The parasitic nematode Oesophagostomum dentatum synthesizes unusual glycosaminoglycan-like O-glycans

The parasitic nematode Oesophagostomum dentatum synthesizes unusual glycosaminoglycan-like O-glycans Abstract O-glycosylation is probably one of the most varied sets of post-translational modifications across all organisms, but amongst the most refractory to analyze. In animals, O-xylosylation of serine residues represents the first stage in the synthesis of glycosaminoglycans, whose repeat regions are generally analyzed as fragments resulting from enzymatic or chemical degradation, whereas their core regions can be isolated by β-elimination or endo-β-xylosidase digestion. In the present study, we show that hydrazinolysis can be employed for release of glycosaminoglycan-type oligosaccharides from nematodes prior to fluorescent labeling with 2-aminopyridine. While various [HexNAcHexA]nGal2Xyl oligosaccharides were isolated from the model organism Caenorhabditis elegans, more unusual glycosaminoglycan-type glycans were found to be present in the porcine parasite Oesophagostomum dentatum. In this case, as judged by MS/MS before and after hydrofluoric acid or β-galactosidase digestion, core sequences with extra galactose and phosphorylcholine residues were detected as [(±PC)HexNAcHexA]n(±PC)Galβ3-(±Galβ4)Galβ4Xyl. Thus, hydrazinolysis and fluorescent labeling can be combined to analyze unique forms of O-xylosylation, including new examples of zwitterionic glycan modifications. glycosaminoglycan-like O-glycans, hydrazinolysis, mass spectrometry, nematode Introduction Glycosaminoglycans (GAGs), based on repeats of N-acetylhexosamine and hexuronic acid residues, are a hallmark of the metazoa and play key roles in animal development. In mammals, they are primarily classified as chondroitin, dermatan, heparan, hyaluronan or keratan sulfates, depending on the exact sequence of the repeating units and the type (or lack thereof) of the covalent linkage to protein (Li et al. 2012; Prydz 2015). However, glycosaminoglycan chains are challenging to analyze due to their heterogeneous and anionic nature. Rarely are intact GAGs analyzed, exceptions being bikunin and decorin (Ly et al. 2011; Yu et al. 2017). Most commonly individual endoglycosidase-generated repeat units or, in the case of chondroitin and heparan, the GlcAβ3Galβ3Galβ4XylβOSer tetrasaccharide core are examined (Kon et al. 1991; Li et al. 2012); also chondroitinase-treated chains, trimmed down to a residual hexasaccharide but still attached to the core protein, have been analyzed (Noborn et al. 2018). Phosphorylation and sulphation of core xylose and galactose residues are known (Tone et al. 2008), but other modifications of this region are rare; however, Siaα2,3Galβ1,4Xyl sequences can be generated in mammalian cells if glycosaminoglycan biosynthesis is impaired (Bai et al. 1999; Wen et al. 2014). In nematodes, it has been shown that the chondroitin/heparan common tetrasaccharide core is conserved in the model nematode Caenorhabditis elegans, but also that the chondroitin chains are primarily not sulphated in this organism (Yamada et al. 1999; Guerardel et al. 2001; Dierker et al. 2016; Izumikawa et al. 2016). On the other hand, C. elegans has been valuable in elucidating the role of GAGs in animal development as each of the sqv (squashed vulva) mutants affects a different gene required for chondroitin sulfate biosynthesis, including formation of the tetrasaccharide core (Hwang et al. 2003). There is apparently little information regarding GAGs of nematodes other than C. elegans, even though many are parasitic species. In contrast, there is relatively rich information available on N-glycomes from this phylum, whereby we have recently reported novel N-glycan structures from C. elegans (Yan, Brecker et al. 2015; Yan, Jin et al. 2015; Yan et al. 2018) as well as from Oesophagostomum dentatum (Jimenez-Castells et al. 2017). As part of these studies, we employed hydrazinolysis (Jimenez-Castells et al. 2017; Yan et al. 2018). This method is primarily used for release of N-glycans, but in case of altered conditions can be applied to prepare mucin-type O-glycans (Fukuda et al. 1976; Patel et al. 1993). Unexpectedly, upon applying “N-glycan-specific” hydrazinolysis conditions on nematode glycopeptides, we also released a set of structures which, due to the fragmentation pattern, were concluded to be based on xylose as the reducing terminus. Thereby, we could analyze short glycosaminoglycan-type O-glycans from both C. elegans and O. dentatum by off-line LC–MS in combination with enzymatic and chemical treatments. While the core structure of the unsulphated GAG-like O-glycans in C. elegans was as expected, related oligosaccharides from O. dentatum were decorated with extra galactose and phosphorylcholine residues, which is indicative of species-specific aspects of nematode glycosylation. A selection of the resulting data is discussed in order to illustrate the difference in the GAG-like O-glycomes between C. elegans and O. dentatum. Results Hydrazinolysis as a method to release glycosaminoglycan chains As part of our ongoing studies on the N-glycosylation of the model nematode C. elegans, we performed hydrazinolysis under “N-glycan-specific” conditions at 100°C for 5 h (Yan et al. 2018), which is not recommended for mucin-type O-glycans due to “peeling” of the reducing terminus. After hydrazinolysis and pyridylamination, we employed an off-line HPLC-MS approach to analyze the released glycans; thereby, we were surprised that we identified some glycans in the early fractions with a fragmentation pattern indicative of a reducing-terminal pentose (m/z 229; i.e., 132 + PA-label) as opposed to N-acetylglucosamine (m/z 300; i.e., 203 + PA-label) occurring on 2-aminopyridine-labeled N-glycans. Thus we surmised that GAG-like O-glycans could also be released by hydrazinolysis at 100°C and that these could be easily separated from N-glycans by RP-HPLC. Indeed, MALDI-TOF MS and MS/MS of all fractions suggested that the former eluted between 2 and 5 min (<2.5 g.u.; Figure 1A) while the latter were isolated in the subsequent fractions (7–35 min; >2.5 g.u.). Like other chemical glycan-release methods, hydrazinolysis is known to result in peeling (loss of the reducing-terminal residue), but these artefactual products are indistinguishable from those of endogenous endoglycosidases. However, either are easily identified due to intense positive-mode MS/MS Y-ions at m/z 300 and 462 (Man0–1GlcNAc1-PA for N-glycans lacking one reducing-terminal GlcNAc) and 259, 421 and 597 (GlcA0-1Gal1-2-PA for GAG-like O-glycans lacking the xylose residue), which contrasts with the typical Y1-ion at m/z 229 (Xyl1-PA) indicative of an intact glycosaminoglycan reducing terminus. On this basis, we presume a minor degree of peeling, but the majority of the GAG-like structures is concluded to be intact (Supplementary Figure S1). Fig. 1. View largeDownload slide RP-HPLC and MALDI-TOF-MS analyses of GAG-like O-glycans from Caenorhabditis elegans. (A) After hydrazinolysis and pyridylamination, glycans from C. elegans were separated by an Agilent Hypersil ODS-C18 column; the zoom shows the region in which GAG-like O-glycans elute. (B–E) MS of fractions I-IV show increments of m/z 176 (HexA) and 203 (HexNAc) compatible with the basic sequence [HexNAcn,n−1GlcAβ3n]Galβ3Galβ4Xyl-PA; shown are negative (B) or positive MS (C–E). (F–J) Selected positive (F, H–J; [M + H]+) or negative (G; [M–H]−) MS/MS. Fragments in positive mode are annotated for m/z 229 (Y1-ion, Xyl1-PA), 391 (Y2-ion, Gal1Xyl1-PA), 553 (Y3-ion, Gal2Xyl1-PA), 729 (Y4-ion, GlcA1Gal2Xyl1-PA), 932 (Y5-ion, HexNAc1GlcA1Gal2Xyl1-PA) and subsequent Y-ions with serial addition of GlcA and HexNAc. The fragments in negative mode of the m/z 1106 glycan showed specific B/C and Y/Z-ions associated with the HexA residue. MS/MS and glycan annotations (□ HexNAc; HexA; GlcA; Gal; Xyl) are based on standard nomenclatures (Domon and Costello 1988; Varki et al. 2015). Calibration is in terms of glucose units (g.u.). Elution positions of major N-glycans between 6 and 24 min are annotated (Yan et al. 2018); for further and wider-range MS and MS/MS analyses of C. elegans GAG-liked glycans, refer to Supplementary Figures S1 and S2. Fig. 1. View largeDownload slide RP-HPLC and MALDI-TOF-MS analyses of GAG-like O-glycans from Caenorhabditis elegans. (A) After hydrazinolysis and pyridylamination, glycans from C. elegans were separated by an Agilent Hypersil ODS-C18 column; the zoom shows the region in which GAG-like O-glycans elute. (B–E) MS of fractions I-IV show increments of m/z 176 (HexA) and 203 (HexNAc) compatible with the basic sequence [HexNAcn,n−1GlcAβ3n]Galβ3Galβ4Xyl-PA; shown are negative (B) or positive MS (C–E). (F–J) Selected positive (F, H–J; [M + H]+) or negative (G; [M–H]−) MS/MS. Fragments in positive mode are annotated for m/z 229 (Y1-ion, Xyl1-PA), 391 (Y2-ion, Gal1Xyl1-PA), 553 (Y3-ion, Gal2Xyl1-PA), 729 (Y4-ion, GlcA1Gal2Xyl1-PA), 932 (Y5-ion, HexNAc1GlcA1Gal2Xyl1-PA) and subsequent Y-ions with serial addition of GlcA and HexNAc. The fragments in negative mode of the m/z 1106 glycan showed specific B/C and Y/Z-ions associated with the HexA residue. MS/MS and glycan annotations (□ HexNAc; HexA; GlcA; Gal; Xyl) are based on standard nomenclatures (Domon and Costello 1988; Varki et al. 2015). Calibration is in terms of glucose units (g.u.). Elution positions of major N-glycans between 6 and 24 min are annotated (Yan et al. 2018); for further and wider-range MS and MS/MS analyses of C. elegans GAG-liked glycans, refer to Supplementary Figures S1 and S2. Screening of glycosaminoglycan-like O-glycans from C. elegans The GAG-like O-glycans are based on a GlcAβ3Galβ3Galβ4Xyl tetrasaccharide core sequence common to all chondroitin and heparan sulfates and has been previously found in C. elegans (Yamada et al. 2002). A closer examination of the relevant MS and MS/MS spectra indeed revealed that [HexNAcn,n−1HexAn]GlcA1Gal2Xyl1-PA sequences (m/z 932–4721) were eluted within the first 5 min of the RP-HPLC run, with the larger oligosaccharides tending to elute earlier due to an increasing number of hexuronic acid residues (Figure 1A). All GAG-like O-glycans from C. elegans exhibited intense core-sequence Y-ions in positive MS/MS mode (Figure 1F and H–J) at m/z: 229 (Y1; Xyl-PA), 391 (Y2; Gal1Xyl-PA), 553 (Y3; Gal2Xyl-PA), 729 (Y4; GlcA1Gal2Xyl-PA), and subsequent serial addition of N-acetylhexosamine (HexNAc, +203 Da) and hexuronic acid (HexA, +176 Da); B ions were also observed in positive mode at m/z 380–1138 (B2–B6; HexNAc1-3GlcA1-3; Figure 1H–J). However, B-ions were relatively more intense in negative mode with the largest fragments being B16 for the m/z 3584 species (Figure 1G and Supplementary Figure S2). Considering the presence of hexuronic acid (primarily assumed to be glucuronic acid) in GAG-like chains, the relevant RP-HPLC fractions were also subject to MALDI-TOF MS and MS/MS in negative ion mode. As demonstrated for HexNAc1HexA2Gal2Xyl-PA (m/z 1108/1106), these spectra revealed specific BC and YZ-ions associated with its HexA residues including an intense C1-ion at m/z 193 (HexA+H2O, Figure 1F). The MS/MS data are thereby in accordance with previously published data demonstrating isolation from C. elegans of unsulphated chondroitin-like tetra- and pentamers following β-elimination (Guerardel et al. 2001), but here we actually observed up to 25 saccharide units. Screening of glycosaminoglycan-like O-glycans from O. dentatum We recently also employed hydrazinolysis to release N-glycans from O. dentatum (Jimenez-Castells et al. 2017), which is a common parasite of pigs with the severity of the symptoms depending on the parasite load. The workflow was similar as for C. elegans, but the glycans were separated on a fused core RP-amide column, as well as on ODS-C18 and AS11 columns (Supplementary Figure 3). Again, a range of glycans with m/z 229 Y1-fragments was detected in early-eluting fractions within the first 5 min (< 2.5 g.u.; Figure 2). However, the range of putative glycosaminoglycan-like O-glycans from O. dentatum contrasted with those from C. elegans as there were Δm/z values suggestive of additional hexose and/or phosphorylcholine residues. Indeed, MS/MS fragmentation of GAG-like O-glycans from O. dentatum confirmed that several structures were decorated by phosphorylcholine and/or an additional hexose, thus forming novel sequences of PC0–4HexNAcn/n−1GlcAnHex3Xyl1. Therefore, we also treated selected GAG-containing fractions with exoglycosidases and hydrofluoric acid to elucidate further aspects of their structure. Fig. 2. View largeDownload slide RP-HPLC and MALDI-TOF-MS analyses of GAG-like O-glycans from Oesophagostomum dentatum. (A and B) After hydrazinolysis and pyridylamination, glycans from male and female O. dentatum were separated on an RP-amide column; the zoom of the chromatograms from females (A) and males (B) focuses on the region in which GAG-like O-glycans elute. (C–H) Positive mode MS of male fractions I–VI is compatible with the basic sequence of [HexNAcn,n−1-GlcAβ3n]-Galβ3-Galβ4-Xyl-PA as well as unprecedented core decoration with phosphorylcholine (PC) and/or galactose. Proposed structures (all [M + H]+) were confirmed by MS/MS as well as by chemical or enzymatic digestions. Calibration is in terms of glucose units (g.u.). Elution positions of major N-glycans between 5 and 24 min are annotated (Jimenez-Castells et al. 2017). For the ODS-C18 RP-HPLC and HIAX NP-HPLC chromatograms of O. dentatum GAG-like O-glycans, refer to Supplementary Figure S3. Fig. 2. View largeDownload slide RP-HPLC and MALDI-TOF-MS analyses of GAG-like O-glycans from Oesophagostomum dentatum. (A and B) After hydrazinolysis and pyridylamination, glycans from male and female O. dentatum were separated on an RP-amide column; the zoom of the chromatograms from females (A) and males (B) focuses on the region in which GAG-like O-glycans elute. (C–H) Positive mode MS of male fractions I–VI is compatible with the basic sequence of [HexNAcn,n−1-GlcAβ3n]-Galβ3-Galβ4-Xyl-PA as well as unprecedented core decoration with phosphorylcholine (PC) and/or galactose. Proposed structures (all [M + H]+) were confirmed by MS/MS as well as by chemical or enzymatic digestions. Calibration is in terms of glucose units (g.u.). Elution positions of major N-glycans between 5 and 24 min are annotated (Jimenez-Castells et al. 2017). For the ODS-C18 RP-HPLC and HIAX NP-HPLC chromatograms of O. dentatum GAG-like O-glycans, refer to Supplementary Figure S3. Anionic and neutral glycosaminoglycan-like O-glycans from O. dentatum The basic core sequences were investigated in both O. dentatum male and female O-glycomes by positive and negative MS/MS (Figure 3 and Supplementary Figure S4) modes to confirm the presence of GAG-like O-glycans. Their fragmentation patterns were rather similar to those observed in C. elegans in terms of the Y/B-ion intensities and distributions. However, the maximum number of HexNAc1HexA1 repeat units detected for O. dentatum male and female (n = 3) was lower than for C. elegans (n = 10). Fig. 3. View largeDownload slide Positive and negative MS/MS of GAG-like O-glycans from Oesophagostomum dentatum. The positive and negative MS/MS of anionic GAG-like O-glycans from O. dentatum (A–I) show data on structures from m/z 715 to 1487 (HexNAc0–2GlcA0-3Gal2–3Xyl1-PA). Most of these structures have the basic GAG-like O-glycan core sequence and share some of the same Y-fragmentation features as described in C. elegans (Figure 1); however, a few possess a trigalactosylated core sequence (D) as indicated by a specific Y3-ion in positive mode at m/z 715 (Gal3Xyl1-PA). The positive MS/MS of phosphorylcholine-modified GAG-like O-glycans from O. dentatum (J–R) exhibit key PC-fragments at m/z 328 (PC1Hex1) and/or 369 (PC1HexNAc1) while the ion-loss of 59 Da is due to an inherent PC breakdown. The fragment signature of a PC and galactosylated core is an Y3-ion of PC1Hex3Xyl-PA at m/z 880. *Contaminant fragments derived from coeluting PC-containing glycans; for MS/MS of m/z 1056 and 1435 in HIAX fractions lacking the m/z 369 cofragment, refer to Supplementary Figure S4A and D. Fig. 3. View largeDownload slide Positive and negative MS/MS of GAG-like O-glycans from Oesophagostomum dentatum. The positive and negative MS/MS of anionic GAG-like O-glycans from O. dentatum (A–I) show data on structures from m/z 715 to 1487 (HexNAc0–2GlcA0-3Gal2–3Xyl1-PA). Most of these structures have the basic GAG-like O-glycan core sequence and share some of the same Y-fragmentation features as described in C. elegans (Figure 1); however, a few possess a trigalactosylated core sequence (D) as indicated by a specific Y3-ion in positive mode at m/z 715 (Gal3Xyl1-PA). The positive MS/MS of phosphorylcholine-modified GAG-like O-glycans from O. dentatum (J–R) exhibit key PC-fragments at m/z 328 (PC1Hex1) and/or 369 (PC1HexNAc1) while the ion-loss of 59 Da is due to an inherent PC breakdown. The fragment signature of a PC and galactosylated core is an Y3-ion of PC1Hex3Xyl-PA at m/z 880. *Contaminant fragments derived from coeluting PC-containing glycans; for MS/MS of m/z 1056 and 1435 in HIAX fractions lacking the m/z 369 cofragment, refer to Supplementary Figure S4A and D. The novel hexose-substituted core sequence was observed in both O. dentatum male and female O-glycomes and correlated with an intense Y3-ion at m/z 715 corresponding to Hex3Xyl-PA (Figure 3D). To investigate the nature and position of the additional hexose, we treated an example fraction with either β3-galactosidase, β3/4-galactosidase or jack bean α-mannosidase (Figure 4A–C); only the β3/4-galactosidase was able to remove one hexose from HexNAc1GlcA1Hex3Xyl1-PA (m/z 1094), which suggested that the hexose branch is a β4-linked galactose. Furthermore, the positive MS/MS of this structure (Figure 3D; m/z 1094) indicated that the branching galactose was linked to the first galactose of the core sequence as judged by the intense Y2 and Y3 ions at m/z 553 and 715 (Gal2-3Xyl1-PA), while the Y2 + Y2′ ion at m/z 391 (Gal1Xyl1-PA) is much less intense as it results from a double fragmentation event. Upon degalactosylation to HexNAc1GlcA1Gal2Xyl1-PA (m/z 932; Figure 4C), the Y2 ion at m/z 391 (Gal1Xyl1-PA) is of increased intensity as this time it results from a single fragmentation. The conclusion regarding the position of the extra hexose was further confirmed in PC-containing structures (see below). Fig. 4. View largeDownload slide Identification of new core sequences of GAG-like O-glycans from O. dentatum by enzymatic and chemical digestions (A–G). The HexNAc1GlcA1Hex3Xyl1-PA (m/z 1094, see Figure 3D) was analyzed by positive MS and MS/MS before (A) and after β3-galactosidase (B) or β3/4-galactosidase (C) digestions; only the β3/4-galactosidase was able to remove a single hexose, as also seen by the alteration in the MS/MS spectra (see insets), thus indicating that the third hexose is a branching β4-linked galactose. The PC1-2HexNAc1GlcA1Hex2Xyl1-PA glycans (m/z 1097 and 1262; see Figures 2E/G and 3 M/N) were analyzed by positive MS before (D, F) and after (E, G) hydrofluoric acid (HF) treatment which removed one or two phosphorylcholine residues to yield HexNAc1GlcA1Hex2Xyl1-PA at m/z 932; shifts in the MS/MS spectra (see insets) show the absence of the PC-containing B-ions (e.g. PC-HexNAc at m/z 369) and the dominance of the PA-containing Y-ions (e.g., Xyl-PA at m/z 229) after HF treatment. Some partial loss of methyl groups from the PC units during the release and work-up procedure resulted in the observation of ions at m/z 1248 in addition to the bona fide structure at m/z 1262 (F) Fig. 4. View largeDownload slide Identification of new core sequences of GAG-like O-glycans from O. dentatum by enzymatic and chemical digestions (A–G). The HexNAc1GlcA1Hex3Xyl1-PA (m/z 1094, see Figure 3D) was analyzed by positive MS and MS/MS before (A) and after β3-galactosidase (B) or β3/4-galactosidase (C) digestions; only the β3/4-galactosidase was able to remove a single hexose, as also seen by the alteration in the MS/MS spectra (see insets), thus indicating that the third hexose is a branching β4-linked galactose. The PC1-2HexNAc1GlcA1Hex2Xyl1-PA glycans (m/z 1097 and 1262; see Figures 2E/G and 3 M/N) were analyzed by positive MS before (D, F) and after (E, G) hydrofluoric acid (HF) treatment which removed one or two phosphorylcholine residues to yield HexNAc1GlcA1Hex2Xyl1-PA at m/z 932; shifts in the MS/MS spectra (see insets) show the absence of the PC-containing B-ions (e.g. PC-HexNAc at m/z 369) and the dominance of the PA-containing Y-ions (e.g., Xyl-PA at m/z 229) after HF treatment. Some partial loss of methyl groups from the PC units during the release and work-up procedure resulted in the observation of ions at m/z 1248 in addition to the bona fide structure at m/z 1262 (F) Zwitterionic glycosaminoglycan-like O-glycans from O. dentatum Modifications with phosphorylcholine are associated with a mass increment of 165 Da and occur, e.g. on N-glycans of nematodes, Lepidoptera, cestodes and a mollusc as well as on lipopolysaccharides of some bacteria and glycolipids of some invertebrates; this modification can be detected in positive MS mode and has a proven sensitivity to hydrofluoric acid treatment (Paschinger et al. 2012; Paschinger and Wilson 2015; Yan, Wilson et al. 2015; Eckmair et al. 2016). In contrast to the ODS-C18 HPLC column, the presence of phosphorylcholine reduces retention times on the RP-amide HPLC column, an effect even more pronounced for multi-PC containing structures occurring in the 2.5 min HPLC fractions of O. dentatum male and female glycomes (<2 g.u.; Figure 2A–C and Supplementary Figure S3). The MS/MS fragmentation patterns of PC-containing structures of GAG-like O-glycans from O. dentatum demonstrated a range of structures from PC1Hex2Xyl1-PA to PC3HexNAc3GlcA3Hex2Xyl1 (m/z 718–2185; Figure 3J–R and Supplementary Figure S4) and revealed intense PC-fragments at m/z 328 (PC1Hex1) and m/z 369 (PC1HexNAc1) which indicated that PC either substitutes a HexNAc or Gal. Also, the presence of PC on a galactosylated core sequence was deduced in the case of PC1GlcA1Gal3Xyl-PA and PC1HexA1–2HexNAc1–2GlcA1Gal3Xyl-PA (m/z 1056, 1435 and 1814; see Figure 3L and Supplementary Figure S4D and F) which showed Y3-ions at m/z 718 (PC1Gal2Xyl-PA) and 880 (PC1Gal3Xyl-PA). In the case of PC2HexNAc1GlcA1Hex2Xyl-PA (m/z 1262; Figure 3N), fragments were observed at m/z 872 (B3; PC2HexNAc1GlcA1Hex1), 391 (Y2; Hex1Xyl-PA) and 718 (Y3; PC1Hex2Xyl-PA), but not at m/z 556 (theoretically PC1Hex1Xyl1-PA). Thus, the MS/MS data demonstrate that one phosphorylcholine is linked to HexNAc, while the other one substitutes the second core galactose and so it is concluded that the structure is PCHexNAcGlcAβ3(PC)Galβ3Galβ4Xyl. The PC position is similar in the case of an additional galactose (Figure 3L and P) as judged by positive fragments at m/z 880 (Y3; PC1Gal3Xyl-PA) and 718 (Y3 + Y2′; PC1Gal2Xyl-PA), which excludes the possibility that the branching galactose is substituted with PC. Furthermore, the PC1-2HexNAc1GlcA1Gal2Xyl1-PA structures (m/z 1097–1262; Figure 4D and F) were treated with HF which specifically removed all phosphodiester substitutions to yield HexNAc1GlcA1Gal2Xyl1-PA (m/z 932; Figure 4E and G), thus confirming the presence of the PC residues. The products of HF treatment also displayed an MS/MS pattern dominated by Y-fragments, rather than by the comparatively very easily ionizing PC-containing B-fragments present in the case of the original glycans (compare insets in Figure 4D and E). Discussion In this study, we found that short GAG-like O-glycans were efficiently coreleased with N-glycans by hydrazinolysis, which enabled their subsequent labeling by pyridylamination (PA) and analysis by an off-line LC–MS strategy. In other studies, the core regions of chondroitin and heparan chains have been analyzed after release by either β-elimination which excludes the possibility of subsequent labeling by reductive amination. Alternative strategies have been to use either an endo-β-xylosidase (apparently not commercially available) or lithium hydroxide, both of which enable subsequent labeling (Kon et al. 1991; Yamada et al. 2002). Previously, hydrazinolysis of proteoglycans was apparently only used to de-N-acetylate the HexNAc residues prior to nitrous acid deamination which is a method for isolation of modified forms of the individual disaccharide repeats (Shaklee and Conrad 1986). Thus, we can demonstrate a new use for hydrazine in the study of glycosaminoglycan-like structures. The GAG-like O-glycans isolated from C. elegans and from O. dentatum were separated from N-glycans by RP-HPLC which enabled subsequent MALDI-TOF MS and MS/MS as well as chemical or enzymatic treatments. Thereby, it is concluded that hydrazinolysis at high temperature (“N-glycan-specific” conditions) is able to release simultaneously GAG-like O-glycans and N-glycans, but destroys most of the mucin-type O-glycans. Although there is a degree of artefactual “peeling” of the natural reducing terminal monosaccharide (i.e. loss of xylose) or demethylation of phosphorylcholine (Δm/z 14), this was probably no more than 20% in our hands when performing hydrazinolysis on these samples, but an exact figure is difficult to estimate on the basis of fluorescence and ionization intensities of HPLC fractions containing multiple components. In terms of separation, a trend to earlier RP-HPLC elution of the longer oligosaccharides was observed, which is in keeping with the increasing number of hexuronic acid residues. The results for both organisms were surprisingly different with larger and unmodified core sequences being present in C. elegans, while male and female O. dentatum expressed GAG-like structures with new core sequences with an additional galactose and/or phosphorylcholine. Furthermore, no significant difference was observed between male and female in O. dentatum samples in terms of quality and quantity of GAG-like glycans. Based on previous studies, including data on the single pentasaccharide containing xylose at the reducing terminus identified by NMR after β-elimination (Guerardel et al. 2001), we presume that we have isolated unsulphated chondroitin chains based on GalNAcβ4GlcAβ3Galβ3Galβ4Xyl. On the other hand, the strategy of using hydrazinolysis followed by fluorescent labeling allowed us to isolate and identify more and larger chondroitin-like O-glycans than previously found in nematodes (up to almost 5 kDa in C. elegans; Figure 1), but structures larger than 25 saccharide units were not observed. Furthermore, chondroitin in its sulphated form is rare in C. elegans and possibly represents less than 1% of total GAG in this organism (Izumikawa et al. 2016); thus, it would be a challenge to detect such chains. Several glycosyltransferases genes (sqv-1, -2, -3 and -8) are required to synthesize the core sequence of GAG-like O-glycans in C. elegans, all of which are essential for its development (Hwang et al. 2003). However, the biosynthetic origin of the additional β4-linked galactose and phosphorylcholine residues on the O. dentatum [PCGalNAcβ4GlcAβ3]nPCGalβ3(Galβ4)Galβ4Xyl structures is unknown. On the other hand, similar to its N-glycans (Jimenez-Castells et al. 2017), the degree of phosphorylcholinylation on O. dentatum glycosaminoglycan-like sequences is relatively high as compared to the total of the identified structures, but also represents the first zwitterionic modification to be detected on this class of glycan. Thus, our study is another indication of the high variability of glycoconjugate structures in invertebrates, which can only be uncovered by the use of an extended methodological portfolio and in-depth analysis of the glycomic data. Materials and methods Nematode culture and isolation Caenorhabditis elegans (wild‐type N2 strain) was cultivated in our laboratory using standard conditions with Escherichia coli strain OP50 (i.e. maintained on nematode growth medium agar plates, grown in larger scale in liquid S complete medium and isolated by sucrose density centrifugation prior to freezing at −80°C). Oesophagostomum dentatum adults (OD-Hann strain) were recovered from the large intestines of infected pigs by agar-gel migration and visually separated into males and females prior to washing in 0.9% NaCl at 38°C, sedimentation, snap freezing and storage at −80°C until use (Slotved et al. 1996). Hydrazinolysis of nematode glycopeptides Worms were homogenized and subject to proteolysis with pepsin (overnight at 37°C; for C. elegans) or thermolysin (2 h at 70°C; for O. dentatum) prior to hydrazinolysis and pyridylamination; based on previous procedures (Patel et al. 1993; Jimenez-Castells et al. 2017). Briefly, 1 mg protease per gram of wet weight material was employed to prepare glycopeptides, which were purified by cation exchange (50 W × 8 Dowex, BioRad; elution with 0.5 M ammonium acetate, pH 6) and gel filtration (G25, GE Healthcare; elution with 0.5% acetic acid) prior to transfer into a glass reaction tube and drying overnight. Hydrazinolysis was then performed in 500 μL of anhydrous hydrazine (prepared from hydrazine monohydrate, Sigma) at 100°C for 5 h and removal of unreacted reagent by centrifugal evaporation. Glycans were re-N-acetylated using 450 μL of 100 mM sodium bicarbonate and 21 μL of acetic anhydride at 0°C for 1 h. Then, the reducing ends were liberated using 600 μL of 5% trifluoroacetic acid at 4°C for 1 h. Thereafter, the free glycans were purified by cation exchange (50 W × 8 Dowex; eluted with 2% acetic acid), nonporous graphitized carbon (eluted with 40% acetonitrile containing 0.1% TFA) and C18 columns (eluted with water). Finally, glycans were fluorescently labeled using 2-aminopyridine (Hase et al. 1984). For a fuller description of these methods, refer to the Supplement. HPLC purification of O-glycans Oligosaccharides with xylose at the reducing terminus were separated from N-glycans by RP-HPLC using a Shimadzu Nexera HPLC system equipped with a fluorescence detector (RF 20 AXS) and reversed phase columns (either Ascentis Express RP-Amide, Sigma-Aldrich, or Hypersil C18, Agilent). Glycans were eluted using a buffer system of 100 mM ammonium acetate (pH 4.0) and 30% (v/v) methanol at a flow rate of either 0.8 or 1.5 mL/min (Hykollari et al. 2017). Glycans were detected by fluorescence with excitation/emission wavelengths of 320/400 nm. RP-HPLC columns were calibrated daily in terms of glucose units using a pyridylaminated dextran hydrolysate; the order of elution of the standards was confirmed by MALDI-TOF MS of collected calibrant fractions. In the case of O. dentatum, the glycans eluting early from the RP-amide column were repooled and applied to an AS11 HIAX/NP-HPLC column using a set of oligomannosidic glycans for calibration (see Supplement). MALDI-TOF MS analysis Glycans in individual RP-HPLC fractions were analyzed by MALDI-TOF MS (Autoflex Speed, Bruker Daltonics, Germany) in positive and negative ion mode using FlexControl 3.4 software and 6-aza-2-thiothymine as matrix; MS/MS to confirm the composition of all proposed structures was performed by laser-induced dissociation (precursor ion selector was generally set to ±0.6%). The detector voltage was typically 1977 V for MS and 2133 V for MS/MS; 1000–3000 shots from different regions of the sample spots were summed. Spectra were processed with the manufacturer’s software (Bruker Flexanalysis 3.3.80) using the SNAP algorithm with a signal/noise threshold of 6 for MS (unsmoothed) and 3 for MS/MS (four times smoothed). All MS and MS/MS spectra were manually interpreted on the basis of the mass fragmentation pattern and results of chemical and enzymatic treatments; O-xylose-based glycans were distinguished from N-glycans by the respective presence in positive MS/MS of Y1-ions at m/z 229 (Xyl1-PA) or 300 (HexNAc1-PA). Structural elucidation using exoglycosidases and chemical treatment Exoglycosidase treatment was performed using either recombinant β3/4-galactosidase from Aspergillus niger prepared in house (Dragosits et al. 2014) or recombinant β3-galactosidase from Xanthomonas manihotis from New England Biolabs (Wong-Madden and Landry 1995). In general, a 1–2 μL aliquot of a lyophilysed and redissolved HPLC fraction into 10 μL was incubated with 0.2 μL exoglycosidase and 0.5 μL 100 mM ammonium acetate solution, pH 5.0, overnight at 37°C. For removal of phosphorylcholine, aliquots of O-glycan fractions were dried in a Speed-Vac and then incubated with 3 μL of 48% (w/v) hydrofluoric acid (HF) on ice for 24 h prior to drying again (Dennis et al. 1998). Chemically or enzymatically treated glycans were reanalyzed by MALDI-TOF MS and MS/MS without further purification. Supplementary data Supplementary data is available at GLYCOBIOLOGY online. Funding This work was supported by the Austrian Science Fund (FWF) and by the European Union; S.Y. and K.P. are FWF fellows (Grants P30021 and P25058); J.V. was an experienced researcher within the Glycopar EU FP7 Marie Curie Initial Training Network (PITN-GA-2013-608295). Acknowledgements We also thank the staff of Ludger Ltd for an introduction to hydrazinolysis, especially Radoslaw Kozak and Daniel Spencer, as well as Markus Blaukopf for further help with hydrazinolysis and Bärbel Ruttkowski for nematode collection. 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Comparison of RP-HPLC modes to analyse the N-glycome of the free-living nematode Pristionchus pacificus . Electrophoresis . 36 ( 11–12 ): 1314 – 1329 . Google Scholar CrossRef Search ADS PubMed Yu Y , Duan J , Leach FE 3rd , Toida T , Higashi K , Zhang H , Zhang F , Amster IJ , Linhardt RJ . 2017 . Sequencing the dermatan sulfate chain of decorin . J Am Chem Soc . 139 ( 46 ): 16986 – 16995 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Glycobiology Oxford University Press

The parasitic nematode Oesophagostomum dentatum synthesizes unusual glycosaminoglycan-like O-glycans

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Abstract

Abstract O-glycosylation is probably one of the most varied sets of post-translational modifications across all organisms, but amongst the most refractory to analyze. In animals, O-xylosylation of serine residues represents the first stage in the synthesis of glycosaminoglycans, whose repeat regions are generally analyzed as fragments resulting from enzymatic or chemical degradation, whereas their core regions can be isolated by β-elimination or endo-β-xylosidase digestion. In the present study, we show that hydrazinolysis can be employed for release of glycosaminoglycan-type oligosaccharides from nematodes prior to fluorescent labeling with 2-aminopyridine. While various [HexNAcHexA]nGal2Xyl oligosaccharides were isolated from the model organism Caenorhabditis elegans, more unusual glycosaminoglycan-type glycans were found to be present in the porcine parasite Oesophagostomum dentatum. In this case, as judged by MS/MS before and after hydrofluoric acid or β-galactosidase digestion, core sequences with extra galactose and phosphorylcholine residues were detected as [(±PC)HexNAcHexA]n(±PC)Galβ3-(±Galβ4)Galβ4Xyl. Thus, hydrazinolysis and fluorescent labeling can be combined to analyze unique forms of O-xylosylation, including new examples of zwitterionic glycan modifications. glycosaminoglycan-like O-glycans, hydrazinolysis, mass spectrometry, nematode Introduction Glycosaminoglycans (GAGs), based on repeats of N-acetylhexosamine and hexuronic acid residues, are a hallmark of the metazoa and play key roles in animal development. In mammals, they are primarily classified as chondroitin, dermatan, heparan, hyaluronan or keratan sulfates, depending on the exact sequence of the repeating units and the type (or lack thereof) of the covalent linkage to protein (Li et al. 2012; Prydz 2015). However, glycosaminoglycan chains are challenging to analyze due to their heterogeneous and anionic nature. Rarely are intact GAGs analyzed, exceptions being bikunin and decorin (Ly et al. 2011; Yu et al. 2017). Most commonly individual endoglycosidase-generated repeat units or, in the case of chondroitin and heparan, the GlcAβ3Galβ3Galβ4XylβOSer tetrasaccharide core are examined (Kon et al. 1991; Li et al. 2012); also chondroitinase-treated chains, trimmed down to a residual hexasaccharide but still attached to the core protein, have been analyzed (Noborn et al. 2018). Phosphorylation and sulphation of core xylose and galactose residues are known (Tone et al. 2008), but other modifications of this region are rare; however, Siaα2,3Galβ1,4Xyl sequences can be generated in mammalian cells if glycosaminoglycan biosynthesis is impaired (Bai et al. 1999; Wen et al. 2014). In nematodes, it has been shown that the chondroitin/heparan common tetrasaccharide core is conserved in the model nematode Caenorhabditis elegans, but also that the chondroitin chains are primarily not sulphated in this organism (Yamada et al. 1999; Guerardel et al. 2001; Dierker et al. 2016; Izumikawa et al. 2016). On the other hand, C. elegans has been valuable in elucidating the role of GAGs in animal development as each of the sqv (squashed vulva) mutants affects a different gene required for chondroitin sulfate biosynthesis, including formation of the tetrasaccharide core (Hwang et al. 2003). There is apparently little information regarding GAGs of nematodes other than C. elegans, even though many are parasitic species. In contrast, there is relatively rich information available on N-glycomes from this phylum, whereby we have recently reported novel N-glycan structures from C. elegans (Yan, Brecker et al. 2015; Yan, Jin et al. 2015; Yan et al. 2018) as well as from Oesophagostomum dentatum (Jimenez-Castells et al. 2017). As part of these studies, we employed hydrazinolysis (Jimenez-Castells et al. 2017; Yan et al. 2018). This method is primarily used for release of N-glycans, but in case of altered conditions can be applied to prepare mucin-type O-glycans (Fukuda et al. 1976; Patel et al. 1993). Unexpectedly, upon applying “N-glycan-specific” hydrazinolysis conditions on nematode glycopeptides, we also released a set of structures which, due to the fragmentation pattern, were concluded to be based on xylose as the reducing terminus. Thereby, we could analyze short glycosaminoglycan-type O-glycans from both C. elegans and O. dentatum by off-line LC–MS in combination with enzymatic and chemical treatments. While the core structure of the unsulphated GAG-like O-glycans in C. elegans was as expected, related oligosaccharides from O. dentatum were decorated with extra galactose and phosphorylcholine residues, which is indicative of species-specific aspects of nematode glycosylation. A selection of the resulting data is discussed in order to illustrate the difference in the GAG-like O-glycomes between C. elegans and O. dentatum. Results Hydrazinolysis as a method to release glycosaminoglycan chains As part of our ongoing studies on the N-glycosylation of the model nematode C. elegans, we performed hydrazinolysis under “N-glycan-specific” conditions at 100°C for 5 h (Yan et al. 2018), which is not recommended for mucin-type O-glycans due to “peeling” of the reducing terminus. After hydrazinolysis and pyridylamination, we employed an off-line HPLC-MS approach to analyze the released glycans; thereby, we were surprised that we identified some glycans in the early fractions with a fragmentation pattern indicative of a reducing-terminal pentose (m/z 229; i.e., 132 + PA-label) as opposed to N-acetylglucosamine (m/z 300; i.e., 203 + PA-label) occurring on 2-aminopyridine-labeled N-glycans. Thus we surmised that GAG-like O-glycans could also be released by hydrazinolysis at 100°C and that these could be easily separated from N-glycans by RP-HPLC. Indeed, MALDI-TOF MS and MS/MS of all fractions suggested that the former eluted between 2 and 5 min (<2.5 g.u.; Figure 1A) while the latter were isolated in the subsequent fractions (7–35 min; >2.5 g.u.). Like other chemical glycan-release methods, hydrazinolysis is known to result in peeling (loss of the reducing-terminal residue), but these artefactual products are indistinguishable from those of endogenous endoglycosidases. However, either are easily identified due to intense positive-mode MS/MS Y-ions at m/z 300 and 462 (Man0–1GlcNAc1-PA for N-glycans lacking one reducing-terminal GlcNAc) and 259, 421 and 597 (GlcA0-1Gal1-2-PA for GAG-like O-glycans lacking the xylose residue), which contrasts with the typical Y1-ion at m/z 229 (Xyl1-PA) indicative of an intact glycosaminoglycan reducing terminus. On this basis, we presume a minor degree of peeling, but the majority of the GAG-like structures is concluded to be intact (Supplementary Figure S1). Fig. 1. View largeDownload slide RP-HPLC and MALDI-TOF-MS analyses of GAG-like O-glycans from Caenorhabditis elegans. (A) After hydrazinolysis and pyridylamination, glycans from C. elegans were separated by an Agilent Hypersil ODS-C18 column; the zoom shows the region in which GAG-like O-glycans elute. (B–E) MS of fractions I-IV show increments of m/z 176 (HexA) and 203 (HexNAc) compatible with the basic sequence [HexNAcn,n−1GlcAβ3n]Galβ3Galβ4Xyl-PA; shown are negative (B) or positive MS (C–E). (F–J) Selected positive (F, H–J; [M + H]+) or negative (G; [M–H]−) MS/MS. Fragments in positive mode are annotated for m/z 229 (Y1-ion, Xyl1-PA), 391 (Y2-ion, Gal1Xyl1-PA), 553 (Y3-ion, Gal2Xyl1-PA), 729 (Y4-ion, GlcA1Gal2Xyl1-PA), 932 (Y5-ion, HexNAc1GlcA1Gal2Xyl1-PA) and subsequent Y-ions with serial addition of GlcA and HexNAc. The fragments in negative mode of the m/z 1106 glycan showed specific B/C and Y/Z-ions associated with the HexA residue. MS/MS and glycan annotations (□ HexNAc; HexA; GlcA; Gal; Xyl) are based on standard nomenclatures (Domon and Costello 1988; Varki et al. 2015). Calibration is in terms of glucose units (g.u.). Elution positions of major N-glycans between 6 and 24 min are annotated (Yan et al. 2018); for further and wider-range MS and MS/MS analyses of C. elegans GAG-liked glycans, refer to Supplementary Figures S1 and S2. Fig. 1. View largeDownload slide RP-HPLC and MALDI-TOF-MS analyses of GAG-like O-glycans from Caenorhabditis elegans. (A) After hydrazinolysis and pyridylamination, glycans from C. elegans were separated by an Agilent Hypersil ODS-C18 column; the zoom shows the region in which GAG-like O-glycans elute. (B–E) MS of fractions I-IV show increments of m/z 176 (HexA) and 203 (HexNAc) compatible with the basic sequence [HexNAcn,n−1GlcAβ3n]Galβ3Galβ4Xyl-PA; shown are negative (B) or positive MS (C–E). (F–J) Selected positive (F, H–J; [M + H]+) or negative (G; [M–H]−) MS/MS. Fragments in positive mode are annotated for m/z 229 (Y1-ion, Xyl1-PA), 391 (Y2-ion, Gal1Xyl1-PA), 553 (Y3-ion, Gal2Xyl1-PA), 729 (Y4-ion, GlcA1Gal2Xyl1-PA), 932 (Y5-ion, HexNAc1GlcA1Gal2Xyl1-PA) and subsequent Y-ions with serial addition of GlcA and HexNAc. The fragments in negative mode of the m/z 1106 glycan showed specific B/C and Y/Z-ions associated with the HexA residue. MS/MS and glycan annotations (□ HexNAc; HexA; GlcA; Gal; Xyl) are based on standard nomenclatures (Domon and Costello 1988; Varki et al. 2015). Calibration is in terms of glucose units (g.u.). Elution positions of major N-glycans between 6 and 24 min are annotated (Yan et al. 2018); for further and wider-range MS and MS/MS analyses of C. elegans GAG-liked glycans, refer to Supplementary Figures S1 and S2. Screening of glycosaminoglycan-like O-glycans from C. elegans The GAG-like O-glycans are based on a GlcAβ3Galβ3Galβ4Xyl tetrasaccharide core sequence common to all chondroitin and heparan sulfates and has been previously found in C. elegans (Yamada et al. 2002). A closer examination of the relevant MS and MS/MS spectra indeed revealed that [HexNAcn,n−1HexAn]GlcA1Gal2Xyl1-PA sequences (m/z 932–4721) were eluted within the first 5 min of the RP-HPLC run, with the larger oligosaccharides tending to elute earlier due to an increasing number of hexuronic acid residues (Figure 1A). All GAG-like O-glycans from C. elegans exhibited intense core-sequence Y-ions in positive MS/MS mode (Figure 1F and H–J) at m/z: 229 (Y1; Xyl-PA), 391 (Y2; Gal1Xyl-PA), 553 (Y3; Gal2Xyl-PA), 729 (Y4; GlcA1Gal2Xyl-PA), and subsequent serial addition of N-acetylhexosamine (HexNAc, +203 Da) and hexuronic acid (HexA, +176 Da); B ions were also observed in positive mode at m/z 380–1138 (B2–B6; HexNAc1-3GlcA1-3; Figure 1H–J). However, B-ions were relatively more intense in negative mode with the largest fragments being B16 for the m/z 3584 species (Figure 1G and Supplementary Figure S2). Considering the presence of hexuronic acid (primarily assumed to be glucuronic acid) in GAG-like chains, the relevant RP-HPLC fractions were also subject to MALDI-TOF MS and MS/MS in negative ion mode. As demonstrated for HexNAc1HexA2Gal2Xyl-PA (m/z 1108/1106), these spectra revealed specific BC and YZ-ions associated with its HexA residues including an intense C1-ion at m/z 193 (HexA+H2O, Figure 1F). The MS/MS data are thereby in accordance with previously published data demonstrating isolation from C. elegans of unsulphated chondroitin-like tetra- and pentamers following β-elimination (Guerardel et al. 2001), but here we actually observed up to 25 saccharide units. Screening of glycosaminoglycan-like O-glycans from O. dentatum We recently also employed hydrazinolysis to release N-glycans from O. dentatum (Jimenez-Castells et al. 2017), which is a common parasite of pigs with the severity of the symptoms depending on the parasite load. The workflow was similar as for C. elegans, but the glycans were separated on a fused core RP-amide column, as well as on ODS-C18 and AS11 columns (Supplementary Figure 3). Again, a range of glycans with m/z 229 Y1-fragments was detected in early-eluting fractions within the first 5 min (< 2.5 g.u.; Figure 2). However, the range of putative glycosaminoglycan-like O-glycans from O. dentatum contrasted with those from C. elegans as there were Δm/z values suggestive of additional hexose and/or phosphorylcholine residues. Indeed, MS/MS fragmentation of GAG-like O-glycans from O. dentatum confirmed that several structures were decorated by phosphorylcholine and/or an additional hexose, thus forming novel sequences of PC0–4HexNAcn/n−1GlcAnHex3Xyl1. Therefore, we also treated selected GAG-containing fractions with exoglycosidases and hydrofluoric acid to elucidate further aspects of their structure. Fig. 2. View largeDownload slide RP-HPLC and MALDI-TOF-MS analyses of GAG-like O-glycans from Oesophagostomum dentatum. (A and B) After hydrazinolysis and pyridylamination, glycans from male and female O. dentatum were separated on an RP-amide column; the zoom of the chromatograms from females (A) and males (B) focuses on the region in which GAG-like O-glycans elute. (C–H) Positive mode MS of male fractions I–VI is compatible with the basic sequence of [HexNAcn,n−1-GlcAβ3n]-Galβ3-Galβ4-Xyl-PA as well as unprecedented core decoration with phosphorylcholine (PC) and/or galactose. Proposed structures (all [M + H]+) were confirmed by MS/MS as well as by chemical or enzymatic digestions. Calibration is in terms of glucose units (g.u.). Elution positions of major N-glycans between 5 and 24 min are annotated (Jimenez-Castells et al. 2017). For the ODS-C18 RP-HPLC and HIAX NP-HPLC chromatograms of O. dentatum GAG-like O-glycans, refer to Supplementary Figure S3. Fig. 2. View largeDownload slide RP-HPLC and MALDI-TOF-MS analyses of GAG-like O-glycans from Oesophagostomum dentatum. (A and B) After hydrazinolysis and pyridylamination, glycans from male and female O. dentatum were separated on an RP-amide column; the zoom of the chromatograms from females (A) and males (B) focuses on the region in which GAG-like O-glycans elute. (C–H) Positive mode MS of male fractions I–VI is compatible with the basic sequence of [HexNAcn,n−1-GlcAβ3n]-Galβ3-Galβ4-Xyl-PA as well as unprecedented core decoration with phosphorylcholine (PC) and/or galactose. Proposed structures (all [M + H]+) were confirmed by MS/MS as well as by chemical or enzymatic digestions. Calibration is in terms of glucose units (g.u.). Elution positions of major N-glycans between 5 and 24 min are annotated (Jimenez-Castells et al. 2017). For the ODS-C18 RP-HPLC and HIAX NP-HPLC chromatograms of O. dentatum GAG-like O-glycans, refer to Supplementary Figure S3. Anionic and neutral glycosaminoglycan-like O-glycans from O. dentatum The basic core sequences were investigated in both O. dentatum male and female O-glycomes by positive and negative MS/MS (Figure 3 and Supplementary Figure S4) modes to confirm the presence of GAG-like O-glycans. Their fragmentation patterns were rather similar to those observed in C. elegans in terms of the Y/B-ion intensities and distributions. However, the maximum number of HexNAc1HexA1 repeat units detected for O. dentatum male and female (n = 3) was lower than for C. elegans (n = 10). Fig. 3. View largeDownload slide Positive and negative MS/MS of GAG-like O-glycans from Oesophagostomum dentatum. The positive and negative MS/MS of anionic GAG-like O-glycans from O. dentatum (A–I) show data on structures from m/z 715 to 1487 (HexNAc0–2GlcA0-3Gal2–3Xyl1-PA). Most of these structures have the basic GAG-like O-glycan core sequence and share some of the same Y-fragmentation features as described in C. elegans (Figure 1); however, a few possess a trigalactosylated core sequence (D) as indicated by a specific Y3-ion in positive mode at m/z 715 (Gal3Xyl1-PA). The positive MS/MS of phosphorylcholine-modified GAG-like O-glycans from O. dentatum (J–R) exhibit key PC-fragments at m/z 328 (PC1Hex1) and/or 369 (PC1HexNAc1) while the ion-loss of 59 Da is due to an inherent PC breakdown. The fragment signature of a PC and galactosylated core is an Y3-ion of PC1Hex3Xyl-PA at m/z 880. *Contaminant fragments derived from coeluting PC-containing glycans; for MS/MS of m/z 1056 and 1435 in HIAX fractions lacking the m/z 369 cofragment, refer to Supplementary Figure S4A and D. Fig. 3. View largeDownload slide Positive and negative MS/MS of GAG-like O-glycans from Oesophagostomum dentatum. The positive and negative MS/MS of anionic GAG-like O-glycans from O. dentatum (A–I) show data on structures from m/z 715 to 1487 (HexNAc0–2GlcA0-3Gal2–3Xyl1-PA). Most of these structures have the basic GAG-like O-glycan core sequence and share some of the same Y-fragmentation features as described in C. elegans (Figure 1); however, a few possess a trigalactosylated core sequence (D) as indicated by a specific Y3-ion in positive mode at m/z 715 (Gal3Xyl1-PA). The positive MS/MS of phosphorylcholine-modified GAG-like O-glycans from O. dentatum (J–R) exhibit key PC-fragments at m/z 328 (PC1Hex1) and/or 369 (PC1HexNAc1) while the ion-loss of 59 Da is due to an inherent PC breakdown. The fragment signature of a PC and galactosylated core is an Y3-ion of PC1Hex3Xyl-PA at m/z 880. *Contaminant fragments derived from coeluting PC-containing glycans; for MS/MS of m/z 1056 and 1435 in HIAX fractions lacking the m/z 369 cofragment, refer to Supplementary Figure S4A and D. The novel hexose-substituted core sequence was observed in both O. dentatum male and female O-glycomes and correlated with an intense Y3-ion at m/z 715 corresponding to Hex3Xyl-PA (Figure 3D). To investigate the nature and position of the additional hexose, we treated an example fraction with either β3-galactosidase, β3/4-galactosidase or jack bean α-mannosidase (Figure 4A–C); only the β3/4-galactosidase was able to remove one hexose from HexNAc1GlcA1Hex3Xyl1-PA (m/z 1094), which suggested that the hexose branch is a β4-linked galactose. Furthermore, the positive MS/MS of this structure (Figure 3D; m/z 1094) indicated that the branching galactose was linked to the first galactose of the core sequence as judged by the intense Y2 and Y3 ions at m/z 553 and 715 (Gal2-3Xyl1-PA), while the Y2 + Y2′ ion at m/z 391 (Gal1Xyl1-PA) is much less intense as it results from a double fragmentation event. Upon degalactosylation to HexNAc1GlcA1Gal2Xyl1-PA (m/z 932; Figure 4C), the Y2 ion at m/z 391 (Gal1Xyl1-PA) is of increased intensity as this time it results from a single fragmentation. The conclusion regarding the position of the extra hexose was further confirmed in PC-containing structures (see below). Fig. 4. View largeDownload slide Identification of new core sequences of GAG-like O-glycans from O. dentatum by enzymatic and chemical digestions (A–G). The HexNAc1GlcA1Hex3Xyl1-PA (m/z 1094, see Figure 3D) was analyzed by positive MS and MS/MS before (A) and after β3-galactosidase (B) or β3/4-galactosidase (C) digestions; only the β3/4-galactosidase was able to remove a single hexose, as also seen by the alteration in the MS/MS spectra (see insets), thus indicating that the third hexose is a branching β4-linked galactose. The PC1-2HexNAc1GlcA1Hex2Xyl1-PA glycans (m/z 1097 and 1262; see Figures 2E/G and 3 M/N) were analyzed by positive MS before (D, F) and after (E, G) hydrofluoric acid (HF) treatment which removed one or two phosphorylcholine residues to yield HexNAc1GlcA1Hex2Xyl1-PA at m/z 932; shifts in the MS/MS spectra (see insets) show the absence of the PC-containing B-ions (e.g. PC-HexNAc at m/z 369) and the dominance of the PA-containing Y-ions (e.g., Xyl-PA at m/z 229) after HF treatment. Some partial loss of methyl groups from the PC units during the release and work-up procedure resulted in the observation of ions at m/z 1248 in addition to the bona fide structure at m/z 1262 (F) Fig. 4. View largeDownload slide Identification of new core sequences of GAG-like O-glycans from O. dentatum by enzymatic and chemical digestions (A–G). The HexNAc1GlcA1Hex3Xyl1-PA (m/z 1094, see Figure 3D) was analyzed by positive MS and MS/MS before (A) and after β3-galactosidase (B) or β3/4-galactosidase (C) digestions; only the β3/4-galactosidase was able to remove a single hexose, as also seen by the alteration in the MS/MS spectra (see insets), thus indicating that the third hexose is a branching β4-linked galactose. The PC1-2HexNAc1GlcA1Hex2Xyl1-PA glycans (m/z 1097 and 1262; see Figures 2E/G and 3 M/N) were analyzed by positive MS before (D, F) and after (E, G) hydrofluoric acid (HF) treatment which removed one or two phosphorylcholine residues to yield HexNAc1GlcA1Hex2Xyl1-PA at m/z 932; shifts in the MS/MS spectra (see insets) show the absence of the PC-containing B-ions (e.g. PC-HexNAc at m/z 369) and the dominance of the PA-containing Y-ions (e.g., Xyl-PA at m/z 229) after HF treatment. Some partial loss of methyl groups from the PC units during the release and work-up procedure resulted in the observation of ions at m/z 1248 in addition to the bona fide structure at m/z 1262 (F) Zwitterionic glycosaminoglycan-like O-glycans from O. dentatum Modifications with phosphorylcholine are associated with a mass increment of 165 Da and occur, e.g. on N-glycans of nematodes, Lepidoptera, cestodes and a mollusc as well as on lipopolysaccharides of some bacteria and glycolipids of some invertebrates; this modification can be detected in positive MS mode and has a proven sensitivity to hydrofluoric acid treatment (Paschinger et al. 2012; Paschinger and Wilson 2015; Yan, Wilson et al. 2015; Eckmair et al. 2016). In contrast to the ODS-C18 HPLC column, the presence of phosphorylcholine reduces retention times on the RP-amide HPLC column, an effect even more pronounced for multi-PC containing structures occurring in the 2.5 min HPLC fractions of O. dentatum male and female glycomes (<2 g.u.; Figure 2A–C and Supplementary Figure S3). The MS/MS fragmentation patterns of PC-containing structures of GAG-like O-glycans from O. dentatum demonstrated a range of structures from PC1Hex2Xyl1-PA to PC3HexNAc3GlcA3Hex2Xyl1 (m/z 718–2185; Figure 3J–R and Supplementary Figure S4) and revealed intense PC-fragments at m/z 328 (PC1Hex1) and m/z 369 (PC1HexNAc1) which indicated that PC either substitutes a HexNAc or Gal. Also, the presence of PC on a galactosylated core sequence was deduced in the case of PC1GlcA1Gal3Xyl-PA and PC1HexA1–2HexNAc1–2GlcA1Gal3Xyl-PA (m/z 1056, 1435 and 1814; see Figure 3L and Supplementary Figure S4D and F) which showed Y3-ions at m/z 718 (PC1Gal2Xyl-PA) and 880 (PC1Gal3Xyl-PA). In the case of PC2HexNAc1GlcA1Hex2Xyl-PA (m/z 1262; Figure 3N), fragments were observed at m/z 872 (B3; PC2HexNAc1GlcA1Hex1), 391 (Y2; Hex1Xyl-PA) and 718 (Y3; PC1Hex2Xyl-PA), but not at m/z 556 (theoretically PC1Hex1Xyl1-PA). Thus, the MS/MS data demonstrate that one phosphorylcholine is linked to HexNAc, while the other one substitutes the second core galactose and so it is concluded that the structure is PCHexNAcGlcAβ3(PC)Galβ3Galβ4Xyl. The PC position is similar in the case of an additional galactose (Figure 3L and P) as judged by positive fragments at m/z 880 (Y3; PC1Gal3Xyl-PA) and 718 (Y3 + Y2′; PC1Gal2Xyl-PA), which excludes the possibility that the branching galactose is substituted with PC. Furthermore, the PC1-2HexNAc1GlcA1Gal2Xyl1-PA structures (m/z 1097–1262; Figure 4D and F) were treated with HF which specifically removed all phosphodiester substitutions to yield HexNAc1GlcA1Gal2Xyl1-PA (m/z 932; Figure 4E and G), thus confirming the presence of the PC residues. The products of HF treatment also displayed an MS/MS pattern dominated by Y-fragments, rather than by the comparatively very easily ionizing PC-containing B-fragments present in the case of the original glycans (compare insets in Figure 4D and E). Discussion In this study, we found that short GAG-like O-glycans were efficiently coreleased with N-glycans by hydrazinolysis, which enabled their subsequent labeling by pyridylamination (PA) and analysis by an off-line LC–MS strategy. In other studies, the core regions of chondroitin and heparan chains have been analyzed after release by either β-elimination which excludes the possibility of subsequent labeling by reductive amination. Alternative strategies have been to use either an endo-β-xylosidase (apparently not commercially available) or lithium hydroxide, both of which enable subsequent labeling (Kon et al. 1991; Yamada et al. 2002). Previously, hydrazinolysis of proteoglycans was apparently only used to de-N-acetylate the HexNAc residues prior to nitrous acid deamination which is a method for isolation of modified forms of the individual disaccharide repeats (Shaklee and Conrad 1986). Thus, we can demonstrate a new use for hydrazine in the study of glycosaminoglycan-like structures. The GAG-like O-glycans isolated from C. elegans and from O. dentatum were separated from N-glycans by RP-HPLC which enabled subsequent MALDI-TOF MS and MS/MS as well as chemical or enzymatic treatments. Thereby, it is concluded that hydrazinolysis at high temperature (“N-glycan-specific” conditions) is able to release simultaneously GAG-like O-glycans and N-glycans, but destroys most of the mucin-type O-glycans. Although there is a degree of artefactual “peeling” of the natural reducing terminal monosaccharide (i.e. loss of xylose) or demethylation of phosphorylcholine (Δm/z 14), this was probably no more than 20% in our hands when performing hydrazinolysis on these samples, but an exact figure is difficult to estimate on the basis of fluorescence and ionization intensities of HPLC fractions containing multiple components. In terms of separation, a trend to earlier RP-HPLC elution of the longer oligosaccharides was observed, which is in keeping with the increasing number of hexuronic acid residues. The results for both organisms were surprisingly different with larger and unmodified core sequences being present in C. elegans, while male and female O. dentatum expressed GAG-like structures with new core sequences with an additional galactose and/or phosphorylcholine. Furthermore, no significant difference was observed between male and female in O. dentatum samples in terms of quality and quantity of GAG-like glycans. Based on previous studies, including data on the single pentasaccharide containing xylose at the reducing terminus identified by NMR after β-elimination (Guerardel et al. 2001), we presume that we have isolated unsulphated chondroitin chains based on GalNAcβ4GlcAβ3Galβ3Galβ4Xyl. On the other hand, the strategy of using hydrazinolysis followed by fluorescent labeling allowed us to isolate and identify more and larger chondroitin-like O-glycans than previously found in nematodes (up to almost 5 kDa in C. elegans; Figure 1), but structures larger than 25 saccharide units were not observed. Furthermore, chondroitin in its sulphated form is rare in C. elegans and possibly represents less than 1% of total GAG in this organism (Izumikawa et al. 2016); thus, it would be a challenge to detect such chains. Several glycosyltransferases genes (sqv-1, -2, -3 and -8) are required to synthesize the core sequence of GAG-like O-glycans in C. elegans, all of which are essential for its development (Hwang et al. 2003). However, the biosynthetic origin of the additional β4-linked galactose and phosphorylcholine residues on the O. dentatum [PCGalNAcβ4GlcAβ3]nPCGalβ3(Galβ4)Galβ4Xyl structures is unknown. On the other hand, similar to its N-glycans (Jimenez-Castells et al. 2017), the degree of phosphorylcholinylation on O. dentatum glycosaminoglycan-like sequences is relatively high as compared to the total of the identified structures, but also represents the first zwitterionic modification to be detected on this class of glycan. Thus, our study is another indication of the high variability of glycoconjugate structures in invertebrates, which can only be uncovered by the use of an extended methodological portfolio and in-depth analysis of the glycomic data. Materials and methods Nematode culture and isolation Caenorhabditis elegans (wild‐type N2 strain) was cultivated in our laboratory using standard conditions with Escherichia coli strain OP50 (i.e. maintained on nematode growth medium agar plates, grown in larger scale in liquid S complete medium and isolated by sucrose density centrifugation prior to freezing at −80°C). Oesophagostomum dentatum adults (OD-Hann strain) were recovered from the large intestines of infected pigs by agar-gel migration and visually separated into males and females prior to washing in 0.9% NaCl at 38°C, sedimentation, snap freezing and storage at −80°C until use (Slotved et al. 1996). Hydrazinolysis of nematode glycopeptides Worms were homogenized and subject to proteolysis with pepsin (overnight at 37°C; for C. elegans) or thermolysin (2 h at 70°C; for O. dentatum) prior to hydrazinolysis and pyridylamination; based on previous procedures (Patel et al. 1993; Jimenez-Castells et al. 2017). Briefly, 1 mg protease per gram of wet weight material was employed to prepare glycopeptides, which were purified by cation exchange (50 W × 8 Dowex, BioRad; elution with 0.5 M ammonium acetate, pH 6) and gel filtration (G25, GE Healthcare; elution with 0.5% acetic acid) prior to transfer into a glass reaction tube and drying overnight. Hydrazinolysis was then performed in 500 μL of anhydrous hydrazine (prepared from hydrazine monohydrate, Sigma) at 100°C for 5 h and removal of unreacted reagent by centrifugal evaporation. Glycans were re-N-acetylated using 450 μL of 100 mM sodium bicarbonate and 21 μL of acetic anhydride at 0°C for 1 h. Then, the reducing ends were liberated using 600 μL of 5% trifluoroacetic acid at 4°C for 1 h. Thereafter, the free glycans were purified by cation exchange (50 W × 8 Dowex; eluted with 2% acetic acid), nonporous graphitized carbon (eluted with 40% acetonitrile containing 0.1% TFA) and C18 columns (eluted with water). Finally, glycans were fluorescently labeled using 2-aminopyridine (Hase et al. 1984). For a fuller description of these methods, refer to the Supplement. HPLC purification of O-glycans Oligosaccharides with xylose at the reducing terminus were separated from N-glycans by RP-HPLC using a Shimadzu Nexera HPLC system equipped with a fluorescence detector (RF 20 AXS) and reversed phase columns (either Ascentis Express RP-Amide, Sigma-Aldrich, or Hypersil C18, Agilent). Glycans were eluted using a buffer system of 100 mM ammonium acetate (pH 4.0) and 30% (v/v) methanol at a flow rate of either 0.8 or 1.5 mL/min (Hykollari et al. 2017). Glycans were detected by fluorescence with excitation/emission wavelengths of 320/400 nm. RP-HPLC columns were calibrated daily in terms of glucose units using a pyridylaminated dextran hydrolysate; the order of elution of the standards was confirmed by MALDI-TOF MS of collected calibrant fractions. In the case of O. dentatum, the glycans eluting early from the RP-amide column were repooled and applied to an AS11 HIAX/NP-HPLC column using a set of oligomannosidic glycans for calibration (see Supplement). MALDI-TOF MS analysis Glycans in individual RP-HPLC fractions were analyzed by MALDI-TOF MS (Autoflex Speed, Bruker Daltonics, Germany) in positive and negative ion mode using FlexControl 3.4 software and 6-aza-2-thiothymine as matrix; MS/MS to confirm the composition of all proposed structures was performed by laser-induced dissociation (precursor ion selector was generally set to ±0.6%). The detector voltage was typically 1977 V for MS and 2133 V for MS/MS; 1000–3000 shots from different regions of the sample spots were summed. Spectra were processed with the manufacturer’s software (Bruker Flexanalysis 3.3.80) using the SNAP algorithm with a signal/noise threshold of 6 for MS (unsmoothed) and 3 for MS/MS (four times smoothed). All MS and MS/MS spectra were manually interpreted on the basis of the mass fragmentation pattern and results of chemical and enzymatic treatments; O-xylose-based glycans were distinguished from N-glycans by the respective presence in positive MS/MS of Y1-ions at m/z 229 (Xyl1-PA) or 300 (HexNAc1-PA). Structural elucidation using exoglycosidases and chemical treatment Exoglycosidase treatment was performed using either recombinant β3/4-galactosidase from Aspergillus niger prepared in house (Dragosits et al. 2014) or recombinant β3-galactosidase from Xanthomonas manihotis from New England Biolabs (Wong-Madden and Landry 1995). In general, a 1–2 μL aliquot of a lyophilysed and redissolved HPLC fraction into 10 μL was incubated with 0.2 μL exoglycosidase and 0.5 μL 100 mM ammonium acetate solution, pH 5.0, overnight at 37°C. For removal of phosphorylcholine, aliquots of O-glycan fractions were dried in a Speed-Vac and then incubated with 3 μL of 48% (w/v) hydrofluoric acid (HF) on ice for 24 h prior to drying again (Dennis et al. 1998). Chemically or enzymatically treated glycans were reanalyzed by MALDI-TOF MS and MS/MS without further purification. Supplementary data Supplementary data is available at GLYCOBIOLOGY online. Funding This work was supported by the Austrian Science Fund (FWF) and by the European Union; S.Y. and K.P. are FWF fellows (Grants P30021 and P25058); J.V. was an experienced researcher within the Glycopar EU FP7 Marie Curie Initial Training Network (PITN-GA-2013-608295). Acknowledgements We also thank the staff of Ludger Ltd for an introduction to hydrazinolysis, especially Radoslaw Kozak and Daniel Spencer, as well as Markus Blaukopf for further help with hydrazinolysis and Bärbel Ruttkowski for nematode collection. 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GlycobiologyOxford University Press

Published: May 11, 2018

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