Core Richness of N-Glycans of Caenorhabditis elegans: A Case Study on Chemical and Enzymatic Release

Despite years of research, the glycome of the model nematode Caenorhabditis elegans is still not fully understood. Certainly, data over the years have indicated that this organism synthesizes unusual N-glycans with a range of galactose and fucose modifications on the Man2–3GlcNAc2 core region. Previously, up to four fucose residues were detected on its N-glycans, despite these lacking the fucosylated antennae typical of many other eukaryotes; some of these fucose residues are capped with hexose residues as shown by the studies of us and others. There have, though, been contrasting reports regarding the maximal number of fucose substitutions in C. elegans, which in part may be due to different methodological approaches, including use of either peptide:N-glycosidases F and A (PNGase F and A) or anhydrous hydrazine to cleave the N-glycans from glycopeptides. Here we compare the use of hydrazine with that of a new enzyme (rice PNGase Ar) and show that both enable release of glycans with more sugar residues on the proximal GlcNAc than previously resolved. By use of exoglycosidase sequencing, in conjunction with high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), we now reveal that actually up to five fucose residues modify the core region of C. elegans N-glycans and that the α1,3-fucose on the reducing terminus can be substituted by an α-linked galactose. Thus, traditional PNGase F and A release may be insufficient for release of the more highly core-modified N-glycans, especially those occurring in C. elegans, but novel enzymes can compete against chemical methods in terms of safety, ease of cleanup, and quality of resulting glycomic data.


Glycopeptide preparation:
Harvested worms were boiled in water for 10 minutes to heat-inactivate glycosidases prior to homogenisation. Nematode homogenates were transferred into glass flasks and treated either with pepsin (Sigma) in 5% formic acid overnight at 37 °C or with thermolysin (Promega) in 50 mM ammonium bicarbonate buffer (pH 8) supplemented with 0.5 mM CaCl 2 for 2 hours at 70 °C. Typically, 2 g (wet weight) of worms were proteolysed with 2 mg of protease. After proteolysis, the mixtures were centrifuged and the glycopeptides were purified by cation exchange chromatography (Dowex 50W×8; BioRad): in the case of a thermolysin digest, the proteolysate was acidified, while the pepsin digests required no pH adjustment prior to incubation with 10 ml of the pre-washed chromatography medium. After 1 hr, the resin and supernatant were poured into a polypropylene column and unbound material washed with 2% acetic acid; glycopeptides were eluted with 0.5 M ammonium acetate, pH 6. The orcinol-positive fractions were pooled, lyophilised and glycopeptides were desalted on a Sephadex G25 column (GE Healthcare; column volume of 80 ml) using 0.5% acetic acid as eluent; again orcinol-positive fractions were pooled, lyophilised and remaining protease activity heat-inactivated at 95 °C for 10 minutes.
N-glycan release with peptide:N-glycosidases: Enzymatic release of N-glycans from worm peptic glycopeptides was done using three different peptide:N-glycosidases: (i) recombinant bacterial PNGase F (from Flavobacterium [Elizabethkingia] meningosepticum, Roche; 3 U) under alkaline conditions overnight (50 mM ammonium bicarbonate, pH 8), (ii) native almond PNGase A under acidic conditions overnight (from Prunus amygdalus, Roche; in 50 mM ammonium acetate, pH 5, 0.25 mU) and (iii) recombinant rice PNGase Ar (from Oryza sativa expressed in Pichia pastoris and Endo H treated, New England Biolabs; also at pH 5 overnight, 15 U). In the first experiments, digestion was either with PNGase F followed by PNGase A then PNGase Ar or with PNGase A followed by PNGase Ar of peptic peptides. In another experiment, thermolysin was used for proteolysis followed by two rounds of PNGase Ar release (each overnight with 25 U) without the use of the other two PNGases. Chemical release of glycans from peptic glycopeptides by hydrazine was performed as described below. Thereby, all three enzymes (PNGase F, A and Ar) as well as hydrazine were tested with peptic peptides, but a newer proteolysis protocol with thermolysin was followed when using PNGase Ar alone. Between each enzymatic step, the released glycans were separated from the remaining glycopeptides Dowex 50W×8 (5 ml resin, with glycans in the 2% acetic acid 'filtrate' and glycopeptides in the 0.5 M ammonium acetate eluate). When the remaining glycopeptides were to be treated with another peptide:N-glycosidase, these were generally desalted by gel filtration on G25 as described above.

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The glycans were then further purified by solid-phase extraction using non-porous graphitised carbon (nPGC; 250 mg ENVI-Carb, Supelco). The glycans were dissolved in water, the nPGC material washed with water and the glycans eluted with 40 % acetonitrile; in the case of C. elegans, a subsequent step with 40% acetonitrile and 0.5% trifluoroacetic acid resulted in elution of no further glycans as judged by MALDI-TOF MS. Finally, the glycans were purified by solid-phase extraction on C18 material (100 mg LiChroprep® RP-18, Merck; elution with water) prior to pyridylamination as previously described. In case of glycans released by two rounds of PNGase Ar digestion, solely C18 purification was performed after Dowex chromatography prior to pyridylamination. In general, it can be noted that two rounds of any enzymatic release (regardless of the PNGase) are necessary to release all oligomannosidic and paucimannosidic structures; such 'pre-removal' of the majority of N-glycans then means that the final round of release by PNGase Ar results in isolation of the more unusual structures.

N-glycan release with hydrazine:
For chemical release, 10 mg peptic glycopeptides were transferred into a glass reaction tube and dried overnight prior to adding 500 µL of anhydrous hydrazine (prepared from monohydrate hydrazine; Sigma-Aldrich) and incubated at 100 °C for 5 h. Unreacted anhydrous hydrazine was removed by centrifugal evaporation. Samples were cooled to 0 °C and re-N-acetylated by the addition of 1 M sodium bicarbonate solution (450 μl) and acetic anhydride (21 μl) and incubated at 0 °C for 60 min. The samples were then acidified by addition of 5% (v/v) trifluoroacetic acid (600 μl) to the samples and incubated at 4°C for 60 min in order to liberate the reducing end of the glycans, followed by another round of Dowex 50W×8 chromatography. The glycans were then further purified by solid-phase extraction (as described above for enzymatic release) prior to pyridylamination. Note that hydrazine is a hazardous reagent and must only be used when applying relevant safety procedures. Hydrazinolysis was performed twice on two aliquots of the same glycopeptide preparation and both times yielded a similar set of glycans with evidence for only a minor degree of peeling of the reducing terminus.

Glycan labelling:
Pyridylamination was performed basically as described by Hase. In brief, 100 mg 2aminopyridine (Sigma-Aldrich) was dissolved in 76 µl concentrated HCl and 152 µl water; 80 µl of this solution was added to the dried glycan sample, prior to incubation in boiling water for 15 minutes. Then a solution of 4.4 mg of sodium cyanoborohydride (Sigma-Aldrich) in a mixture of 9 µl of the aforementioned 2-aminopyridine solution and 13 µl water was prepared; 4 µl of this cyanoborohydride-aminopyridine solution was added to the sample and the incubation was continued overnight at 90 °C prior to gel filtration (Sephadex G15; GE Healthcare, 1 × 50 cm). Fluorescence (excitation/emission 320/400 nm) of the fractions was measured using a Tecan microtitre plate reader.

Supplementary Figure 1. RP-HPLC chromatograms of N-glycans released by PNGases and
hydrazine. N-glycans were released from glycopeptides of the N2 wild type C. elegans (i) hydrazine (A), or (ii) PNGase F, PNGase A and PNGase Ar (B-D), (iii) PNGase A and PNGase Ar (E, F), (iv) two rounds of PNGase Ar (G, H) resulting in eight different glycan pools (see also Scheme) which were labelled with 2-aminopyridine and separated on RP-HPLC; the fluorescence intensities are normalised to the highest peak in each chromatogram. A partially-hydrolysed dextran standard was used to calibrate the column and the glucose units are indicated on the chromatograms (some minor day-to-day shifts occur). Dominant glycan structures, including Man 3,8,9 GlcNAc 2 (Man3, Man8B, Man9) are annotated and used as "landmarks" to align the chromatograms run on different days from different nematode cultures. For the two rounds of PNGase Ar release, it is estimated (based on fluorescence intensities) that the second digestion step released less than 5% of the glycan amount as compared to the first.

Supplementary Figure 3. MALDI-TOF MS comparison of RP-HPLC fractions in selected
regions. Different sets of glycans were released from wild type C. elegans using PNGase F, PNGase A post F, PNGase A alone, PNGase Ar alone (first and second rounds) or hydrazine and identified by MALDI-TOF mass spectrometry. Examples are given for three different RP-HPLC regions: (i) glycans substituted with two GalFuc motifs on the proximal core GlcNAc (Hex 6-7 HexNAc 2 Fuc 2-3 Me 0-1 ) eluted between 4.2 to 4.7 g.u., e.g., the m/z 1767 glycan, which was only observed in fractions from PNGase Ar or hydrazine but neither from PNGase F nor native PNGase A; (ii) tetra-fucosylated structures carrying three core GalFuc motifs eluted between 5.0 to 5.5 g.u., e.g., Hex 6-7 HexNAc 2 Fuc 4 Me 1 (m/z 2073 and 2235), which were only observed in fractions from PNGase Ar or hydrazine; (iii) between 6.2 to 6.7 g.u. eluted one of the predominant structures Hex 5 HexNAc 2 Fuc 2 (m/z 1605), which was only observed after treatment with PNGase A or Ar or hydrazine, but was most pronounced in the PNGase Ar alone pool (see Supplementary Figure 1); its sensitivity to β-galactosidase treatment (loss of two hexoses to m/z 1281) and the associated changes in the core Y-fragments (m/z 608/754 to m/z 446/592) indicated that the structure contains β1,4-galactose both on the α1,6fucose and the β1,4-mannose.  1911 (A, B), whereby the latter no longer presented key ions at m/z 770 and 1078, indicating removal of two β1,4-linked galactose residues from the core α1,6-fucose (compare D and E). Subsequent hydrofluoric acid treatment (C) resulted in major products at m/z 1295 (Hex 3 HexNAc 2 Fuc 2 Me 1 ) and 1457 (Hex 4 HexNAc 2 Fuc 2 Me) as well as intermediate ones of 1603 and 1765, which together with the shifts of key ions (from m/z 754 to 446) indicated the loss of both distal and proximal 3linked GalFuc units (F and G). (ii) A second 2D-HPLC fraction (≈21 min on HIAX) contained only one isomer of m/z 2235, whose sensitivity to A. oryzae β-galactosidase (loss of one hexose; H and I) was accompanied by shifts of diagnostic ions from m/z 916 to 754 and from m/z 608 to 446 (K and L). Hydrofluoric acid treatment alone resulted in a sequential loss of two GalFuc units of 308 Da as evidenced by major products at m/z 1927 and 1619 (J) and a shift of the diagnostic ions from m/z 916 to 608 (M and N). The m/z 2059 glycan in the 20 min fraction was converted by serial treatment to structures of m/z 1897 (loss of galactose) and then of 1589 or 1281 (loss of two GalFuc units) concomitantly with the conversion of diagnostic ions at m/z 916 to 754 and m/z 608 to 446 (A-C and O-R). Asterisks indicate nonglycan impurities; red and blue dashed lines indicate the digestion pathway for minor and major m/z 2235 isomers and black for the glycan of m/z 2059, while partial removal of methylfucose from the bisecting galactose is shown by the loss of 160 Da. (iii) The proposed isomeric structures as well as structures of products upon galactosidase or hydrofluoric acid treatments; for data on α-galactosidase digestion as a further structural proof see Figure 4.