Gender and developmental specific N-glycomes of the porcine parasite Oesophagostomum dentatum

Abstract

Background: The porcine nodule worm Oesophagostomum dentatum is a strongylid class V nematode rather closely related to the model organism Caenorhabditis elegans. However, in contrast to the non-parasitic C. elegans, the parasitic O. dentatum is an obligate sexual organism, which makes both a gender and developmental glycomic comparison possible.

Methods: Different enzymatic and chemical methods were used to release N-glycans from male and female O. dentatum as well as from L3 and L4 larvae. Glycans were analysed by MALDI-TOF MS after either 2D-HPLC (normal then reversed phase) or fused core RP-HPLC.

Results: Whereas the L3 N-glycome was simpler and more dominated by phosphorylcholine-modified structures, the male and female worms express a wide range of core fucosylated N-glycans with up to three fucose residues. Seemingly, simple methylated paucimannosidic structures can be considered 'male', while methylation of fucosylated glycans was more pronounced in females. On the other hand, while many of the fucosylated paucimannosidic glycans are identical with examples from other nematode species, but simpler than the tetrafucosylated glycans of C. elegans, there is a wide range of phosphorylcholine-modified glycans with extended HexNAc_2-4PC_2-4 motifs not observed in our previous studies on other nematodes.

Conclusion: The interspecies tendency of class V nematodes to share most, but not all, N-glycans applies also to O. dentatum; furthermore, we establish, for the first time in a parasitic nematode, that glycomes vary upon development and sexual differentiation.

General significance: Unusual methylated, core fucosylated and phosphorylcholine-containing N-glycans vary between stages and genders in a parasitic nematode.

Keywords: Glycomics; HPLC; Mass spectrometry; Nematoda; Strongylida.

Figure 1. Two-dimensional HPLC of O. dentatum N-glycans.

(A) Pyridylaminated PNGase A-released N-glycans from larvae and adults were separated on a Palpak NP-HPLC column and each collected fraction was subjected to RP-HPLC on Hypersil C18 followed by MALDI-TOF MS; the major observed m/z values are indicated together with (in red) abbreviated compositions of mannosidic glycans (HxN2). (B) RP-HPLC analyses of the 5.2 g.u. NP-HPLC fractions (red dashed box in panel A) indicate differences in the co-eluting ‘size-fractionated’ glycans. (C and D) MALDI-TOF MS/MS of four selected glycans (as [M+H]⁺) of differing RP-HPLC retention times (underlined with red in panel B) isolated from larvae and adults; the proposed structures are shown using the Symbol Nomenclature for Glycans. NP- and RP-HPLC columns were externally calibrated daily in terms of glucose units (g.u.) with, in both cases, the smallest glucose oligomers eluting first as proven by MALDI-TOF MS of collected calibrant fractions (the g.u. on the C18 column differ from those for the RP amide column used for 1D-HPLC); fluorescence intensities are given in mV.

Figure 2. RP-amide HPLC of PNGase F-released O. dentatum N-glycans.

Pyridylaminated PNGase F-released N-glycans from male and female adults are shown annotated with the proposed structures (as confirmed by MS/MS and digestion data; see Figures 4-7 and the Supplementary Table); calibration is in terms of glucose units (g.u.). The PNGase F-released glycomes are dominated by oligomannosidic, α1,6-fucosylated, GalFuc-modified (late eluting) as well as phosphorylcholine-modified glycans.

Figure 3. RP-amide HPLC of PNGase A-released O. dentatum N-glycans.

Pyridylaminated PNGase A-released N-glycans (after separation from the PNGase F-released glycans) from male and female adults are shown annotated with the proposed structures (see Figures 4-7 and the Supplementary Table); calibration is in terms of glucose units (g.u.). There are a number of multi-fucosylated glycans as well as small, early-eluting α1,3-fucosylated structures, not present in the PNGase F-released pools.

Figure 4. Analysis of fucosylated O. dentatum N-glycans by MALDI-TOF MS.

(A-H) Four isomers of Hex₃HexNAc₂Fuc₁-PA (m/z 1135; PNGase A-released female pyridylaminated N-glycans) separated by RP-amide HPLC (with the indicated elution times in terms of glucose units) were analysed by MALDI-TOF MS ([M+H]⁺) and MS/MS before and after hydrofluoric acid, bovine α-fucosidase or Aspergillus β-galactosidase treatment; the losses of fucose or hexose are indicated (-F or -H). (I-K) MALDI-TOF MS/MS of RP-amide separated isomers of Hex₂HexNAc₂Fuc₃-PA (m/z 1265) and Hex₃HexNAc₂Fuc₂-PA (m/z 1281; L-P). Key fragments and proposed structures are annotated with a standard symbolic nomenclature or with compositions in terms of fucose (F), hexose (H) and N-acetylhexosamine (N). The presence of m/z 665 or 827 fragments correlates with either one or two mannose residues, whereas the m/z 592, 608 and 754 fragment ions are diagnostic for the modifications of the 2-aminopyridine-tagged reducing terminus (Fuc_1-2Gal_0-1GlcNAc₁-PA).

Figure 5. MALDI-TOF MS analysis of two Hex₃HexNAc₂Fuc₃ isomers.

(A and E) MS/MS of 2D-HPLC separated isomers of Hex₃HexNAc₂Fuc₃ (m/z 1427 as [M+H]⁺; 6.8 and 8.0 g.u. from the PNGase A-released female glycome) with key fragments symbolically annotated; this is the most dominant multiply-fucosylated composition in the O. dentatum N-glycome. (B-D and F-I) MALDI-TOF MS before and after incubation with either Xanthomonas α1,2/3-mannosidase, hydrofluoric acid or Aspergillus β-galactosidase; the effect of these treatments is indicated with the dashed lines and alterations in MS/MS spectra (insets). The first isomer (B) corresponds to the sixth most dominant N-glycan in Haemonchus, whereas the second (F) is a minor structure in Pristionchus.

Figure 6. MALDI-TOF MS/MS analysis of methylated N-glycans including gender-specific structures.

(A-D) MS and MS/MS of 2D-HPLC-separated PNGase A-released glycans specifically detected in either female (A) or male (B-D) N-glycomes. (E-H) MS/MS of selected RP-amide-separated methylated female PNGase A-released glycans highlighting the different methylation positions (on fucose, mannose or galactose) on one isomer of Hex₂HexNAc₂Fuc₃Me₁-PA and three isomers of Hex₃HexNAc₂Fuc₃Me₁-PA; the latter isomer (H) is also found in the male glycome. Example losses of fucose (146), methylfucose (160) or methylgalactose together with fucose (322) are shown. Relevant supporting digestion data (hydrofluoric acid or α-mannosidase) are summarised in the Supplementary Table.

Figure 7. Analysis of PC-containing O. dentatum N-glycans.

(A-J) MALDI-TOF MS/MS spectra of selected phosphorylcholine-modified N-glycans (as [M+H]⁺) separated by RP-amide HPLC. (K-P) MALDI-TOF MS and MS/MS of a phosphorylcholine-containing N-glycan present in the 7.6 g.u. fraction before and after treatment with hydrofluoric acid and chitinase; due to components in the enzyme preparation, sodiated adducts dominate, but only the protonated forms are annotated. In all panels, key fragments are annotated either symbolically or with abbreviations with fragment ions at m/z 369 being a signature for HexNAc₁PC₁, whereas m/z 737, 1105 and 1635 fragments are indicative of phosphorylcholine modifying two, three or four HexNAc residues in series; ions 59 mass units lower than a fragment containing multiple PC moieties are marked with an asterisk and could be due to loss of N(Me)₃ from the choline moiety, whereas ambiguity in terms of the antennal configuration of the m/z 2462 structure is indicated by a bracket. The example data are of PNGase A-, F- or hydrazine-released glycans from female or male adults eluting from the RP-amide HPLC column at the indicated glucose units.

Figure 8. Summary of occurrence and example structures of O. dentatum N-glycans.

Percentage occurrence of categories of N-glycans, together with example structures, are shown; the percentages for male and female adults as well as L3 and L4 larvae are based on the fluorescence intensity of 2D-HPLC fractions and indicate glycomic trends during development and differentiation. For the subcategories ‘methylated with fucose’ and ‘trifucose core’, the percentages in brackets show further distinctions in the gender and stage-specific glycomes. Also shown are examples of glycans with a specific or rather biased expression in either adults (regardless of gender), males, females or larvae.