogenic, is directly related to deimination of arginine to citrulline in MBP.
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20. 20
  Whitaker, J. N. Myelin encephalitogenic protein fragments in cerebrospinal fluid of persons with multiple sclerosis. Neurology 1977, 27, 911– 920, DOI: 10.1212/WNL.27.10.911
  20
  Myelin encephalitogenic protein fragments in cerebrospinal fluid of persons with multiple sclerosis
  Whitaker, John N.
  Neurology (1977), 27 (10), 911-20CODEN: NEURAI; ISSN:0028-3878.
  With a double-antibody radioimmunoassay performed on unconcd. cerebrospinal fluid, 8 of 14 patients in an acute phase of multiple sclerosis had levels of 3.4 to 15.4 ng/mL of the P1 fragment (residues 43-88) of myelin encephalitogenic protein. Encephalitogenic protein-P1 was found only in the acute phase and was present in 6 of 7 persons in the first week of an exacerbation and absent in 29 multiple sclerosis patients who were stable or had a gradually progressive course. Six of 117 controls had detectable cerebrospinal fluid encephalitogenic protein-P1. Only in 2 of these, one with a recent cerebral infarction and one with diabetic nephropathy who was in coma, were the levels in the range encountered in patients in the acute phase of multiple sclerosis. Although not entirely specific for multiple sclerosis, the presence of material in the cerebrospinal fluid of multiple sclerosis patients cross-reacting with encephalitogenic protein-P1 appears to be a characteristic of acute exacerbations.
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21. 21
  Whitaker, J. R.; Granum, P. E. An absolute method for protein determination based on difference in absorbance at 235 and 280 nm. Anal. Biochem. 1980, 109, 156– 159, DOI: 10.1016/0003-2697(80)90024-X
  21
  An absolute method for protein determination based on difference in absorbance at 235 and 280 nm
  Whitaker, John R.; Granum, Per Einar
  Analytical Biochemistry (1980), 109 (1), 156-9CODEN: ANBCA2; ISSN:0003-2697.
  Protein (in mg/mL) is detd. spectrometrically by subtracting the absorbance at 280 nm from the absorbance at 235 nm, then dividing by 2.51 (which is the difference between the av. absorptivities at the 2 wavelengths). This method does not require a std. curve, is not subject to interference by nucleic acids, and is independent of amino acid compn. The sensitivity of this method is 45% that of the Lowry method.
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22. 22
  Pritzker, L. B.; Joshi, S.; Gowan, J. J.; Moscarello, M. A.; Harauz, G. Deimination of myelin basic protein. 1. Effect of deimination of arginyl residues of myelin basic protein on its structure and susceptibility to digestion by cathepsin D. Biochemistry 2000, 39, 5374– 5381, DOI: 10.1021/bi9925569
  22
  Deimination of Myelin Basic Protein. 1. Effect of Deimination of Arginyl Residues of Myelin Basic Protein on Its Structure and Susceptibility to Digestion by Cathepsin D
  Pritzker, Laura B.; Joshi, Shashikant; Gowan, Jessica J.; Harauz, George; Moscarello, Mario A.
  Biochemistry (2000), 39 (18), 5374-5381CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  The effect of deimination of arginyl residues in bovine myelin basic protein (MBP) on its susceptibility to digestion by cathepsin D has been studied. Using bovine component 1 (C-1) of MBP, the most unmodified of the components with all 18 arginyl residues intact, we have generated a no. of citrullinated forms by treatment of the protein with purified peptidylarginine deiminase (PAD) in vitro. We obtained species contg. 0-9.9 mol of citrulline/mol of MBP. These various species were digested with cathepsin D, a metalloproteinase which cleaves proteins at Phe-Phe linkages. The rate of digestion compared to component 1 was only slightly affected when 2.7 or 3.8 mol of citrulline/mol of MBP was present. With 7.0 mol of citrulline/mol of MBP, a large increase in the rate of digestion occurred. No further increase was obsd. with 9.9 mol of citrulline/mol of MBP. The immunodominant peptide 43-88 (bovine sequence) was released slowly when 2.7 and 3.8 mol of citrulline/mol of MBP was present, but it was released rapidly when 7.0 mol of citrulline/mol of MBP was present. The dramatic change in digestion with 7.0 mol of citrulline/mol of MBP or more could be explained by a change in three-dimensional structure. Mol. dynamics simulation showed that increasing the no. of citrullinyl residues above 7 mol/mol of MBP generated a more open structure, consistent with exptl. observations in the literature. We conclude that PAD, which deiminates arginyl residues in proteins, decreases both the charge and compact structure of MBP. This structural change allows better access of the Phe-Phe linkages to cathepsin D. This scheme represents an effective way of generating the immunodominant peptide which sensitizes T-cells for the autoimmune response in demyelinating disease.
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23. 23
  Wood, D. D.; Bilbao, J. M.; O’Connors, P.; Moscarello, M. A. Acute multiple sclerosis (Marburg type) is associated with developmentally immature myelin basic protein. Ann. Neurol. 1996, 40, 18– 24, DOI: 10.1002/ana.410400106
  23
  Acute multiple sclerosis (Marburg type) is associated with developmentally immature myelin basic protein
  Wood D D; Bilbao J M; O'Connors P; Moscarello M A
  Annals of neurology (1996), 40 (1), 18-24 ISSN:0364-5134.
  We have studied a case of acute, fulminating multiple sclerosis (MS) (Marburg type) at the pathological and biochemical levels. Postmortem examination of the brain revealed extensive areas of gross rarefaction in the hemispheric white matter. Histologically, well-demarcated areas of demyelination with a large influx of macrophages and a subtle perivascular infiltration of lymphocytes were seen with relative preservation of the axis cylinders. Myelin basic protein (MBP) was isolated and purified [correction of purifed] from noninvolved white matter. It was slightly larger in molecular weight than MBP from normal brain or from chronic MS brain. The increase in mass was accounted for, in part, by the deimination of 18 of 19 arginyl residues to citrulline, making the patient's MBP much less cationic than MBP from normal white matter. When expressed as the ratio of least cationic form of MBP to the most cationic (C-8/C-1), the normal ratio was 0.82, chronic MS 2.5, and the patient in this study 6.7. Because the ratio of 6.7 was similar to 7.5 found for a 15-month-old infant, MBP was considered to be of the immature form. The data are consistent with a genetic factor influencing the charge microheterogeneity of MBP. The resulting less cationic MBP cannot carry out its normal function of compacting multilayers.
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24. 24
  Mastronardi, F. G.; Wood, D. D.; Mei, J.; Raijmakers, R.; Tseveleki, V.; Dosch, H. M.; Probert, L.; Casaccia-Bonnefil, P.; Moscarello, M. A. Increased citrullination of histone H3 in multiple sclerosis brain and animal models of demyelination: a role for tumor necrosis factor-induced peptidylarginine deiminase 4 translocation. J. Neurosci. 2006, 26, 11387– 11396, DOI: 10.1523/JNEUROSCI.3349-06.2006
  24
  Increased citrullination of histone H3 in multiple sclerosis brain and animal models of demyelination: a role for tumor necrosis factor-induced peptidylarginine deiminase 4 translocation
  Mastronardi, Fabrizio G.; Wood, D. Denise; Mei, Jiang; Raijmakers, Reinout; Tseveleki, Vivian; Dosch, Hans-Michael; Probert, Lesley; Casaccia-Bonnefil, Patrizia; Moscarello, Mario A.
  Journal of Neuroscience (2006), 26 (44), 11387-11396CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
  Modification of arginine residues by citrullination is catalyzed by peptidylarginine deiminases (PADs), of which five are known, generating irreversible protein structural modifications. We have shown previously that enhanced citrullination of myelin basic protein contributed to destabilization of the myelin membrane in the CNS of multiple sclerosis (MS) patients. We now report increased citrullination of nucleosomal histones by PAD4 in normal-appearing white matter (NAWM) of MS patients and in animal models of demyelination. Histone citrullination was attributable to increased levels and activity of nuclear PAD4. PAD4 translocation into the nucleus was attributable to elevated tumor necrosis factor-α (TNF-α) protein. The elevated TNF-α in MS NAWM was not assocd. with CD3+ or CD8+ lymphocytes, nor was it assocd. with CD68+ microglia/macrophages. GFAP, a measure of astrocytosis, was the only cytol. marker that was consistently elevated in the MS NAWM, suggesting that TNF-α may have been derived from astrocytes. In cell cultures of mouse and human oligodendroglial cell lines, PAD4 was predominantly cytosolic but TNF-α treatment induced its nuclear translocation. To address the involvement of TNF-α in targeting PAD4 to the nucleus, we found that transgenic mice overexpressing TNF-α also had increased levels of citrullinated histones and elevated nuclear PAD4 before demyelination. In conclusion, high citrullination of histones consequent to PAD4 nuclear translocation is part of the process that leads to irreversible changes in oligodendrocytes and may contribute to apoptosis of oligodendrocytes in MS.
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25. 25
  Nicholas, A. P.; Whitaker, J. N. Preparation of a monoclonal antibody to citrullinated epitopes: its characterization and some applications to immunohistochemistry in human brain. Glia 2002, 37, 328– 336, DOI: 10.1002/glia.10039
  25
  Preparation of a monoclonal antibody to citrullinated epitopes: its characterization and some applications to immunohistochemistry in human brain
  Nicholas Anthony P; Whitaker John N
  Glia (2002), 37 (4), 328-36 ISSN:0894-1491.
  Using hybridoma technology, an IgM monoclonal antibody (mAb), designated as F95, was developed against a deca-citrullinated peptide (DCP) consisting of 10 citrulline residues and a carboxyl Gly-Gly-Cys through which DCP was covalently linked to an activated carrier protein, keyhole limpet hemocyanin (KLH). Clones were selected on the basis of not reacting with human unmodified and noncitrullinated myelin basic protein (MBP), MBP-C1, but reacting well with human citrullinated MBP (MBP-C8). When tested by ELISA, this mAb demonstrated minimal reactivity with human MBP-C1, varying reactivity with the C2-C5 isomers of human MBP, moderate binding with guinea pig MBP-C8, and strong reactivity with human MBP-C8. By ELISA, mAb F95 was directed predominantly against citrulline, not MBP, as revealed by its binding to DCP linked with activated KLH, bovine serum albumin (BSA), or ovalbumin (OA), but not with KLH, BSA, or OA alone. Immunohistochemistry of normal human brain demonstrated that F95 stained central nervous system myelin and a subset of astrocytes. Given the citrulline-directed features of mAb F95, this immunohistochemical pattern suggests that certain astroglial filaments expressing glial fibrillary acidic protein also contain citrulline-bearing components. These potentially implicate citrullinated proteins, notably in astroglial filaments, in a variety of normal and pathological neurobiological processes.
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26. 26
  Raijmakers, R.; Vogelzangs, J.; Croxford, J. L.; Wesseling, P.; van Venrooij, W. J.; Pruijn, G. J. Citrullination of central nervous system proteins during the development of experimental autoimmune encephalomyelitis. J. Comp. Neurol. 2005, 486, 243– 253, DOI: 10.1002/cne.20529
  26
  Citrullination of central nervous system proteins during the development of experimental autoimmune encephalomyelitis
  Raijmakers Reinout; Vogelzangs Judith; Croxford J Ludovic; Wesseling Pieter; van Venrooij Walther J; Pruijn Ger J M
  The Journal of comparative neurology (2005), 486 (3), 243-53 ISSN:0021-9967.
  Immunization of mammals with central nervous system (CNS)-derived proteins or peptides induces experimental autoimmune encephalomyelitis (EAE), a disease resembling the human autoimmune disease multiple sclerosis (MS). Both diseases are accompanied by destruction of a part of the of the myelin sheaths, which surround neurites in the CNS. Previous studies in MS have described alterations in the citrullination of myelin basic protein, one of the main protein constituents of the myelin sheath. Here, we show that, also during the development of EAE in mice, hypercitrullination occurs in the areas of the spinal cord that show the highest degree of inflammation and that myelin basic protein and glial fibrillary acidic protein are among the hypercitrullinated proteins. We conclude that hypercitrullination of myelin proteins in the CNS is a common phenomenon in demyelinating disease. Hypercitrullination may cause conformational changes in proteins, so the affected proteins may be involved in the pathogenesis of CNS autoimmune disease by acting as autoreactive T-cell epitopes. This is the first report in which hypercitrullination of CNS proteins in EAE is described and in which proteins other than myelin basic protein are reported to be citrullinated during autoimmune-mediated CNS inflammation.
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27. 27
  Cao, L.; Sun, D.; Whitaker, J. N. Citrullinated myelin basic protein induces experimental autoimmune encephalomyelitis in Lewis rats through a diverse T cell repertoire. J. Neuroimmunol. 1998, 88, 21– 29, DOI: 10.1016/S0165-5728(98)00063-0
  27
  Citrullinated myelin basic protein induces experimental autoimmune encephalomyelitis in Lewis rats through a diverse T cell repertoire
  Cao, Ligong; Sun, Deming; Whitaker, John N.
  Journal of Neuroimmunology (1998), 88 (1,2), 21-29CODEN: JNRIDW; ISSN:0165-5728. (Elsevier Science B.V.)
  An increased proportion of citrullinated MBP (MBP-C8) occurs in the brains of multiple sclerosis (MS) patients. In this study, MBP-C8 from guinea pig (GP) brains was isolated and found encephalitogenic in Lewis rats upon immunization. An encephalitogenic T cell line selected with MBP-C8 preferentially reacted with MBP-C8 over unmodified MBP. This T cell line responded weakly to the dominant encephalitogenic epitope, GP-MBP peptide 70-88, and did not display restricted TCR β-chain usage (such as Vβ8.2). The distinctive features of MBP-C8 were also demonstrated by its ability to reinduce active EAE in 70% of rats which had recovered from unmodified MBP induced EAE. These findings raise the possibility that citrullinated MBP may elicit a different pathogenic T cell repertoire for the recurrent phases of inflammatory demyelination.
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28. 28
  Carrillo-Vico, A.; Leech, M. D.; Anderton, S. M. Contribution of myelin autoantigen citrullination to T cell autoaggression in the central nervous system. J. Immunol. 2010, 184, 2839– 2846, DOI: 10.4049/jimmunol.0903639
  28
  Contribution of Myelin Autoantigen Citrullination to T Cell Autoaggression in the Central Nervous System
  Carrillo-Vico, Antonio; Leech, Melanie D.; Anderton, Stephen M.
  Journal of Immunology (2010), 184 (6), 2839-2846CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
  Breakdown in immunol. self tolerance, leading to autoimmune diseases such as multiple sclerosis, might arise from immune recognition of self proteins that have undergone heightened posttranslational modification under pathophysiol. conditions. A posttranslational modification of particular interest is the deimination of Arg to citrulline, catalyzed by peptidylarginyl deiminase (PAD) enzymes. As a CD4+ T cell-driven model of multiple sclerosis, the authors used exptl. autoimmune encephalomyelitis (EAE) induced with the immunodominant 35-55 peptide of myelin oligodendrocyte glycoprotein (pMOG) in C57BL/6 mice to test whether citrullination of a T cell epitope can contribute to disease etiopathol. Immunization with an altered peptide ligand (APL) of pMOG with an Arg citrulline conversion at a TCR contact (residue 41) led to the activation of two populations of APL-responsive T cells that either did, or did not cross-react with the native pMOG peptide. This APL could induce EAE. However, this reflected the activation of T cells that cross-reacted with the native pMOG epitope, because prior tolerization of these T cells using pMOG prevented APL-induced EAE. Using a passive transfer model, the authors found that T cells that responded specifically to the citrullinated form of pMOG were neither necessary, nor sufficient to initiate the EAE lesion. Nevertheless, these cells could provoke exacerbation of pathol. if transferred into mice with ongoing EAE. The PAD2 and PAD4 enzymes were markedly upregulated in the inflamed CNS. Therefore, once inflammation is established, citrullination of target autoantigens can allow an expanded repertoire of T cells to contribute to CNS pathol.
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29. 29
  Quinn, T. A.; Dutt, M.; Shindler, K. S. Optic neuritis and retinal ganglion cell loss in a chronic murine model of multiple sclerosis. Front. Neurol. 2011, 2, 50 DOI: 10.3389/fneur.2011.00050
  29
  Optic neuritis and retinal ganglion cell loss in a chronic murine model of multiple sclerosis
  Quinn Thomas A; Dutt Mahasweta; Shindler Kenneth S
  Frontiers in neurology (2011), 2 (), 50 ISSN:.
  Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are neurodegenerative diseases with characteristic inflammatory demyelination in the central nervous system, including the optic nerve. Neuronal and axonal damage is considered to be the main cause of long-term disability in patients with MS. Neuronal loss, including retinal ganglion cell (RGC) apoptosis in eyes with optic neuritis (ON), also occurs in EAE. However, there is significant variability in the clinical course and level of neuronal damage in MS and EAE. The current studies examine the mechanisms and kinetics of RGC loss in C57/BL6 mice immunized with myelin oligodendrocyte glycoprotein to induce a chronic EAE disease. Clinical progression of EAE was scored daily and vision was assessed by optokinetic responses. At various time points, RGCs were counted and optic nerves were examined for inflammatory cell infiltration. Almost all EAE mice develop ON by day 15 post-immunization; however, RGC loss is delayed in these mice. No RGC loss is detected 25 days post-immunization, whereas RGC numbers in EAE mice significantly and progressively decrease compared to controls from 35 to 50 days post-immunization. The delayed time course of RGC loss is in stark contrast to that reported in relapsing EAE, as well as in rats with chronic EAE. Results suggest that different clinical disease courses of optic nerve inflammation may trigger distinct mechanisms of neuronal damage, or RGCs in different rodent strains may have variable resistance to neuronal degeneration.
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30. 30
  Beniac, D. R.; Luckevich, M. D.; Czarnota, G. J.; Tompkins, T. A.; Ridsdale, R. A.; Ottensmeyer, F. P.; Moscarello, M. A.; Harauz, G. Three-dimensional structure of myelin basic protein. I. Reconstruction via angular reconstitution of randomly oriented single particles. J. Biol. Chem. 1997, 272, 4261– 4268, DOI: 10.1074/jbc.272.7.4261
  30
  Three-dimensional structure of myelin basic protein. I. Reconstruction via angular reconstitution of randomly oriented single particles
  Beniac, Daniel R.; Luckevich, Maria D.; Czarnota, Gregory J.; Tompkins, Thomas A.; Ridsdale, Ross A.; Ottensmeyer, F. Peter; Moscarello, Mario A.; Harauz, George
  Journal of Biological Chemistry (1997), 272 (7), 4261-4268CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  Myelin basic protein (MBP) plays an integral role in the structure and function of the myelin sheath. In humans and cattle, an 18.5-kDa isoform of MBP predominates and exists as a multitude of charge isomers resulting from extensive and varied post-translational modifications. We have purified the least modified isomer (named C1) of the 18.5-kDa isoform of MBP from fresh bovine brain and imaged this protein as neg. stained single particle adsorbed to a lipid monolayer. Under these conditions, MBP/C1 presented diverse projections whose relative orientations were detd. using an iterative quaternion-assisted angular reconstitution scheme. In different buffers, one with a low salt and the other with a high salt concn., the conformation of the protein was slightly different. In low salt buffer, the three-dimensional reconstruction, solved to a resoln. of 4 nm, had an overall "C" shape of outer radius 5.5 nm, inner radius 3 nm, overall circumference 15 nm, and height 4.7 nm. The three-dimensional reconstruction of the protein in high salt buffer, solved to a resoln. of 2.8 nm, was essentially the same in terms of overall dimensions but had a somewhat more compact architecture. These results are the first structures achieved directly for this unusual macromol., which plays a key role in the development of multiple sclerosis.
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31. 31
  Ridsdale, R. A.; Beniac, D. R.; Tompkins, T. A.; Moscarello, M. A.; Harauz, G. Three-dimensional structure of myelin basic protein. II. Molecular modeling and considerations of predicted structures in multiple sclerosis. J. Biol. Chem. 1997, 272, 4269– 4275, DOI: 10.1074/jbc.272.7.4269
  31
  Three-dimensional structure of myelin basic protein. II. Molecular modeling and considerations of predicted structures in multiple sclerosis
  Ridsdale, Ross A.; Beniac, Daniel R.; Tompkins, Thomas A.; Moscarello, Mario A.; Harauz, George
  Journal of Biological Chemistry (1997), 272 (7), 4269-4275CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  A computational model of myelin basic protein (MBP) has been constructed based on the premise of a phylogenetically conserved β-sheet backbone and on electron microscopical three-dimensional reconstructions. Many residues subject to post-translational modification (phosphorylation, methylation, or conversion of arginines to citrullines) were located in loop regions and thus accessible to modifying enzymes. The triproline segment (residues 99-101) is fully exposed on the back surface of the protein in a long crossover connection between two parallel β-strands. The proximity of this region to the underlying β-sheet suggests that post-translational modifications here might have potential synergistic effects on the entire structure. Post-translational modifications that lead to reduced surface charge could result first in a weakened attachment to the myelin membrane rather than in a gross conformational change of the protein itself. Such mechanisms could be operative in demyelinating diseases such as multiple sclerosis.
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32. 32
  Haas, H.; Oliveira, C. L.; Torriani, I. L.; Polverini, E.; Fasano, A.; Carlone, G.; Cavatorta, P.; Riccio, P. Small angle x-ray scattering from lipid-bound myelin basic protein in solution. Biophys. J. 2004, 86, 455– 460, DOI: 10.1016/S0006-3495(04)74122-3
  32
  Small angle X-ray scattering from lipid-bound myelin basic protein in solution
  Haas, H.; Oliveira, C. L. P.; Torriani, I. L.; Polverini, E.; Fasano, A.; Carlone, G.; Cavatorta, P.; Riccio, P.
  Biophysical Journal (2004), 86 (1, Pt. 1), 455-460CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
  The structure of myelin basic protein (MBP), purified from the myelin sheath in both lipid-free (LF-MBP) and lipid-bound (LB-MBP) forms, was investigated in soln. by small angle x-ray scattering. The water-sol. LF-MBP, extd. at pH < 3.0 from defatted brain, is the classical prepn. of MBP, commonly regarded as an intrinsically unfolded protein. LB-MBP is a lipoprotein-detergent complex extd. from myelin with its native lipidic environment at pH > 7.0. Under all conditions, the scattering from the two protein forms was different, indicating different mol. shapes. For the LB-MBP, well-defined scattering curves were obtained, suggesting that the protein had a unique, compact (but not globular) structure. Furthermore, these data were compatible with earlier results from mol. modeling calcns. on the MBP structure which have been refined by us. In contrast, the LF-MBP data were in accordance with the expected open-coil conformation. The results represent the first direct structural information from x-ray scattering measurements on MBP in its native lipidic environment in soln.
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33. 33
  Jahn, O.; Tenzer, S.; Werner, H. Myelin Proteomics: Molecular Anatomy of an Insulating Sheath. Mol. Neurobiol. 2009, 40, 55– 72, DOI: 10.1007/s12035-009-8071-2
  33
  Myelin Proteomics: Molecular Anatomy of an Insulating Sheath
  Jahn, Olaf; Tenzer, Stefan; Werner, Hauke B.
  Molecular Neurobiology (2009), 40 (1), 55-72CODEN: MONBEW; ISSN:0893-7648. (Humana Press Inc.)
  A review. Fast-transmitting vertebrate axons are elec. insulated with multiple layers of nonconductive plasma membrane of glial cell origin, termed myelin. The myelin membrane is dominated by lipids, and its protein compn. has historically been viewed to be of very low complexity. In this review, we discuss an updated ref. compendium of 342 proteins assocd. with central nervous system myelin that represents a valuable resource for analyzing myelin biogenesis and white matter homeostasis. Cataloging the myelin proteome has been made possible by tech. advances in the sepn. and mass spectrometric detection of proteins, also referred to as proteomics. This led to the identification of a large no. of novel myelin-assocd. proteins, many of which represent low abundance components involved in catalytic activities, the cytoskeleton, vesicular trafficking, or cell adhesion. By mass spectrometry-based quantification, proteolipid protein and myelin basic protein constitute 17% and 8% of total myelin protein, resp., suggesting that their abundance was previously overestimated. As the biochem. profile of myelin-assocd. proteins is highly reproducible, differential proteome analyses can be applied to material isolated from patients or animal models of myelin-related diseases such as multiple sclerosis and leukodystrophies.
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34. 34
  Fewster, M. E.; Hirono, H.; Mead, J. F. Lipid composition of myelin in multiple sclerosis. J. Neurol. 1976, 213, 119– 131, DOI: 10.1007/BF00313273
  34
  Lipid composition of myelin in multiple sclerosis
  Fewster, Mona E.; Hirono, H.; Mead, J. F.
  Journal of Neurology (1976), 213 (2), 119-31CODEN: JNRYA9; ISSN:0340-5354.
  Myelin was isolated from histol. normal white matter and plaques from multiple sclerosis (MS) patients and from white matter of neurol. normal controls. No difference was found in the total lipid content. There were no detectable deficits in MS myelin of phosphoglycerides, plasmalogens or sphingolipids. Gangliosides and lysolecithin were not detected. Anal. of the fatty aldehyde compn. of the phosphoglycerides and the fatty acid compn. of the cholesteryl esters, phosphoglycerides and sphingolipids did not show any differences between the normal and MS myelin.
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35. 35
  Wilson, R.; Tocher, D. R. Lipid and fatty acid composition is altered in plaque tissue from multiple sclerosis brain compared with normal brain white matter. Lipids 1991, 26, 9– 15, DOI: 10.1007/BF02544017
  35
  Lipid and fatty acid composition is altered in plaque tissue from multiple sclerosis brain compared with normal brain white matter
  Wilson, Robert; Tocher, Douglas R.
  Lipids (1991), 26 (1), 9-15CODEN: LPDSAP; ISSN:0024-4201.
  Plaques and white matter from brains of multiple sclerosis (MS) patients were analyzed for lipid content, class compn., and fatty acid compn. of total lipid, together with the fatty acid compn. of plaque glycerophospholipids, and the results were compared with white matter from normal brain. Plaques contained less than 30% of the lipid present in normal white matter. Plaque lipid was characterized by significantly increased proportions of glycerophospholipids and decreased cerebrosides and sulfatides. In addn., a subacute plaque contained approx. 10 times the proportion of steryl esters obsd. in chronic plaques or normal white matter. Total lipid from all the MS plaques showed significantly increased percentages of satd. fatty acids, n-6, n-3 and total polyunsatd. fatty acids and decreased percentages of monoenes and alk-1-enyl ethers in comparison with normal brains. These results were consistent with increased cellularity and astrogliosis assocd. with MS plaques. However, anal. of plaque glycerophospholipids showed that the fatty acid changes obsd. in total lipid were not simply due to the increased myelin lipids, but that the fatty acid compn. of the individual glycerophospholipids was different.
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36. 36
  Alling, C.; Vanier, M. T.; Svennerholm, L. Lipid alterations in apparently normal white matter in multiple sclerosis. Brain Res. 1971, 35, 325– 336, DOI: 10.1016/0006-8993(71)90478-1
  36
  Lipid alterations in apparently normal white matter in multiple sclerosis
  Alling, Christer; Vanier, Marie T.; Svennerholm, Lars
  Brain Research (1971), 35 (2), 325-36CODEN: BRREAP; ISSN:0006-8993.
  Apparently normal white matter from 6 subjects with multiple sclerosis (MS) of 5-41 years duration was compared with roughly age-matched normal tissue. Cholesterol, phospholipids, and galactolipids were slightly lower in MS brains, but significantly lower values were found only for serine phosphoglycerides and sulfatides. The fatty acid patterns of MS brains were strikingly similar to those of normal brains. Nevertheless, the concns. of some of the fatty acids characteristic of white matter were slightly lower in MS brains. There was no evidence for a primary lipid defect of myelin. The abnormalities in the lipid and the fatty acid compn. were explained by the presence of small lesions overlooked at isolation of apparently normal white matter.
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37. 37
  Woelk, H.; Borri, P. Lipid and fatty acid composition of myelin purified from normal and MS brains. Eur. Neurol. 1973, 10, 250– 260, DOI: 10.1159/000114281
  37
  Lipid and fatty acid composition of myelin purified from normal and MS [multiple sclerosis] brains
  Woelk, Helmut; Borri, Piero
  European Neurology (1973), 10 (4), 250-60CODEN: EUNEAP; ISSN:0014-3022.
  The concns. of phospholipids and galactolipids and their fatty acid compns. were detd. in purified myelin from apparently normal white matter of 11 MS and 11 control brains. Myelin from MS brains showed a significant decrease in serine phosphatide and a slight decrease in the ethanolamine phosphatide fraction when compared with normal myelin; increases were found in the lysocholine and lysoethanolamine phosphoglycerides. In the galactolipids, lower figures were obtained for sulfatides, resulting in a higher cerebroside/sulfatide ratio for MS myelin. Anal. of the fatty acid pattern of phospholipids in MS brains revealed lower values for 18:1 and, except for the lyso compds., slightly higher amts. for 20:4 and 22:6. Lower levels for the sum of 18:3 and 20:1 fatty acids were found for the ethanolamine and inositol phosphoglyceride fractions.
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38. 38
  Gerstl, B.; Eng, L. F.; Tavaststjerna, M.; Smith, J. K.; Kruse, S. L. Lipids and proteins in multiple sclerosis white matter. J. Neurochem. 1970, 17, 677– 689, DOI: 10.1111/j.1471-4159.1970.tb00547.x
  38
  Lipids and proteins in multiple sclerosis white matter
  Gerstl, Bruno; Eng, Lawrence F.; Tavaststjerna, M.; Smith, James Knox; Kruse, S. L.
  Journal of Neurochemistry (1970), 17 (5), 677-89CODEN: JONRA9; ISSN:0022-3042.
  Quant. anals. of white matter from four brains of patients with multiple sclerosis (MS) and four control brains were carried out for total and sol. proteins, individual lipid fractions, and their corresponding fatty acids. In three specimens from two of the MS brains there were redns. of cerebrosides and of the C20:1 acid in the ethanolamine glycerophosphatide (EGP) fraction and a slight increase of tetraenes and trienes, while all other components were present in concns. similar to those in the controls. In three other samples from two of the MS brains, galactolipids were deficient to a greater extent than cholesterol, EGP, or CGP (choline glycerophosphatide), while proteins were within the control range. In samples where thinning of myelin was observed in Luxol-blue stained sections, there were proportional decreases of all components. The percentage of C20:1 acid in the EGP fraction was reduced in two of three myelin prepns. from corresponding samples of MS white matter, and that of C24:1 acid in the cerebroside fraction was reduced in all three MS myelin prepns. when compared with the two controls. The data suggest that inadequacy of the fatty acid elongation process together with deficits of cerebrosides represent one of the early biochem. lesions in the white matter of the MS brain.
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39. 39
  Arnetoli, G.; Pazzagli, A.; Amaducci, L. Fatty acid and aldehyde changes in choline- and ethanolamine-containing phospholipids in the white matter of multiple sclerosis brains. J. Neurochem. 1969, 16, 461– 463, DOI: 10.1111/j.1471-4159.1969.tb10387.x
  39
  Fatty acid and aldehyde changes in choline- and ethanolamine-containing phospholipids in the white matter of multiple sclerosis brains
  Arnetoli, G.; Pazzagli, Adolfo; Amaducci, Luigi
  Journal of Neurochemistry (1969), 16 (3), 461-3CODEN: JONRA9; ISSN:0022-3042.
  As detd. by gas chromatog. of the fatty acid methyl esters, the fatty acid compn. of the choline-contg. phospholipids from central white matter of 5 multiple sclerosis brains was not different from that in 4 control brains. In multiple sclerosis brains the percentage of monounsatd. C18 fatty acids in ethanolamine-contg. phospholipids from central white matter was decreased and the percentage of satd. C18 fatty acids and (or) monounsatd. C18 fatty aldehydes was increased compared to controls. Thus, the ratio of satd. C18 acid and (or) monounsatd. C18-aldehyde to monounsatd. C18-fatty acid in ethanolamine-contg. phospholipids was 1.51 in multiple sclerosis brains compared to 0.82 in controls. Thus ethanolamine-contg. phospholipids may be affected earlier or to a greater extent than choline-contg. phospholipids in this type of disease.
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40. 40
  Göpfert, E.; Pytlik, S.; Debuch, H. 2′,3′-Cyclic nucleotide 3′-phosphohydrolase and lipids of myelin from multiple sclerosis and normal brains. J. Neurochem. 1980, 34, 732– 739, DOI: 10.1111/j.1471-4159.1980.tb11205.x
  40
  2',3'-Cyclic nucleotide 3'-phosphohydrolase and lipids of myelin from multiple sclerosis and normal brains
  Gopfert E; Pytlik S; Debuch H
  Journal of neurochemistry (1980), 34 (3), 732-9 ISSN:0022-3042.
  There is no expanded citation for this reference.
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41. 41
  Neu, I.; Woelk, H. Investigations of the lipid metabolism of the white matter in multiple sclerosis Changes in glycero-phosphatides and lipid-splitting enzymes. Neurochem. Res. 1982, 7, 727– 735, DOI: 10.1007/BF00965525
  41
  Investigations of the lipid metabolism of the white matter in multiple sclerosis: changes in glycero-phosphatides and lipid-splitting enzymes
  Neu I; Woelk H
  Neurochemical research (1982), 7 (6), 727-35 ISSN:0364-3190.
  Phospho- and galacto- lipids and lipidhydrolyzing enzymes have been determined in the white matter of a young patient with a subacute course of multiple sclerosis (MS). Significant changes were observed for the concentration of glycerophosphatides and the fatty acid pattern of the normal appearing with matter surrounding MS-plaques. Among the individual glycerophosphatides a significant decrease of phosphatidylserine and phosphatidylinositol was found, whereas the ethanolamine containing phosphatides showed lower figures (non significant). The fatty acid pattern of the ethanolamine-phosphatide-fraction of the diseased tissue a decrease of the 18:1 and the sum of 20:1 and 18:3 fatty acids as compared to the normal control, whereas the highly unsaturated, long-chained fatty acids 20:4 (arachidonic acid) and 22:6 (docosahexaenic acid) were elevated. The measurement of lipidhydrolyzing enzymes resulted in an increased phospholipase A1 activity in the diseased tissue. The experimental data point to a decreased activity of the fatty acid elongation system in the course of MS. The decrease of the acidic glycerophosphatides might be due to the increased phospholipase A1 activity.
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42. 42
  Del Boccio, P.; Pieragostino, D.; Di Ioia, M.; Petrucci, F.; Lugaresi, A.; De Luca, G.; Gambi, D.; Onofrj, M.; Di Ilio, C.; Sacchetta, P.; Urbani, A. Lipidomic investigations for the characterization of circulating serum lipids in multiple sclerosis. J. Proteomics 2011, 74, 2826– 2836, DOI: 10.1016/j.jprot.2011.06.023
  42
  Lipidomic investigations for the characterization of circulating serum lipids in multiple sclerosis
  Del Boccio, Piero; Pieragostino, Damiana; Di Ioia, Maria; Petrucci, Francesca; Lugaresi, Alessandra; De Luca, Giovanna; Gambi, Domenico; Onofrj, Marco; Di Ilio, Carmine; Sacchetta, Paolo; Urbani, Andrea
  Journal of Proteomics (2011), 74 (12), 2826-2836CODEN: JPORFQ; ISSN:1874-3919. (Elsevier B.V.)
  Multiple Sclerosis (MS) is a neurodegenerative autoimmune demyelinating disease affecting young adults. The etiol. still remains a mystery and diagnosis is impaired by the lack of defined mol. markers. Autoimmune response remains the main topic under investigation and recent studies suggest addnl. non-proteic mediators of brain inflammation such as lipids. We carried out an LC-MS based lipidomics approach to highlight serum lipids profiling in MS. Method was optimized and applied in a preliminary clin. cross-sectional investigation of MS patients vs Healthy Controls (HC) and patients with Other Neurol. Diseases (OND). Ten significant metabolites were highlighted and tentatively identified by accurate mass and MS/MS expts. Our most relevant data show altered level of lyso-glycerophosphatidylcholine (lysoPC) and glycerophosphatidylcholine (PC) species. Total lysoPC/PC ratio showed significant decrease in pathol. groups (MS, OND) and, in addn., MS subjects had a relevant decrease of this ratio also in respect to OND. These findings suggest that there may be an altered phospholipid metab. in MS that can be evaluated in serum. Some of these features are distinctive and may be considered specific for MS. Our lipidomics data show, for the first time, evidence in serum of a relationship between LysoPC/PC ratio and MS.
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43. 43
  Boggs, J. M. Myelin Basic Protein; Nova Science Publishers, Incorporated: New York, 2008.
  There is no corresponding record for this reference.
44. 44
  Procaccini, C.; De Rosa, V.; Pucino, V.; Formisano, L.; Matarese, G. Animal models of Multiple Sclerosis. Eur. J. Pharmacol. 2015, 759, 182– 191, DOI: 10.1016/j.ejphar.2015.03.042
  44
  Animal models of Multiple Sclerosis
  Procaccini, Claudio; De Rosa, Veronica; Pucino, Valentina; Formisano, Luigi; Matarese, Giuseppe
  European Journal of Pharmacology (2015), 759 (), 182-191CODEN: EJPHAZ; ISSN:0014-2999. (Elsevier B.V.)
  Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) which involves a complex interaction between immune system and neural cells. Animal modeling has been crit. for addressing MS pathogenesis. The three most characterized animal models of MS are (1) the exptl. autoimmune/allergic encephalomyelitis (EAE); (2) the virally-induced chronic demyelinating disease, known as Theiler's murine encephalomyelitis virus (TMEV) infection and (3) the toxin-induced demyelination. All these models, in a complementary way, have allowed to reach a good knowledge of the pathogenesis of MS. Specifically, EAE is the model which better reflects the autoimmune pathogenesis of MS and is extremely useful to study potential exptl. treatments. Furthermore, both TMEV and toxin-induced demyelination models are suitable for characterizing the role of the axonal injury/repair and the remyelination process in MS. In conclusion, animal models, despite their limitations, remain the most useful instrument for implementing the study of MS.
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45. 45
  Niroomand, H.; Pamu, R.; Mukherjee, D.; Khomami, B. Microenvironment alterations enhance photocurrents from photosystem I confined in supported lipid bilayers. J. Mater. Chem. A 2018, 6, 12281– 12290, DOI: 10.1039/C8TA00898A
  45
  Microenvironment alterations enhance photocurrents from photosystem I confined in supported lipid bilayers
  Niroomand, Hanieh; Pamu, Ravi; Mukherjee, Dibyendu; Khomami, Bamin
  Journal of Materials Chemistry A: Materials for Energy and Sustainability (2018), 6 (26), 12281-12290CODEN: JMCAET; ISSN:2050-7496. (Royal Society of Chemistry)
  Transmembrane photosynthetic proteins, photosystem I (PSI), are nano-scale biol. photodiodes that enable light-activated unidirectional electron flow. The robust photochem. properties of PSI make it a promising candidate for harnessing solar energy. However, the role of natural membrane confinements of PSI in orchestrating this photoactivated charge sepn. with near unity quantum efficiency, which is central to the rational design of PSI-based energy conversion systems, is still ill-understood. Motivated by this lack of fundamental understanding, herein the authors investigate the photoactivity of biomimetic constructs of cyanobacterial PSI encapsulated within solid-supported lipid bilayers (SLB) assembled on electrodes. PSI confined in SLBs is assembled from PSI-proteoliposomes that are synthesized from the recently developed facile routes for engineering neg. charged phospholipid (DPhPG) bilayer membranes. Specifically, detailed chronoamperometry measurements have been used to investigate photocurrent variations arising from the SLBs supported on self-assembled monolayer (SAM) substrates. These measurements, in conjunction with cryo-TEM, at. force microscopy imaging and force spectroscopy, allow for direct visualization and detection of the SLBs of PSI-proteoliposomes on the substrates. The results indicate the crit. role of microenvironment alterations, heretofore not considered, in achieving ∼4-5-fold enhancements in photocurrents generated from PSI complexes under SLB confinements as compared to those from a dense monolayer of equiv. concns. of PSI on SAM substrates.
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46. 46
  Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. Theory of self-assembly of lipid bilayers and vesicles. Biochim. Biophys. Acta, Biomembr. 1977, 470, 185– 201, DOI: 10.1016/0005-2736(77)90099-2
  46
  Theory of self-assembly of lipid bilayers and vesicles
  Israelachvili, Jacob N.; Mitchell, D. John; Ninham, Barry W.
  Biochimica et Biophysica Acta, Biomembranes (1977), 470 (2), 185-201CODEN: BBBMBS; ISSN:0005-2736.
  A simple theory is developed that explains the formation of bilayers and vesicles and accounts quant. for many of their phys. properties. Properties including vesicle size distributions and bilayer elasticity, emerge from a unified theory that links thermodn., interaction free energy, and mol. geometry. The theory may be applied to the anal. of more complicated membrane structures and mechanisms.
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47. 47
  Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press, 2015.
  There is no corresponding record for this reference.
48. 48
  Wood, D. D.; Moscarello, M. A. The isolation, characterization, and lipid-aggregating properties of a citrulline containing myelin basic protein. J. Biol. Chem. 1989, 264, 5121– 5127
  48
  The isolation, characterization, and lipid-aggregating properties of a citrulline containing myelin basic protein
  Wood, D. D.; Moscarello, M. A.
  Journal of Biological Chemistry (1989), 264 (9), 5121-7CODEN: JBCHA3; ISSN:0021-9258.
  Human myelin basic protein was fractionated into its various charge isomers by CM52 cation exchange chromatog. Of the total charge applied to the column, ∼25-30% appeared in the void vol. This material, termed C-8, was further purified by reversed phase HPLC. Amino acid analyses of C-8 revealed low arginine (Arg) (7 residue % in C-8 compared to 11-12 residue % in C-1) and increased glutamic acid or glutamine residues. The low Arg was accounted for by a corresponding amt. of citrulline. Sequence anal. after chem. fragmentation (CNBr and BNPS-skatole) and enzymic (cathepsin D and carboxypeptidase S-1) digestion localized the citrulline at residues 25, 31, 122, 130, 159, and 170 of the amino acid sequence. The effect of this loss of pos. charge on the ability of the protein to aggregate lipid vesicles was demonstrated with vesicles composed of phosphatidylcholine (92.2 mol %) and phosphatidylserine (7.8 mol %). C-1 was the most effective charge isomer, and C-8 was the least effective. The ability of these charge isomers to aggregate vesicles correlated with the net pos. charge on each. Vesicles composed of phosphatidylcholine alone were not aggregated by lipophilin or any of the charge isomers. However, when lipophilin was incorporated into phosphatidylcholine vesicles (50%), small, optically clear suspensions of vesicles were formed. None of C-1, C-2, or C-3 aggregated these vesicles, but C-8 produced rapid vesicle aggregation. Since the substitution of citrulline for Arg would generate several relatively long apolar sequences, these would enhance the ability of C-8 to interact with the hydrophobic lipophilin mol., promoting vesicle aggregation by hydrophobic interactions.
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49. 49
  Harauz, G.; Musse, A. A Tale of Two Citrullines—Structural and Functional Aspects of Myelin Basic Protein Deimination in Health and Disease. Neurochem. Res. 2007, 32, 137– 158, DOI: 10.1007/s11064-006-9108-9
  49
  A Tale of Two Citrullines-Structural and Functional Aspects of Myelin Basic Protein Deimination in Health and Disease
  Harauz, George; Musse, Abdiwahab A.
  Neurochemical Research (2007), 32 (2), 137-158CODEN: NEREDZ; ISSN:0364-3190. (Springer)
  A review. Myelin basic protein (MBP) binds to neg. charged lipids on the cytosolic surface of oligodendrocyte membranes and is responsible for adhesion of these surfaces in the multilayered myelin sheath. The pattern of extensive post-translational modifications of MBP is dynamic during normal central nervous system (CNS) development and during myelin degeneration in multiple sclerosis (MS), affecting its interactions with the myelin membranes and with other mols. In particular, the degree of deimination (or citrullination) of MBP is correlated with the severity of MS, and may represent a primary defect that precedes neurodegeneration due to autoimmune attack. That the degree of MBP deimination is also high in early CNS development indicates that this modification plays major physiol. roles in myelin assembly. In this review, we describe the structural and functional consequences of MBP deimination in healthy and diseased myelin.
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50. 50
  Matsumoto, T.; Kobayashi, T.; Kamata, K. Role of lysophosphatidylcholine (LPC) in atherosclerosis. Curr. Med. Chem. 2007, 14, 3209– 3220, DOI: 10.2174/092986707782793899
  50
  Role of lysophosphatidylcholine (LPC) in atherosclerosis
  Matsumoto, Takayuki; Kobayashi, Tsuneo; Kamata, Katsuo
  Current Medicinal Chemistry (2007), 14 (30), 3209-3220CODEN: CMCHE7; ISSN:0929-8673. (Bentham Science Publishers Ltd.)
  A review. Lysophosphatidylcholine (LPC) is a bioactive proinflammatory lipid generated by pathol. activities. LPC is also a major phospholipid component of oxidized low-d. lipoprotein (Ox-LDL) and is implicated as a crit. factor in the atherogenic activity of Ox-LDL. LPC is believed to play an important role in atherosclerosis and inflammatory diseases by altering various functions in a no. of cell-types, including endothelial cells, smooth muscle cells, monocytes, macrophages, and T-cells. LPC activates several second messengers - including protein kinase C, extracellular-signal-regulated kinases, protein tyrosine kinases, and Ca2+ -- implicating the engagement of transduction mechanisms in its obsd. actions. Moreover, recent evidence suggests that in several cell-types, cloned orphan G-protein-coupled receptors may serve as the specific receptors via which LPC modulates second messenger pathways (although LPC may not be a direct ligand of such receptors). In addn., current evidence suggests that LPC impairs the endothelium-dependent relaxations mediated by endothelium-derived relaxing factors and directly modulates contractile responses in vascular smooth muscle. However, despite all this, and although elevated levels of LPC have been linked to the cardiovascular complications assocd. with atherosclerosis, ischemia, and diabetes, the precise pathophysiol. roles played by LPC in several states remain to be established. In this review, we focus in some detail on the entirety of the signal-transduction system for LPC, its pathophysiol. implications, and the vascular abnormalities assocd. with it.
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51. 51
  Muramatsu, R.; Kuroda, M.; Matoba, K.; Lin, H.; Takahashi, C.; Koyama, Y.; Yamashita, T. Prostacyclin prevents pericyte loss and demyelination induced by lysophosphatidylcholine in the central nervous system. J. Biol. Chem. 2015, 290, 11515– 11525, DOI: 10.1074/jbc.M114.587253
  51
  Prostacyclin Prevents Pericyte Loss and Demyelination Induced by Lysophosphatidylcholine in the Central Nervous System
  Muramatsu, Rieko; Kuroda, Mariko; Matoba, Ken; Lin, Hsiaoyun; Takahashi, Chisato; Koyama, Yoshihisa; Yamashita, Toshihide
  Journal of Biological Chemistry (2015), 290 (18), 11515-11525CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
  Pericytes play pivotal roles in physiol. and pathophysiol. conditions in the central nervous system. As pericytes prevent vascular leakage, they can halt neuronal damage stemming from a compromised blood-brain barrier. Therefore, pericytes may be a good target for the treatment of neurodegenerative disorders, although evidence is lacking. In this study, we show that prostacyclin attenuates lysophosphatidylcholine (LPC)-mediated vascular dysfunction through pericyte protection in the adult mouse spinal cord. LPC decreased the no. of pericytes in an in vitro blood-brain barrier model, and this decrease was prevented by iloprost treatment, a prostacyclin analog. Intrathecal administration of iloprost attenuated vascular barrier disruption after LPC injection in the mouse spinal cord. Furthermore, iloprost treatment diminished demyelination and motor function deficits in mice injected with LPC. These results support the notion that prostacyclin acts on pericytes to maintain vascular barrier integrity.
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52. 52
  Plemel, J. R.; Michaels, N. J.; Weishaupt, N.; Caprariello, A. V.; Keough, M. B.; Rogers, J. A.; Yukseloglu, A.; Lim, J.; Patel, V. V.; Rawji, K. S.; Jensen, S. K.; Teo, W.; Heyne, B.; Whitehead, S. N.; Stys, P. K.; Yong, V. W. Mechanisms of lysophosphatidylcholine-induced demyelination: A primary lipid disrupting myelinopathy. Glia 2018, 66, 327– 347, DOI: 10.1002/glia.23245
  52
  Mechanisms of lysophosphatidylcholine-induced demyelination: A primary lipid disrupting myelinopathy
  Plemel Jason R; Michaels Nathan J; Caprariello Andrew V; Keough Michael B; Rogers James A; Yukseloglu Aran; Lim Jaehyun; Rawji Khalil S; Jensen Samuel K; Teo Wulin; Stys Peter K; Yong V Wee; Weishaupt Nina; Patel Vikas V; Whitehead Shawn N; Heyne Belinda
  Glia (2018), 66 (2), 327-347 ISSN:.
  For decades lysophosphatidylcholine (LPC, lysolecithin) has been used to induce demyelination, without a clear understanding of its mechanisms. LPC is an endogenous lysophospholipid so it may cause demyelination in certain diseases. We investigated whether known receptor systems, inflammation or nonspecific lipid disruption mediates LPC-demyelination in mice. We found that LPC nonspecifically disrupted myelin lipids. LPC integrated into cellular membranes and rapidly induced cell membrane permeability; in mice, LPC injury was phenocopied by other lipid disrupting agents. Interestingly, following its injection into white matter, LPC was cleared within 24 hr but by five days there was an elevation of endogenous LPC that was not associated with damage. This elevation of LPC in the absence of injury raises the possibility that the brain has mechanisms to buffer LPC. In support, LPC injury in culture was significantly ameliorated by albumin buffering. These results shed light on the mechanisms of LPC injury and homeostasis.
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53. 53
  Birgbauer, E.; Rao, T. S.; Webb, M. Lysolecithin induces demyelination in vitro in a cerebellar slice culture system. J. Neurosci. Res. 2004, 78, 157– 166, DOI: 10.1002/jnr.20248
  53
  Lysolecithin induces demyelination in vitro in a cerebellar slice culture system
  Birgbauer, Eric; Rao, Tadimeti S.; Webb, Michael
  Journal of Neuroscience Research (2004), 78 (2), 157-166CODEN: JNREDK; ISSN:0360-4012. (Wiley-Liss, Inc.)
  Demyelination is a hallmark of several human diseases, including multiple sclerosis. To understand better the process of demyelination and remyelination, we explored the use of an in vitro organotypic cerebellar slice culture system. Para-sagittal slices of postnatal Day 10 (P10) rat cerebellar cultured in vitro demonstrated significant myelination after 1 wk in culture. Treatment of the cultures at 7 days in vitro (DIV) with the bioactive lipid lysolecithin (lysophosphatidylcholine) for 15-17 h in vitro produced marked demyelination. This demyelination was obsd. by immunostaining for the myelin components myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). After a transient demyelinating insult with lysolecithin in vitro, the cultures recovered with oligodendrocyte differentiation recapitulating a normal time course; there was initially re-expression of CNPase and MBP during this recovery, and this was followed by MOG. In addn., there seemed to be some limited remyelination during the recovery phase. Lysolecithin thus induces demyelination in an in vitro organotypic cerebellar slice culture system, providing a model system for studying myelination, demyelination, and remyelination in vitro.
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54. 54
  Boggs, J. M. Myelin basic protein: a multifunctional protein. Cell. Mol. Life Sci. 2006, 63, 1945– 1961, DOI: 10.1007/s00018-006-6094-7
  54
  Myelin basic protein: a multifunctional protein
  Boggs, J. M.
  Cellular and Molecular Life Sciences (2006), 63 (17), 1945-1961CODEN: CMLSFI; ISSN:1420-682X. (Birkhaeuser Verlag)
  A review. Myelin basic protein (MBP), the 2nd most abundant protein in central nervous system myelin, is responsible for adhesion of the cytosolic surfaces of multilayered compact myelin. A member of the 'intrinsically disordered' or conformationally adaptable protein family, it also appears to have several other functions. It can interact with a no. of polyanionic proteins including actin, tubulin, Ca2+-calmodulin, and clathrin, and neg. charged lipids, and acquires structure on binding to them. It may act as a membrane actin-binding protein, which might allow it to participate in transmission of extracellular signals to the cytoskeleton in oligodendrocytes and tight junctions in myelin. Some size isoforms of MBP are transported into the nucleus and thus they may also bind polynucleotides. Extracellular signals received by myelin or cultured oligodendrocytes cause changes in the phosphorylation of MBP, suggesting that MBP is also involved in signaling. Further study of this very abundant protein will reveal how it is utilized by the oligodendrocyte and myelin for different purposes.
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55. 55
  Harauz, G.; Ishiyama, N.; Hill, C. M.; Bates, I. R.; Libich, D. S.; Fares, C. Myelin basic protein-diverse conformational states of an intrinsically unstructured protein and its roles in myelin assembly and multiple sclerosis. Micron 2004, 35, 503– 542, DOI: 10.1016/j.micron.2004.04.005
  55
  Myelin basic protein - diverse conformational states of an intrinsically unstructured protein and its roles in myelin assembly and multiple sclerosis
  Harauz, George; Ishiyama, Noboru; Hill, Christopher M. D.; Bates, Ian R.; Libich, David S.; Fares, Christophe
  Micron (2004), 35 (7), 503-542CODEN: MCONEN; ISSN:0968-4328. (Elsevier Science B.V.)
  A review. The 18.5-kDa isoform of myelin basic protein (MBP) is a major component of the myelin sheath in the central nervous system of higher vertebrates, and a member of a larger family of proteins with a multiplicity of forms and post-translational modifications (PTMs). The 18.5-kDa protein is the exemplar of the family, being most abundant in adult myelin, and thus the most-studied. It is peripherally membrane-assocd., but has generally been investigated in isolated form. MBP is an 'intrinsically unstructured' protein with a high proportion (∼75%) of random coil, but postulated to have core elements of β-sheet and α-helix. Here, the authors review the properties of the MBP family, esp. of the 18.5-kDa isoform, and discuss how its 3-dimensional (3D) structure may be resolved by direct techniques available to them, viz., x-ray and electron crystallog., and soln. and solid-state NMR spectrometry. In particular, it is emphasized that creating an appropriate environment in which the protein can adopt a physiol. relevant fold is crucial to such endeavors. By solving the 3D structure of 18.5-kDa MBP and the effects of PTMs, a better understanding of myelin architecture, and of the mol. mechanisms that transpire in demyelinating diseases such as multiple sclerosis, will be attained.
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56. 56
  D’Souza, C. A.; Wood, D. D.; She, Y.-M.; Moscarello, M. A. Autocatalytic cleavage of myelin basic protein: An alternative to molecular mimicry. Biochemistry 2005, 44, 12905– 12913, DOI: 10.1021/bi051152f
  56
  Autocatalytic Cleavage of Myelin Basic Protein: An Alternative to Molecular Mimicry
  D'Souza, Cheryl A.; Wood, D. Denise; She, Yi-Min; Moscarello, Mario A.
  Biochemistry (2005), 44 (38), 12905-12913CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  Although multiple sclerosis (MS) is thought to be an autoimmune disease, the mechanisms by which immunodominant epitopes are generated and lymphocytes are activated are not known. Here, myelin basic protein-component 1 (MBP-C1) from MS tissue was shown to undergo autocatalytic cleavage at slightly alk. pH. Importantly, one of the major peptides released contained the immunodominant epitope 84-89. Interestingly, MBP isolated from MS patients showed a faster time course of cleavage and a more robust release of epitope 84-89 than MBP isolated from normal individuals. The cleavage reaction was not inhibited by protease inhibitors, except for phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor. Since PMSF inhibition suggested a role for a serine residue in the cleavage, we labeled myelin basic protein with diisopropyl fluorophosphate (DFP), known to bind active site serine residues. Mass spectrometry was used to identify the labeled peptide, which consisted of residues 140-152. Since this peptide contained a single serine residue, we concluded it to be the active serine. The importance of this cleavage mechanism is that it provides for a ready source of the immunodominant peptide for sensitization of T-cells. It is not necessary to invoke other mechanisms such as mol. mimicry.
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57. 57
  Zhou, S. R.; Moscarello, M. A.; Whitaker, J. N. The effects of citrullination or variable amino-terminus acylation on the encephalitogenicity of human myelin basic protein in the PL/J mouse. J. Neuroimmunol. 1995, 62, 147– 152, DOI: 10.1016/0165-5728(95)00112-3
  57
  The effects of citrullination or variable amino-terminus acylation on the encephalitogenicity of human myelin basic protein in the PL/J mouse
  Zhou, Shan-Ren; Moscarello, Mario; Whitaker, John N.
  Journal of Neuroimmunology (1995), 62 (2), 147-152CODEN: JNRIDW; ISSN:0165-5728. (Elsevier)
  The post-translational modifications of myelin basic protein (MBP) in the form of citrullination and varying length of amino-terminus acylation may modify the biol. functions and immunol. features of MBP. Both modifications influence the reaction of antibodies and specific T cells recognizing MBP. The present study was undertaken to compare the encephalitogenicity of the citrullinated isomer of MBP (MBP-C8) with the unmodified isomer of MBP (MBP-C1) and to det. if the length of amino-terminal acylation of MBP peptide 1-21 altered an encephalitogenic epitope. MBP-C8, whether from patients with or without multiple sclerosis (MS), and MBP-C1 could induce active exptl. allergic encephalomyelitis (EAE) in PL/J mice. A trend of reduced severity of EAE was obsd. in MBP-C8-injected animals. An increase in the length of amino-terminus fatty acid decreased the encephalitogenicity of MBP peptide 1-21 for both active and adoptive EAE in PL/J mice. Only lymph node cells sensitive to MBP peptide acetyl 1-21 and Bu 1-21 could transfer clin. EAE. In adoptive EAE, MBP peptides hexyl and octyl 1-21 induced moderate histopathol. but no clin. change, whereas MBP peptide decyl 1-21 caused neither. A broadening in the antibody response could be detected in the sera of mice with active EAE induced by MBP-acylated peptides 1-21. Our findings demonstrate that encephalitogenicity is retained in the presence of citrullination but that the length of amino-terminus acylation diminishes the encephalitogenicity of MBP in the PL/J mouse. These findings may be relevant to MS where central nervous system myelin shows differences from normal in both MBP-C8 content and MBP amino-terminus acylation.
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58. 58
  Madsen, P. M.; Motti, D.; Karmally, S.; Szymkowski, D. E.; Lambertsen, K. L.; Bethea, J. R.; Brambilla, R. Oligodendroglial TNFR2 Mediates Membrane TNF-Dependent Repair in Experimental Autoimmune Encephalomyelitis by Promoting Oligodendrocyte Differentiation and Remyelination. J. Neurosci. 2016, 36, 5128– 5143, DOI: 10.1523/JNEUROSCI.0211-16.2016
  58
  Oligodendroglial TNFR2 Mediates Membrane TNF-Dependent Repair in Experimental Autoimmune Encephalomyelitis by Promoting Oligodendrocyte Differentiation and Remyelination
  Madsen Pernille M; Motti Dario; Karmally Shaffiat; Brambilla Roberta; Szymkowski David E; Lambertsen Kate Lykke; Bethea John R
  The Journal of neuroscience : the official journal of the Society for Neuroscience (2016), 36 (18), 5128-43 ISSN:.
  UNLABELLED: Tumor necrosis factor (TNF) is associated with the pathophysiology of various neurological disorders, including multiple sclerosis. It exists as a transmembrane form tmTNF, signaling via TNF receptor 2 (TNFR2) and TNFR1, and a soluble form, solTNF, signaling via TNFR1. Multiple sclerosis is associated with the detrimental effects of solTNF acting through TNFR1, while tmTNF promotes repair and remyelination. Here we demonstrate that oligodendroglial TNFR2 is a key mediator of tmTNF-dependent protection in experimental autoimmune encephalomyelitis (EAE). CNP-cre:TNFR2(fl/fl) mice with TNFR2 ablation in oligodendrocytes show exacerbation of the disease with increased axon and myelin pathology, reduced remyelination, and increased loss of oligodendrocyte precursor cells and mature oligodendrocytes. The clinical course of EAE is not improved by the solTNF inhibitor XPro1595 in CNP-cre:TNFR2(fl/fl) mice, indicating that for tmTNF to promote recovery TNFR2 in oligodendrocytes is required. We show that TNFR2 drives differentiation of oligodendrocyte precursor cells, but not proliferation or survival. TNFR2 ablation leads to dysregulated expression of microRNAs, among which are regulators of oligodendrocyte differentiation and inflammation, including miR-7a. Our data provide the first direct in vivo evidence that TNFR2 in oligodendrocytes is important for oligodendrocyte differentiation, thereby sustaining tmTNF-dependent repair in neuroimmune disease. Our studies identify TNFR2 in the CNS as a molecular target for the development of remyelinating agents, addressing the most pressing need in multiple sclerosis therapy nowadays. SIGNIFICANCE STATEMENT: Our study, using novel TNF receptor 2 (TNFR2) conditional KO mice with selective TNFR2 ablation in oligodendrocytes, provides the first direct evidence that TNFR2 is an important signal for oligodendrocyte differentiation. Following activation by transmembrane TNF, TNFR2 initiates pathways that drive oligodendrocytes into a reparative mode contributing to remyelination following disease. This identifies TNFR2 as a new molecular target for the development of therapeutic agents in multiple sclerosis.
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59. 59
  Porciatti, V.; Saleh, M.; Nagaraju, M. The pattern electroretinogram as a tool to monitor progressive retinal ganglion cell dysfunction in the DBA/2J mouse model of glaucoma. Invest. Ophthalmol. Visual Sci. 2007, 48, 745– 751, DOI: 10.1167/iovs.06-0733
  59
  The pattern electroretinogram as a tool to monitor progressive retinal ganglion cell dysfunction in the DBA/2J mouse model of glaucoma
  Porciatti Vittorio; Saleh Maher; Nagaraju Mahesh
  Investigative ophthalmology & visual science (2007), 48 (2), 745-51 ISSN:0146-0404.
  PURPOSE: To determine the baseline characteristics, reliability, and dynamic range of the pattern electroretinogram (PERG) as a tool to monitor progressive RGC dysfunction in the DBA/2J mouse model of glaucoma with spontaneously elevated intraocular pressure (IOP). METHODS: PERGs were recorded from 56 undilated eyes of 28 anesthetized (ketamine-xylazine-acepromazine) DBA/2J mice of different ages (2-4 months, n = 44 eyes; 12-14 months, n = 12 eyes) in response to contrast reversal of gratings that maximize PERG amplitude (95% contrast, 1-Hz reversal, 0.05 cyc/deg spatial frequency, 50 degrees x 56 degrees field size). Robust averaging (1800 sweeps) was used to isolate PERG from background noise. Cone-driven ERGs in response to diffuse light flashes superimposed on a rod-adapting background (FERG) were also recorded. RESULTS: PERGs had consistent waveforms and were reproducible across batches of mice and operators. In 2- to 4-month-old mice (prehypertensive stage), the PERG amplitude (mean, 8.15 +/- 0.4 microV [SEM]) was considerably larger than the noise (mean 1.18 +/- 0.1 microV). The test-retest variability (two different sessions 1 week apart) and interocular asymmetry of PERG amplitude was approximately 30%, and that of PERG latency was approximately 17%. In 12- to 14-month-old mice (advanced hypertensive stage) the PERG amplitude (mean, 1.29 +/- 0.12 microV) was close to that of noise. In 12- to 14-month-old mice the FERG was reduced to a lesser extent compared with the PERG. CONCLUSIONS: The PERG has an adequate signal-to-noise ratio, reproducibility, and dynamic range to monitor the progression of functional changes in the inner retina in DBA/2J mice.
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60. 60
  Menon, K.; Rasband, M. N.; Taylor, C. M.; Brophy, P.; Bansal, R.; Pfeiffer, S. E. The myelin–axolemmal complex: biochemical dissection and the role of galactosphingolipids. J. Neurochem. 2003, 87, 995– 1009, DOI: 10.1046/j.1471-4159.2003.02075.x
  60
  The myelin-axolemmal complex: Biochemical dissection and the role of galactosphingolipids
  Menon, Krishna; Rasband, Matthew N.; Taylor, Christopher M.; Brophy, Peter; Bansal, Rashmi; Pfeiffer, Steven E.
  Journal of Neurochemistry (2003), 87 (4), 995-1009CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)
  Myelin-axolemmal interactions regulate many cellular and mol. events, including gene expression, oligodendrocyte survival and ion channel clustering. Here we report the biochem. fractionation and enrichment of distinct subcellular domains from myelinated nerve fibers. Using antibodies against proteins found in compact myelin, non-compact myelin and axolemma, we show that a rigorous procedure designed to purify myelin also results in the isolation of the myelin-axolemmal complex, a high-affinity protein complex consisting of axonal and oligodendroglial components. Further, the isolation of distinct subcellular domains from galactolipid-deficient mice with disrupted axoglial junctions is altered in a manner consistent with the delocalization of axolemmal proteins obsd. in these animals. These results suggest a paradigm for identification of proteins involved in neuroglial signaling.
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61. 61
  Valdivia, A. O.; Myer, C.; Suarez, M. F.; Bhattacharya, S. K. Protein–Lipid Complex Separation Utilizing a Capillary Electrophoresis System. In Metabolomics: Methods and Protocols; Bhattacharya, S. K., Ed.; Springer: New York, 1996; pp 95– 100.
  There is no corresponding record for this reference.
62. 62
  Chauhan, M. Z.; Valencia, A. K.; Piqueras, M. C.; Enriquez-Algeciras, M.; Bhattacharya, S. K. Optic Nerve Lipidomics Reveal Impaired Glucosylsphingosine Lipids Pathway in Glaucoma. Invest. Ophthalmol. Visual Sci. 2019, 60, 1789– 1798, DOI: 10.1167/iovs.18-25802
  62
  Optic nerve lipidomics reveal impaired glucosylsphingosine lipids pathway in glaucoma
  Chauhan, Muhammad Zain; Valencia, Ann-Katrin; Piqueras, Maria Carmen; Enriquez-Algeciras, Mabel; Bhattacharya, Sanjoy K.
  Investigative Ophthalmology & Visual Science (2019), 60 (5), 1789-1798CODEN: IOVSDA; ISSN:1552-5783. (Association for Research in Vision and Ophthalmology)
  To det. major differences in lipid profile between human control and glaucomatous optic nerve. To assess major enzymes in lipid pathway if aberration is revealed for a lipid class by profiling. Optic nerve (ON) samples were obtained from human cadaveric donors [control (n = 11) and primary open-angle glaucoma (POAG; n = 12)]; the lipids were extd. using Bligh and Dyer methods. Control and glaucoma donors were all Caucasians age 72.3 ± 5.9 and 70.3 ± 10.5 (inclusive of both sexes), resp. Lipids were extd. after weighing the tissue; the protein amts. in the corresponding aq. phase of org. solvent extn. were recorded. High-resoln. mass spectrometry was performed using a Qexactive mass spectrometer coupled with an EASY-nLC 1000 liq. chromatograph instrument. Bioinformatics and statistical anal. were performed using LipidSearch v.4.1 and MetaboAnalyst 4.0/STATA 14.2. Protein amts. were detd. using Bradford's method. Western blot, ELISA, and immunohistochem. utilized established protocols and were performed for protein quantification and localization, resp. Addnl. donor tissues were utilized for Western blot, ELISA, and immunohistochem. Principal component anal. (PCA) placed control and glaucomatous ONs in two distinct groups based on anal. of lipid profiles. Total lipid, total phospholipids, total ceramide, and total sphingolipids were similar (without significant difference) between control and glaucoma. However, we found a significant increase in glucosylsphingosine in glaucoma compared to control samples. We found similar levels of glucocerebrosidase (GBA), ceramide glucosyltransferase (UGCG), decreased nonlysosomal glucocerebrosidase (GBA2), and increased lysosomal and nonlysosomal acylsphingosine amidohydrolase (ASAH1 and ASAH2) levels in glaucomatous ON compared to control. We found significant differences in glucosylsphingosine lipids, consistent with decreased GBA and GBA2 and increased ASAH1 and ASAH2 immunoreactivity in glaucoma, suggesting the potential impairment of sphingolipid enzymic pathways in lysosomal and nonlysosomal cellular compartments.
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63. 63
  Travers, T. S.; Harlow, L.; Rosas, I. O.; Gochuico, B. R.; Mikuls, T. R.; Bhattacharya, S. K.; Camacho, C. J.; Ascherman, D. P. Extensive Citrullination Promotes Immunogenicity of HSP90 through Protein Unfolding and Exposure of Cryptic Epitopes. J. Immunol. 2016, 197, 1926– 1936, DOI: 10.4049/jimmunol.1600162
  63
  Extensive Citrullination Promotes Immunogenicity of HSP90 through Protein Unfolding and Exposure of Cryptic Epitopes
  Travers, Timothy S.; Harlow, Lisa; Rosas, Ivan O.; Gochuico, Bernadette R.; Mikuls, Ted R.; Bhattacharya, Sanjoy K.; Camacho, Carlos J.; Ascherman, Dana P.
  Journal of Immunology (2016), 197 (5), 1926-1936CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
  Post-translational protein modifications such as citrullination have been linked to the breach of immune tolerance and clin. autoimmunity. Previous studies from our lab. support this concept, demonstrating that autoantibodies targeting citrullinated isoforms of heat shock protein 90 (HSP90) are assocd. with rheumatoid arthritis complicated by interstitial lung disease. To further explore the relationship between citrullination and structural determinants of HSP90 immunogenicity, we employed a combination of ELISA-based epitope profiling, computational modeling, and mass-spectrometric sequencing of peptidylarginine deiminase (PAD)-modified protein. Remarkably, ELISAs involving selected citrullinated HSP90β/α peptides identified a key epitope corresponding to an internal Arg residue (R502 [HSP90β]/R510 [HSP90α]) that is normally buried within the crystal structure of native/unmodified HSP90. In vitro time/dose-response expts. reveal an ordered pattern of PAD-mediated deimination events culminating in citrullination of R502/R510. Conventional as well as scaled mol. dynamics simulations further demonstrate that citrullination of selected Arg residues leads to progressive disruption of HSP90 tertiary structure, promoting exposure of R502/R510 to PAD modification and subsequent autoantibody binding. Consistent with this process, ELISAs incorporating variably deiminated HSP90 as substrate Ag indicate a direct relationship between the degree of citrullination and the level of ex vivo Ab recognition. Overall, these data support a novel structural paradigm whereby citrullination-induced shifts in protein structure generate cryptic epitopes capable of bypassing B cell tolerance in the appropriate genetic context.
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64. 64
  Wiedemann, C.; Bellstedt, P.; Gorlach, M. CAPITO--a web server-based analysis and plotting tool for circular dichroism data. Bioinformatics 2013, 29, 1750– 1757, DOI: 10.1093/bioinformatics/btt278
  64
  CAPITO-a web server-based analysis and plotting tool for circular dichroism data
  Wiedemann, Christoph; Bellstedt, Peter; Goerlach, Matthias
  Bioinformatics (2013), 29 (14), 1750-1757CODEN: BOINFP; ISSN:1367-4803. (Oxford University Press)
  Motivation: CD (CD) spectroscopy is one of the most versatile tools to study protein folding and to validate the proper fold of purified proteins. Here, we aim to provide a readily accessible, user-friendly and platform-independent tool capable of analyzing multiple CD datasets of virtually any format and returning results as high-quality graphical output to the user. Results: CAPITO (CD Anal. and Plotting Tool) is a novel web server-based tool for analyzing and plotting CD data. It allows reliable estn. of secondary structure content utilizing different approaches. CAPITO accepts multiple CD datasets and, hence, is well suited for a wide application range such as the anal. of temp. or pH-dependent (un)folding and the comparison of mutants.
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65. 65
  Ahmed, M. A.; De Avila, M.; Polverini, E.; Bessonov, K.; Bamm, V. V.; Harauz, G. Solution nuclear magnetic resonance structure and molecular dynamics simulations of a murine 18.5 kDa myelin basic protein segment (S72-S107) in association with dodecylphosphocholine micelles. Biochemistry 2012, 51, 7475– 7487, DOI: 10.1021/bi300998x
  65
  Solution Nuclear Magnetic Resonance Structure and Molecular Dynamics Simulations of a Murine 18.5 kDa Myelin Basic Protein Segment (S72-S107) in Association with Dodecylphosphocholine Micelles
  Ahmed, Mumdooh A. M.; De Avila, Miguel; Polverini, Eugenia; Bessonov, Kyrylo; Bamm, Vladimir V.; Harauz, George
  Biochemistry (2012), 51 (38), 7475-7487CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  The 18.5 kDa myelin basic protein (MBP), the most abundant splice isoform in adult mammalian myelin, is a multifunctional, intrinsically disordered protein involved in the development and compaction of the myelin sheath in the central nervous system. A highly conserved central segment comprises a membrane-anchoring amphipathic α-helix followed by a proline-rich segment that represents a ligand for SH3 domain-contg. proteins. Here, we have detd. using soln. NMR spectroscopy the structure of a 36-residue peptide fragment of MBP (murine 18.5 kDa residues S72-S107, denoted the α2-peptide) comprising these two structural motifs, in assocn. with dodecylphosphocholine (DPC) micelles. The structure was calcd. using CS-ROSETTA (version 1.01) because the nuclear Overhauser effect restraints were insufficient for this protein. The exptl. studies were complemented by mol. dynamics (MD) simulations of a corresponding 24-residue peptide fragment (murine 18.5 kDa residues E80-G103, denoted the MD-peptide), also in assocn. with a DPC micelle in silico. The exptl. and theor. results agreed well with one another, despite the independence of the starting structures and analyses, both showing membrane assocn. via the amphipathic α-helix, and a sharp bend in the vicinity of the Pro93 residue (murine 18.5 kDa sequence numbering). Overall, the conformations elucidated here show how the SH3 ligand is presented to the cytoplasm for interaction with SH3 domain-contg. proteins such as Fyn and contribute to our understanding of myelin architecture at the mol. level.
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66. 66
  Ishida, T.; Kinoshita, K. PrDOS: prediction of disordered protein regions from amino acid sequence. Nucleic Acids Res. 2007, 35, W460– W464, DOI: 10.1093/nar/gkm363
  There is no corresponding record for this reference.
67. 67
  Song, Y.; DiMaio, F.; Wang, R. Y.; Kim, D.; Miles, C.; Brunette, T.; Thompson, J.; Baker, D. High-resolution comparative modeling with RosettaCM. Structure 2013, 21, 1735– 1742, DOI: 10.1016/j.str.2013.08.005
  67
  High-Resolution Comparative Modeling with RosettaCM
  Song, Yifan; DiMaio, Frank; Wang, Ray Yu-Ruei; Kim, David; Miles, Chris; Brunette, T. J.; Thompson, James; Baker, David
  Structure (Oxford, United Kingdom) (2013), 21 (10), 1735-1742CODEN: STRUE6; ISSN:0969-2126. (Elsevier Ltd.)
  We describe an improved method for comparative modeling, RosettaCM, which optimizes a phys. realistic all-atom energy function over the conformational space defined by homologous structures. Given a set of sequence alignments, RosettaCM assembles topologies by recombining aligned segments in Cartesian space and building unaligned regions de novo in torsion space. The junctions between segments are regularized using a loop closure method combining fragment superposition with gradient-based minimization. The energies of the resulting models are optimized by all-atom refinement, and the most representative low-energy model is selected. The CASP10 expt. suggests that RosettaCM yields models with more accurate side-chain and backbone conformations than other methods when the sequence identity to the templates is greater than ∼15%.
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68. 68
  Berendsen, H. J. C.; Grigera, J. R.; Straatsma, T. P. The missing term in effective pair potentials. J. Phys. Chem. A 1987, 91, 6269– 6271, DOI: 10.1021/j100308a038
  68
  The missing term in effective pair potentials
  Berendsen, H. J. C.; Grigera, J. R.; Straatsma, T. P.
  Journal of Physical Chemistry (1987), 91 (24), 6269-71CODEN: JPCHAX; ISSN:0022-3654.
  Effective pair potentials used for simulations of polar liqs. include the av. effects of polarization. Such potentials are generally adjusted to produce the exptl. heat of vaporization. It has not been recognized before that the self-energy term inherent in any polarizable model should be included in effective pair potentials as well. Inclusion of the self-energy correction with a consequent reparametrization of the simple point charge model of water yields an improvement of the effective pair potential for water, as exemplified by d., radial distribution functions, and diffusion const.
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69. 69
  Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J. Chem. Theory Comput. 2015, 11, 3696– 3713, DOI: 10.1021/acs.jctc.5b00255
  69
  ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB
  Maier, James A.; Martinez, Carmenza; Kasavajhala, Koushik; Wickstrom, Lauren; Hauser, Kevin E.; Simmerling, Carlos
  Journal of Chemical Theory and Computation (2015), 11 (8), 3696-3713CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
  Mol. mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Av. errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a phys. motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reprodn. of NMR χ1 scalar coupling measurements for proteins in soln. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.
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70. 70
  Duff, M. R., Jr.; Borreguero, J. M.; Cuneo, M. J.; Ramanathan, A.; He, J.; Kamath, G.; Chennubhotla, S. C.; Meilleur, F.; Howell, E. E.; Herwig, K. W.; Myles, D. A. A.; Agarwal, P. K. Modulating Enzyme Activity by Altering Protein Dynamics with Solvent. Biochemistry 2018, 57, 4263– 4275, DOI: 10.1021/acs.biochem.8b00424
  70
  Modulating Enzyme Activity by Altering Protein Dynamics with Solvent
  Duff, Michael R.; Borreguero, Jose M.; Cuneo, Matthew J.; Ramanathan, Arvind; He, Junhong; Kamath, Ganesh; Chennubhotla, S. Chakra; Meilleur, Flora; Howell, Elizabeth E.; Herwig, Kenneth W.; Myles, Dean A. A.; Agarwal, Pratul K.
  Biochemistry (2018), 57 (29), 4263-4275CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  Optimal enzyme activity depends on a no. of factors, including structure and dynamics. The role of enzyme structure is well recognized, however, the linkage between protein dynamics and enzyme activity has given rise to a contentious debate. We have developed an approach that uses an aq. mixt. of org. solvent to control the functionally relevant enzyme dynamics (without changing the structure), which in turn modulates the enzyme activity. Using this approach, we predicted that the hydride transfer reaction catalyzed by the enzyme dihydrofolate reductase (DHFR) from Escherichia coli in aq. mixts. of isopropanol (IPA) with water will decrease by ∼3 fold at 20% (vol./vol.) IPA concn. Stop flow kinetic measurements find that the pH-independent khydride rate decreases by 2.2 fold. X-ray crystallog. enzyme structures shows no noticeable differences, while computational studies indicate that the transition-state and electrostatic effects were identical for water and mixed solvent conditions; and quasi-elastic neutron scattering studies show that the dynamical enzyme motions are suppressed. Our approach provides a unique avenue to modulating enzyme activity through changes in enzyme dynamics. Further it provides vital insights that show the altered motions of DHFR cause significant changes in the enzyme's ability to access its functionally relevant conformational sub-states, explaining the decreased khydride rate. This approach has important implications for obtaining fundamental insights into the role of rate-limiting dynamics in catalysis and as well as for enzyme engineering.
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71. 71
  Martínez, L.; Andrade, R.; Birgin, E. G.; Martinez, J. M. PACKMOL: a package for building initial configurations for molecular dynamics simulations. J. Comput. Chem. 2009, 30, 2157– 2164, DOI: 10.1002/jcc.21224
  71
  PACKMOL: A package for building initial configurations for molecular dynamics simulations
  Martinez, L.; Andrade, R.; Birgin, E. G.; Martinez, J. M.
  Journal of Computational Chemistry (2009), 30 (13), 2157-2164CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
  Adequate initial configurations for mol. dynamics simulations consist of arrangements of mols. distributed in space in such a way to approx. represent the system's overall structure. In order that the simulations are not disrupted by large van der Waals repulsive interactions, atoms from different mols. must keep safe pairwise distances. Obtaining such a mol. arrangement can be considered a packing problem: Each type mol. must satisfy spatial constraints related to the geometry of the system, and the distance between atoms of different mols. must be greater than some specified tolerance. We have developed a code able to pack millions of atoms, grouped in arbitrarily complex mols., inside a variety of three-dimensional regions. The regions may be intersections of spheres, ellipses, cylinders, planes, or boxes. The user must provide only the structure of one mol. of each type and the geometrical constraints that each type of mol. must satisfy. Building complex mixts., interfaces, solvating biomols. in water, other solvents, or mixts. of solvents, is straightforward. In addn., different atoms belonging to the same mol. may also be restricted to different spatial regions, in such a way that more ordered mol. arrangements can be built, as micelles, lipid double-layers, etc. The packing time for state-of-the-art mol. dynamics systems varies from a few seconds to a few minutes in a personal computer. The input files are simple and currently compatible with PDB, Tinker, Molden, or Moldy coordinate files. The package is distributed as free software and can be downloaded from . © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009.
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72. 72
  Agarwal, P. K. Cis/trans isomerization in HIV-1 capsid protein catalyzed by cyclophilin A: insights from computational and theoretical studies. Proteins 2004, 56, 449– 463, DOI: 10.1002/prot.20135
  72
  Cis/trans isomerization in HIV-1 capsid protein catalyzed by cyclophilin A: Insights from computational and theoretical studies
  Agarwal, Pratul K.
  Proteins: Structure, Function, and Bioinformatics (2004), 56 (3), 449-463CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
  A network of protein vibrations has recently been identified in the enzyme cyclophilin A (CypA) that is assocd. with its peptidylprolyl cis/trans isomerization activity of small peptide substrates. It has been suggested that this network may have a role in promoting the catalytic step during the isomerization reaction. This work presents the results from the characterization of this network during the isomerization of the Gly89-Pro90 peptide bond in the N-terminal domain of the capsid protein (CAN) from human immunodeficiency virus type 1 (HIV-1), which is a naturally occurring, biol. relevant protein substrate for CypA. A variety of computational and theor. studies are utilized to investigate the protein dynamics of the CypA-CAN complex, at multiple time scales, during the isomerization step. The results provide insights into the detailed mechanism of isomerization and confirm the presence of previously reported network of protein vibrations coupled to the reaction. Conserved CypA residues at the complex interface and at positions distal to the interface form parts of this network. There is HIV-1 related medical interest in CypA; incorporation of CypA, complexed with the capsid protein, into the virion is required for the infectious activity of HIV-1. Interaction energy and dynamical cross-correlation calcns. are used for a detailed investigation of the protein-protein interactions in the CypA-CAN complex. The results show that CAN residues His87-Ala-Gly-Pro-Ile-Ala92 form the majority of the interactions with CypA residues. New protein-protein interactions distal to the active site (CypA Arg148-CAN Gln95 and CypA Arg148-CAN Asn121) are also identified.
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73. 73
  Beck, D. A.; Daggett, V. Methods for molecular dynamics simulations of protein folding/unfolding in solution. Methods 2004, 34, 112– 120, DOI: 10.1016/j.ymeth.2004.03.008
  73
  Methods for molecular dynamics simulations of protein folding/unfolding in solution
  Beck, David A. C.; Daggett, Valerie
  Methods (San Diego, CA, United States) (2004), 34 (1), 112-120CODEN: MTHDE9; ISSN:1046-2023. (Elsevier)
  All atom mol. dynamics simulations have become a std. method for mapping equil. protein dynamics and nonequil. events like folding and unfolding. Here, we present detailed methods for performing such simulations. Generic protocols for minimization, solvation, simulation, and anal. derived from previous studies are also presented. As a measure of validation, our water model is compared with expt. An example of current applications of these methods, simulations of the ultrafast folding protein Engrailed Homeodomain are presented including the exptl. evidence used to verify their results. Ultrafast folders are an invaluable tool for studying protein behavior as folding and unfolding events measured by expt. occur on timescales accessible with the high-resoln. mol. dynamics methods we describe. Finally, to demonstrate the prospect of these methods for folding proteins, a temp. quench simulation of a thermal unfolding intermediate of the Engrailed Homeodomain is described.
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74. 74
  Roe, D. R.; Cheatham, T. E., 3rd PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. J. Chem. Theory Comput. 2013, 9, 3084– 3095, DOI: 10.1021/ct400341p
  74
  PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data
  Roe, Daniel R.; Cheatham, Thomas E.
  Journal of Chemical Theory and Computation (2013), 9 (7), 3084-3095CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
  We describe PTRAJ and its successor CPPTRAJ, two complementary, portable, and freely available computer programs for the anal. and processing of time series of three-dimensional at. positions (i.e., coordinate trajectories) and the data therein derived. Common tools include the ability to manipulate the data to convert among trajectory formats, process groups of trajectories generated with ensemble methods (e.g., replica exchange mol. dynamics), image with periodic boundary conditions, create av. structures, strip subsets of the system, and perform calcns. such as RMS fitting, measuring distances, B-factors, radii of gyration, radial distribution functions, and time correlations, among other actions and analyses. Both the PTRAJ and CPPTRAJ programs and source code are freely available under the GNU General Public License version 3 and are currently distributed within the AmberTools 12 suite of support programs that make up part of the Amber package of computer programs (see http://ambermd.org). This overview describes the general design, features, and history of these two programs, as well as algorithmic improvements and new features available in CPPTRAJ.
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75. 75
  Shukla, S.; Bafna, K.; Gullett, C.; Myles, D. A. A.; Agarwal, P. K.; Cuneo, M. J. Differential Substrate Recognition by Maltose Binding Proteins Influenced by Structure and Dynamics. Biochemistry 2018, 57, 5864– 5876, DOI: 10.1021/acs.biochem.8b00783
  75
  Differential Substrate Recognition by Maltose Binding Proteins Influenced by Structure and Dynamics
  Shukla, Shantanu; Bafna, Khushboo; Gullett, Caeley; Myles, Dean A. A.; Agarwal, Pratul K.; Cuneo, Matthew J.
  Biochemistry (2018), 57 (40), 5864-5876CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  The genome of the hyperthermophile Thermotoga maritima contains three isoforms of maltose binding protein (MBP) that are high-affinity receptors for di-, tri-, and tetrasaccharides. Two of these proteins (tmMBP1 and tmMBP2) share significant sequence identity, approx. 90%, while the third (tmMBP3) shares less than 40% identity. MBP from Escherichia coli (ecMBP) shares 35% sequence identity with the tmMBPs. This subset of MBP isoforms offers an interesting opportunity to investigate the mechanisms underlying the evolution of substrate specificity and affinity profiles in a genome where redundant MBP genes are present. In this study, the X-ray crystal structures of tmMBP1, tmMBP2, and tmMBP3 are reported in the absence and presence of oligosaccharides. tmMBP1 and tmMBP2 have binding pockets that are larger than that of tmMBP3, enabling them to bind to larger substrates, while tmMBP1 and tmMBP2 also undergo substrate-induced hinge bending motions (∼52°) that are larger than that of tmMBP3 (∼35°). Small-angle X-ray scattering was used to compare protein behavior in soln., and computer simulations provided insights into dynamics of these proteins. Comparing quant. protein-substrate interactions and dynamical properties of tmMBPs with those of the promiscuous ecMBP and disaccharide selective Thermococcus litoralis MBP provides insights into the features that enable selective binding. Collectively, the results provide insights into how the structure and dynamics of tmMBP homologues enable them to differentiate between a myriad of chem. entities while maintaining their common fold.
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Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c01590.
- Confirmation of the EAE model, variation of hydrocarbon chain, computational modeling of MBP, and five model structures of MBP and MBP sequence alignment (Figures S1–S5); mass spectrometry results for arginine residues and confirmation of MBP presence in gel-excised band (Table S1); and computer simulations of MBP (Movies S1–S3) (ZIP)
- ao0c01590_si_001.zip (22.62 MB)
Accession Codes
MBP (UniProt accession: P02686-5 and P02687). Mass spectrometry data (PRIDE archive accession: PXD015494).

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Myelin Basic Protein Phospholipid Complexation Likely Competes with Deimination in Experimental Autoimmune Encephalomyelitis Mouse ModelClick to copy article linkArticle link copied!

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Abstract

1. Introduction

2. Results and Discussion

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

2.1. Confirmation of the Experimental Autoimmune Encephalomyelitis (EAE) Mouse Model

2.2. Isolation of the MBP–Phospholipid Complex and Deimination in EAE

2.3. MBP–Phospholipid Complexation

2.4. LPC 18:1 Likely Competes for Hyper-deimination of MBP

2.5. Structural Analysis of LPC 18:1–MBP Complex

Figure 6

Figure 7

3. Conclusions

4. Experimental Section

4.1. Generation of Experimental Autoimmune Encephalomyelitis (EAE) Model

4.2. Electroretinogram Recordings

4.3. Optical Coherence Tomography (OCT)

4.4. Enzyme-Linked Immunosorbent Assay (ELISA)

4.5. Immunohistochemistry

4.6. Tissue Preparation and Density Ultracentrifugation

4.7. Gel Electrophoresis and Immunoblotting

4.8. Capillary Electrophoresis (CE)

4.9. Lipid and Protein Extraction

4.10. Sample Preparation for Protein Mass Spectrometry

4.11. High-Performance Liquid Chromatography (HPLC)–Mass Spectrometry

4.12. Liposome Extrusion and Liposome Flotation Assay

4.13. Protein–Lipid Overlay Assay

4.14. MBP–Lipid Deimination Assay

4.15. Circular Dichroism

4.16. Computational Modeling

4.17. Model Preparation

4.18. MD Simulations

4.19. Computational Modeling Analysis

4.20. Statistics

Supporting Information

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Acknowledgments

Abbreviations

References

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Abstract

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Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

References

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Supporting Information

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Myelin Basic Protein Phospholipid Complexation Likely Competes with Deimination in Experimental Autoimmune Encephalomyelitis Mouse Model
Click to copy article linkArticle link copied!