Plasmalogens Alter the Aggregation Rate of Transthyretin and Lower Toxicity of Transthyretin Fibrils

Heart tissue can experience a progressive accumulation of transthyretin (TTR), a small four subunit protein that transports holoretinol binding protein and thyroxine. This severe pathology is known as transthyretin amyloid cardiomyopathy. Numerous experimental studies indicated that the aggregation rate and toxicity of TTR fibrils could be altered by the presence of lipids; however, the role of plasmalogens in this process remains unknown. In this study, we investigate the effect of choline plasmalogens (CPs) with different lengths and saturations of fatty acids (FAs) on TTR aggregation. We found that CPs with saturated and unsaturated FAs strongly suppressed TTR aggregation. We also found that CPs with saturated FAs did not change the morphology of TTR fibrils; however, much thicker fibrillar species were formed in the presence of CPs with unsaturated FAs. Finally, we found that CPs with C16:0, C18:0, and C18:1 FAs substantially lowered the cytotoxicity of TTR fibrils that were formed in their presence.

AFM-IR: Protein samples were deposited onto a 70 nm gold-coated silicon wafer at a volume of 3-6 µL.The deposited sample was left to dry at room temperature for 15-20 minutes, then rinsed with DI water, and lastly dried with a N 2 air flow.AFM-IR imaging and spectral acquisition was acquired by using a nanoIR3 system (Bruker, Santa Barbara, CA, USA), the source of the IR laser is from a QCL laser.AFM imaging was collected through contact mode using AFM tips (ContGB-G AFM probe, NanoAndMore).The contact-mode tip was optimized using a polymethyl acrylate standard sample for the following wavenumbers: 1400-1800 cm -1 .Images were taken at a scan rate of 0.8 Hz with a height and width ranging from 1-10 µm, resolution of 256 for both X and Y parameters, and an I and P gain of 2 and 4 respectively.Laser parameters include a starting power of 25.49%, polarization at 90 deg., a pulse rate around 870 kHz, and an IR focus at 70136 pt.A total of 30 spectra were collected per sample with a co-average of 3 for each spectrum acquired.The spectra were zapped at the 1648-1652 range to remove the artifact caused by the chip-to-chip transition of the instrument.The spectra resolution is 2 cm -1 /pt.The spectra were processed by applying a smoothing of Savitzky-Golay at a polynomial order of 0, using MATLAB as the programming language application.Spectral analysis: Fitting of AFM-IR spectra was performed in GRAMS/AI™ Spectroscopy Software.After amide I region (1570-1800 cm -1 ) was baselined, automated peak identification was performed.Next, fitting was optimized to reach the best possible overlap of the fitted and experimental spectra.Finally, peak areas were determined.Parallel β-sheet was considered from 1616-1630 cm -1 ; α-helix and random coil from 1640-1670 cm -1 , and anti-parallel β-sheet from 1690-1700 cm -1 .All other peaks were discarded from the quantification of the secondary structures.[3] Cell toxicity assays: The N27 rat dopaminergic neuron cell line was purchased from Sigma-Aldrich (St. Louis, MO).Cells were cultured in 96-well plates with RPMI 1640 Medium supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO 2 .Once the cells reached ~70% confluency after 24 hours, they were used for subsequent experiments.For the LDH assay, 100 μL of the cell culture was replaced with 100 μL of RPMI 1640 Medium containing 5% FBS and 10 μL of the protein samples.After 24 hours of incubation, the amount of lactate dehydrogenase (LDH) released into the cell culture medium was measured using the non-radioactive CytoTox 96 cytotoxicity assay kit (G1781, Promega, Madison, WI, USA).The toxicity of the protein aggregates towards N27 cells was measured by the level of formazan produced, which directly correlated with the amount of LDH released using absorbance read at 490 nm.

FigureFigureFigure
Figure S1 Fitted AFM-IR spectra with the corresponding peak areas for TTR.Each averaged spectrum corresponds to 10 spectra acquired from different fibrils.

Figure S6 .
Figure S6.AFM image of TTR:C16:0 fibrils with marked points at which AFM-IR spectra were acquired (left) together with the intact AFM image of these aggregates (right).

Figure S7 .
Figure S7.AFM image of TTR:C18:0 fibrils with marked points at which AFM-IR spectra were acquired (left) together with the intact AFM image of these aggregates (right)

Figure S10 .Figure
Figure S10.AFM image of TTR fibrils with marked points at which AFM-IR spectra were acquired (left) together with the intact AFM image of these aggregates (right).