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Research

Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans

Shi Yan, Lothar Brecker, Chunsheng Jin, Alexander Titz, Martin Dragosits, View ORCID ProfileNiclas G. Karlsson, View ORCID ProfileVerena Jantsch, View ORCID ProfileIain B. H. Wilson and View ORCID ProfileKatharina Paschinger
Molecular & Cellular Proteomics August 1, 2015, First published on May 22, 2015, 14 (8) 2111-2125; https://doi.org/10.1074/mcp.M115.049817
Shi Yan
From the ‡Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria;
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Lothar Brecker
§Institut für Organische Chemie, Universität Wien, 1090 Wien, Austria;
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Chunsheng Jin
¶Institutionen för Biomedicin, Göteborgs universitet, 405 30 Göteborg, Sweden;
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Alexander Titz
From the ‡Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria;
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Martin Dragosits
From the ‡Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria;
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Niclas G. Karlsson
¶Institutionen för Biomedicin, Göteborgs universitet, 405 30 Göteborg, Sweden;
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  • ORCID record for Niclas G. Karlsson
Verena Jantsch
‖Department für Chromosomenbiologie, Max F. Perutz Laboratories, Universität Wien, 1030 Wien, Austria
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Iain B. H. Wilson
From the ‡Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria;
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Katharina Paschinger
From the ‡Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria;
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Abstract

The N-glycosylation of the model nematode Caenorhabditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that “simple” organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N′-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain. By constructing a triple mutant with deletions in the three genes responsible for core fucosylation (fut-1, fut-6 and fut-8), we have produced a nematode strain lacking products of these enzymes, but still retaining maximally one fucose residue on its N-glycans. Using mass spectrometry and HPLC in conjunction with chemical and enzymatic treatments as well as NMR, we examined a set of α-mannosidase-resistant N-glycans. Within this glycomic subpool, we can reveal that the core β-mannose can be trisubstituted and so carries not only the ubiquitous α1,3- and α1,6-mannose residues, but also a “bisecting” β-galactose, which is substoichiometrically modified with fucose or methylfucose. In addition, the α1,3-mannose can also be α-galactosylated. Our data, showing the presence of novel N-glycan modifications, will enable more targeted studies to understand the biological functions and interactions of nematode glycans.

Nematodes represent, along with arthropods, one of the largest groups of animals to exist on the planet; 25.000 species are described, but the existence of up to one million has been estimated (1, 2). They have various ecological niches and include free-living “worms” in the soil, fungivorous, entomopathogenic, and necromenic species as well as parasites of plants and mammals, which share the basic conserved body plan (more-or-less a digestive tube surrounded with muscle, whether larger or smaller). There are five major clades (Rhabditina, Enoplia, Spirurina, Tylenchina, and Dorylaimia) (2), yet the glycosylation of only a few nematode species has been studied with an inevitable focus on the model nematode Caenorhabditis elegans and parasitic species (3). Thereby, the use of C. elegans mutants has been highly valuable in dissecting aspects of nematode N-glycan biosynthesis and revealing the in vivo substrates for certain glycosyltransferases (4).

As many nematodes are parasites, their interactions with the immune systems of their hosts have attracted attention; particularly, there are relationships between autoimmunity, allergy, vaccination, and helminth infections. The “old friends” hypothesis seeks to understand the evolutionary factors that have shaped the immune system and to explain correlations between lifestyles in the developed world and modern “epidemics,” which are due to immunological misbalance (5⇓–7). Promising data have suggested that “worm therapy” may bring advantages to some patients with Crohn's disease or allergies (8, 9); however, such approaches are controversial. Nevertheless, crude extracts even of Caenorhabditis elegans were shown to induce a glycan-dependent Th2 response (10), whereas the excretory-secretory products of some nematodes also have immunomodulatory activity (11). Furthermore, the native glycoproteins of some nematodes have proven effective in vaccination trials, whereas recombinant forms are not, which is suggestive that post-translational modifications may have a role in an efficacious immune response (12).

As at least some of the molecules relevant to nematode immunomodulation or vaccination are glycoproteins, a proper understanding of nematode glycosylation is of biomedical and veterinary relevance. Over the years, it has become apparent that the core chitobiosyl region of nematode N-glycans is subject to a range of modifications, with up to three core fucose residues being present (α1,3- and α1,6-linked on the reducing-terminal “proximal” GlcNAc and α1,3-linked on the second “distal” GlcNAc). However, up to four fucose residues have been detected on C. elegans N-glycans and the exact nature of the linkage of the fourth fucose has remained obscure despite work in our own and other laboratories (3, 13⇓–15). Combined with the latest knowledge regarding the specificity of C. elegans core fucosyltransferases (13, 16, 17) as well as our recent data regarding the exact structures of N-glycans from the C. elegans double hexosaminidase mutant and other nematodes (18⇓–20), we concluded that some models for the tri- and tetrafucosylated N-glycans were incorrect. By preparing a triple mutant unable to core fucosylate its N-glycans, we generated a C. elegans strain containing maximally one fucose residue on the N-linked oligosaccharides. Thereby a pool of unusual mannosidase-resistant N-glycans was identified and, using mass spectrometry (MS) and NMR, we reveal their modification with bisecting galactose frequently capped with fucose or methylfucose.

EXPERIMENTAL PROCEDURES

Preparation of the C. elegans Triple Mutant

Wild-type C. elegans (N2) and single mutants fut-1(ok892), fut-6(ok475) and fut-8(ok2558) were obtained from the Caenorhabditis Genetics Centre (CGC), University of Minnesota, USA. All C. elegans strains were cultured under standard conditions at 20 °C (21). A fut-1;fut-6 double mutant was generated by standard crossings (22). Briefly, the ok892 single mutant was first crossed with N2 wild-type males in order to produce male progeny for subsequent fertilization of the ok475 single mutant. Hermaphrodites from the F2 generation were isolated and allowed to produce eggs prior to examination of the maternal genotypes by PCR. F3 progeny from the heterozygotes carrying both mutations were isolated in a large amount and their genotypes were also examined 48 h after isolation. Additional isolations of heterozygotes and genotyping PCR were carried out until homozygotes with both mutations appeared. To generate the triple mutant, progeny of the ok2558 single mutant and N2 were crossed to fut-1;fut-6 double mutants prior to genotype screening. Following the workflow mentioned above, homozygous mutations in the three fucosyltransferase genes were accumulated over a few generations until a genome of a triple mutant was obtained. Genotypes of each generation were followed using the primers described below.

PCR for Detection of Genomic Deletions

A triplex PCR strategy was used for a time-efficient genotype screening. Three primers were used as a group in one PCR reaction mixture to amplify DNA fragments with distinguishable lengths within a short elongation step (40 s): fut-1, 270 bp (wild-type; wt) and 320 bp (mutant; mut); fut-6, 200 bp (wt) and 158 bp (mut); fut-8, 574 bp (wt) and 469 bp (mut). Primers were ordered from Sigma-Aldrich: fut-1, 5′-CTAAATTGGCATCCACAACCT-3′, 5′-GCCATTTATTAACAGTTCTCAT-3′ and 5′-CCGGAGTAATTAGACCTGC-3′; fut-6, 5′-GAATGCCACCATGCAACAT-3′, 5′-GAATTACCCATGATACTAGAT-3′ and 5′-GCCCCAAATATCAATCTGC-3′; fut-8, 5′-TCAGTCTTCGCCAATCAT-3′, 5′-AATTACCGCATTTGCTAC-3′, and 5′-TAAAAGGAGTGTCCATTG-3′. Single worms were lysed by incubation in 7 μl of lysis buffer (50 mm KCl, 10 mm Tris/HCl, pH 8.3, 2.5 mm MgCl2, 0.45% (w/v) Nonidet P-40, 0.45% (w/v) Tween-20, 60 μg/ml Proteinase K) at 60 °C for 1 h prior to heat inactivation; PCR was carried out in the presence of 0.2 μl (4 pmol) of each primer, 1 μl genomic DNA from worm lysates, 10 μl of GoTaq®Green Master Mix (Promega, Madison, WI) and 8.4 μl of sterile water. PCR fragments were electrophoresed in 3% (w/v) agarose gel mixed with 0.05‰ (w/v) of ethidium bromide and visualized on a UV transilluminator (Bio-Rad, Hercules, CA). 0.5 μg of 100 bp DNA ladder (New England Biolabs, Ipswich, MA) were used as a reference to estimate the sizes of the DNA amplicons.

N-glycan Preparation and MALDI TOF MS Analysis

C. elegans were grown in liquid culture with E. coli OP50 in standard S complete medium, harvested after cultivation at room temperature (20 °C) for 4–6 days and purified by sucrose density centrifugation (in two independent preparations, the yield was 5 and 9 g of worms respectively). N-glycans were released from worm peptic peptides using peptide/N-glycosidase F as previously described, with a subsequent digestion of remaining glycopeptides using peptide/N-glycosidase A (23). The N-glycome of the mutant was profiled by MALDI-TOF MS (Autoflex Speed, Bruker Daltonics, Bremen, Germany) in positive ion mode using FlexControl 3.4 software. Free glycans were labeled with 2-aminopyridine prior to fractionation by normal phase high pressure liquid chromatography (NP-HPLC)1 and reversed-phase HPLC (RP-HPLC; see below). All the HPLC peaks were collected and examined by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS, using 6-aza-2-thiothymine as matrix; tandem MS (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 generally set at 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 six for MS (unsmoothed) and three for MS/MS (four-times smoothed). In total ∼1500 MS and MS/MS spectra were manually interpreted on the basis of the mass, fragmentation pattern, and results of chemical and enzymatic treatments; isobaric structures present in different RP-HPLC fractions were defined on the basis of comparisons in the aforementioned parameters. At least five MS/MS fragment ions were used to aid definition of each of the structures.

HPLC Purification of N-glycans

Separation of PA-labeled glycans was carried out on a Shimadzu HPLC system equipped with a fluorescence detector (RF 10 AXL). In case of NP-HPLC, a TSKgel Amide-80 column (Tosoh Bioscience, Tokyo, Japan) was used with 10 mm ammonium formate, pH 7.0 (buffer A) and 95% (v/v) acetonitrile (buffer B). The gradient of buffer B was applied as follows: 0–5 min, 75% B; 5–10 min, 75–70% B; 10–15 min, 70–65% B; 15–55 min, 65–55% B. In case of RP-HPLC, a Hypersil ODS column (Agilent, Santa Clara, CA) was used with 100 mm ammonium acetate, pH 4.0 (buffer C) and 30% (v/v) methanol (buffer D); a gradient of increasing buffer D (1% per minute) was programmed. The columns were calibrated daily in terms of glucose units (g.u.) with a pyridylaminated partial dextran hydrolysate.

Structural Elucidation Using Exoglycosidases and Chemical Treatment

In general, a 1 μl aliquot of a HPLC fraction was mixed with 0.2 μl exoglycosidase and 0.8 μl 50 mm ammonium acetate solution, pH 5.0; after an overnight incubation at 37 °C, 0.5 μl aliquot of the mixture was analyzed by MALDI-TOF MS. Exoglycosidases employed were: α-galactosidase from green coffee bean (Sigma, 11 mU), recombinant β-galactosidase from Aspergillus niger (24), jack bean α-mannosidase (Sigma-Aldrich, 6.25 mU) and recombinant Xanthomonas manihotis α1,2/3-mannosidase (NEB, 6.4 U). Also, digestions were attempted with α-l-fucosidases from bovine kidney (Sigma-Aldrich, 10 mU), Xanthomonas (α1,2-specific; NEB, 4 mU) and Corynebacterium (α1,2-specific; Takara, 4 μU). For removal of fucose or methylfucose, glycan samples were dried in a SpeedVac and then incubated with 3 μl of 48% (w/v) hydrofluoric acid (HF) on ice for 24 h. The HF was allowed to evaporate overnight. Chemically or enzymatically treated glycans were reanalyzed by MALDI-TOF MS and MS/MS without further purification.

NMR Spectroscopy

The isolated oligosaccharides have been lyophilized, dissolved in D2O (99.996%; Sigma-Aldrich) in concentrations of ∼150 μg in 600 μl and transferred into 5 mm high precision NMR sample tubes (Promochem, Wesel, Germany). All spectra have been recorded on a Bruker AV III-600 AVANCE spectrometer (Bruker, Rheinstetten, Germany) at 600.13 MHz (1H) equipped with a CryoProbe™ Prodigy and were performed using the Bruker Topspin 3.1 software. The 1D proton spectra were recorded with presaturation, acquisition of 32 k data points and a relaxation delay of 1.0 s. After zero filling to 64 k data points and Fourier transformation spectra were performed with a range of 7200 Hz. 2D homonuclear DQF-COSY and TOCSY (100 ms mixing time) spectra have been measured with standard Bruker programs; 128 experiments, each with 2048 data points, were recorded with an appropriate number of scans. Linear forward prediction to 256 data points in the f2 dimension and sinusoidal multiplication in both dimensions and Fourier transformation led to 2D-spectra with a range of 6000 Hz in both dimensions. All measurements have been made at 298.1 K and chemical shifts were referenced to external acetone (δH 2.225 ppm).

LC-MS/MS of HPLC-purified PA-labeled N-glycans

PA-labeled N-glycans were analyzed by LC-MS/MS using a 10 cm × 150 μm I.D. column, prepared in-house, containing 5 μm porous graphitized carbon (PGC) particles (Thermo Scientific, Waltham, MA). Glycans were eluted using a linear gradient from 0–40% acetonitrile in 10 mm ammonium bicarbonate over 40 min at a flow rate of 10 μl/min. The eluted N-glycans were detected using a LTQ ion trap mass spectrometer (Thermo Scientific) in negative-ion mode with an electrospray voltage of 3.5 kV, capillary voltage of −33.0 V and capillary temperature of 300 °C. Air was used as a sheath gas and mass ranges were defined dependent on the specific structure to be analyzed. The data were processed using the Xcalibur software (version 2.0.7, Thermo Scientific). Glycans were identified from their MS/MS spectra by manual annotation; the nomenclature of Domon and Costello for fragment annotation was employed (25).

RESULTS

Impact of a Triple Fucosyltransferase Knock-out on the C. elegans Glycome

Previously, N-glycans with up to four fucose residues have been detected in C. elegans (13, 15, 26⇓⇓⇓–30), whereas only three fucosyltransferases (FUT-1, FUT-6 and FUT-8) required for the trifucosylation of the core chitobiosyl region of N-glycans in C. elegans have been identified (13, 16, 17). With the goal of restricting the N-glycome of this organism to glycans with maximally one fucose, a triple mutant with deletions in the three corresponding genes (fut-1;fut-6;fut-8) was constructed. Deletions of all three genes were confirmed by genomic PCR (supplemental Fig. S1), but no overt major phenotypic defects were detected under laboratory conditions. The N-glycans were prepared after large-scale liquid cultivation of the worms and were subsequently fluorescently labeled with 2-aminopyridine; the overall mass spectrometric profile indicated the presence of a range of N-glycans (m/z 665–1961 as [M+H]+; Fig. 1A and Table I). More exact examination of the spectra showed mass differences of 2 or 14 Da (e.g. m/z 1135, 1149 and 1151 or 1297, 1311 and 1313 as [M+H]+); thereby, some Δm/z values between glycans were of 146 (deoxyhexose), 160 (methyl and deoxyhexose), or 162 (hexose). This was an indication for the presence of fucose on a number of glycans and suggested compositions of Hex3–4HexNAc2Fuc1Me0–1-PA. In addition to the fucosylated and/or methylated glycans, masses corresponding to oligomannosidic and phosphorylcholine-modified oligosaccharides were detected (Table I); the latter modification is defined because of the diagnostic m/z 369 MS/MS fragments (HexNAc1PC1; supplemental Fig. S2). However, as such glycans have been previously described in nematodes (14, 15, 18⇓–20), their analysis was not the focus of the current study.

Fig. 1.
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Fig. 1.

Total N-glycome of the fut-1;fut-6;fut-8 triple knockout and its sensitivity to jack bean α-mannosidase. PA-derivatised N-glycans before and after mannosidase digestion were profiled by MALDI-TOF MS (A and B) and by reversed phase HPLC (C and D); the region of panel B above m/z 900 is magnified fivefold because of the dominance of the m/z 665 mannosidase-digestion product. The major glycan structures identified in this mutant were detected as [M+H]+ and are annotated on the HPLC chromatograms according to the nomenclature of the Consortium for Functional Glycomics. The intensity is in arbitrary units (a.u.).

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Table I Predicted compositions of N-glycans of the C. elegans fut-1;fut-6;fut-8 triple mutant. Observed and calculated m/z values ([M+H]+, MALDI-TOF MS) are given for the protonated forms of pyridylamino-labelled glycans with the composition HexxHexNAcyFuc0–1Me0–2-PA or HexxHexNAcyPC1–3-PA, whereby Me and PC are abbreviations for methyl and phosphorylcholine respectively. The compositions of all glycans were proven by MS/MS and/or digestion data as described in the text; for simplicity, the observed values are rounded to the nearest integer as there is variability in the measured m/z (typically ± 0.1 mass units; see the Figures 1⇓⇓⇓⇓–6 and 9)

Upon incubating the complete N-glycome with jack bean α-mannosidase, the disappearance of the oligomannosidic glycans was accompanied by a large increase in the peak at m/z 665 (Hex1HexNAc2-PA). Nevertheless, a number of glycans were not shifted by this treatment, including the one at m/z 1151, which is predicted to have the composition of Hex4HexNAc2-PA. On the other hand, the glycan with the composition Hex5HexNAc2-PA ([M+H]+ ion of m/z 1313) was now absent (Fig. 1B). Comparable data regarding mannosidase-resistance of some glycans of the mutant strain was obtained by RP-HPLC (Fig. 1C and 1D), which is well-established to also separate isomers of many N-glycans. Compatible with the overall mass spectrometric profiles, the major HPLC peak of 7.2 g.u. containing Man3GlcNAc2-PA and Man5GlcNAc2-PA was replaced, after mannosidase digestion, by a new peak of 6.5 g.u. containing a glycan of m/z 665 corresponding to Man1GlcNAc2-PA; the retention time for this “trimmed-down” product of the oligomannosidic glycans is in keeping with literature values (31). More significantly, a set of glycans eluting between 4.0 and 5.5 glucose units (g.u.) were seemingly resistant to this treatment because their elution position did not shift after incubation with the mannosidase. MALDI-TOF MS of the relevant fractions indicated that they contained glycans with the composition of Hex3–4HexNAc2Fuc0–1Me0–2-PA ([M+H]+ ions of m/z 989 to m/z 1311). As expected from having deletions in all three known core fucosyltransferase genes, MS/MS of these glycans strongly suggested that core fucosylation (which would be shown by a fragment of m/z 446, i.e. Fuc1GlcNAc1-PA) was absent, whereas in general MS/MS spectra of the various glycans primarily confirmed composition (Fig. 2). Treatment of the glycopeptides remaining after PNGase F digestion with PNGase A merely resulted in release of residual glycans of the same m/z as those in the PNGase F pool and that also displayed no sign of core fucosylation as judged by MS/MS (data not shown).

Fig. 2.
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Fig. 2.

MALDI-TOF MS/MS spectra of pyridylamino-labeled N-glycans carrying bisecting β-galactose. N-glycans from the fut-1;fut-6;fut-8 triple mutant separated by RP-HPLC (4.2–5.5 g.u.) were subject to MS/MS. Nonfucosylated forms (A and B), fucosylated forms (C and D) and methyl-fucosylated forms (E–H) displayed sequential “loss” of hexose and (methyl-) fucose residues or methylhexose (the latter only in G). For all (methyl-) fucosylated structures, the absence of key ions such as HexNAc1Fuc1-PA (m/z 446) and Hex1HexNAc1Fuc1-PA (m/z 608) indicated that there is no core fucosylation in this triple knockout. Other key ions such as Hex2HexNAc2Me-PA (m/z 841) and Hex2HexNAc2Fuc1Me-PA (m/z 987) are indicative of the methyl group.

Structure of Mannosidase-resistant Paucihexosidic N-glycans

As a number of glycans in the triple mutant were resistant to α-mannosidase digestion regardless of compositions normally associated with paucimannosidic N-glycans present in many invertebrates, we sought to examine these more closely, including the fucosylated forms. First, a fraction eluting at 4.2 g.u. (see Fig. 1C) containing glycans of Hex3–4HexNAc2-PA (m/z 989 and 1151) and one of Hex4HexNAc2Fuc1-PA (m/z 1297) was analyzed. All three glycans were confirmed to be mannosidase-resistant, but the two glycans lacking fucose lost one hexose upon treatment with recombinant Aspergillus β-galactosidase (Fig. 3). Subsequent incubation with α1,2/3-specific mannosidase resulted in primary products of Hex1–2HexNAc2-PA. Considering previous GC-MS data that 3,4,6-trisubstituted mannose exists in C. elegans (27), whereas on the other hand the relevant GlcNAc-TIII has no homolog in nematodes and that bisecting GlcNAc is known to cause mannosidase-resistance of the two core α-mannose residues (32, 33), we postulated that a bisecting position (C-4) for β-galactose prevented removal of the nonsubstituted α-mannose residues. A C-2 modification of the core β-mannose is unlikely as glycans carrying a substitution on the C-2 position, such as β1,2-xylose, display a different mannosidase sensitivity to the glycans studied here (34).

Fig. 3.
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Fig. 3.

Sequential digestions of the RP-HPLC fraction eluting at 4.2 glucose units. Three co-eluting structures (Hex3–4HexNAc2Fuc0–1-PA, A) from the fut-1;fut-6;fut-8 (F168) triple mutant were resistant to jack bean α-mannosidase digestion (B) but sensitive to recombinant β-galactosidase (C) except for the Hex4HexNAc2Fuc1-PA structure (m/z 1297.5). The β-galactosidase products with m/z 827.4 and 989.5 were further digested by α1,2/3-mannosidase (D) and yielded respective sodiated products with m/z 687.3 and 849.4. Digestion of the m/z 1297 species is shown in Fig. 4. Asterisks indicate nonglycan contaminants.

In contrast, the fucose-containing glycan of m/z 1297 (m/z 1319 as [M+Na]+) in the 4.2 g.u. fraction was resistant to both α-mannosidase and β-galactosidase. A closer examination of the MS/MS spectrum shows that the fucose is two hexose residues distant from the distal GlcNAc as shown by the fragment ions at m/z 973 (or 987 in case of the methylated structures; Fig. 2). To explore the structure of Hex4HexNAc2Fuc1-PA more thoroughly, we performed selected treatments on a 2D-HPLC fraction containing solely this glycan. Initially, we tried a number of fucosidase treatments, but no removal of fucose was observed (data not shown). However, the glycan is partially sensitive to hydrofluoric acid and the defucosylated portion was sensitive to β-galactosidase, but resistant to α-galactosidase (Fig. 4). Subsequently, one mannose could be released from the β-galactosidase-sensitive portion when incubating the glycan with α1,2/3-mannosidase; because of the initial galactosidase resistance, the pattern of digestion after hydrofluoric acid treatment suggests that the glycan has a fucose cap on the β-galactose of a structure the same as the aforementioned Hex4HexNAc2-PA. Regarding the type of linkage, we found in control experiments that hydrofluoric acid can remove α1,2-fucose residues, but not so efficiently as it cleaves α1,3-fucose (supplemental Fig. S3); however, longer incubations resulted in artifacts. Therefore, considering also previous GC-MS data showing the presence of 2-substituted galactose (27) and the multiplicity of α1,2-fucosyltransferase homologs (35) in C. elegans, we propose that the Hex4HexNAc2Fuc1-PA contains an α1,2-fucose linked to the bisecting β-galactose. Further evidence came from NMR and LC-MSn experiments (see below).

Fig. 4.
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Fig. 4.

Sequential digestions of a two-dimensional HPLC fraction containing a Hex4HexNAc2Fuc1-PA variant. A, 2D-HPLC purified form of the α-mannosidase-resistant m/z 1297 glycan shown in Fig. 3 (obtained by NP-HPLC followed by RP-HPLC) was incubated with green coffee bean α-galactosidase, but no hexose residue was removed (B); hydrofluoric acid treatment converted ca. 40% of the structure into a defucosylated form with m/z 1151.5 (C). Afterward, the product was treated with either α-galactosidase (D) or β-galactosidase; only β-galactosidase resulted in a loss of a hexose residue (E). Finally, α1,2/3-mannosidase further trimmed the β-galactosidase product and yield Hex2HexNAc2-PA with m/z 827.3 (F).

Methylated Fucose as a Component of Paucihexosidic N-glycans

We also investigated the nature of N-glycans putatively containing methyl groups. The fraction of 5.2 g.u (see Fig. 1C) contained, other than traces of Man7–9GlcNAc2, a glycan with [M+H]+ ions at m/z 1149, which could correspond to Hex3HexNAc2Fuc1Me1-PA. MS/MS showed a fragment ion at m/z 987, which suggested the presence of methylfucose (Fig. 2F). Thus considering the aforementioned experiments, we treated this fraction with hydrofluoric acid, which resulted in the expected loss of 160 Da (i.e. methylfucose) to yield a glycan of m/z 989 (Hex3HexNAc2-PA; Fig. 5). This glycan was resistant to α-mannosidase (as judged by the ratio of m/z 989 to m/z 1149 remaining constant; the presence of m/z 665 and 827 products was assigned to digestion of the oligomannosidic glycans also present in this fraction). The hydrofluoric acid-treated fraction was then incubated sequentially with β-galactosidase and α1,2/3-mannosidase, which resulted in products of m/z 827 and 665. The portion of Hex3HexNAc2Fuc1Me1-PA, which had not lost the methylfucose residue was, however, resistant to both hexosidases.

Fig. 5.
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Fig. 5.

Structural analysis of a novel methylated Hex3HexNAc2Fuc1Me-PA N-glycan. This α-mannosidase resistant structure was separated by RP-HPLC, eluting at 5.2 glucose unit (A). HF treatment was first applied to the fraction and resulted in a partial conversion to Hex3HexNAc2-PA (m/z 989.4) (B). This product was incubated with either jack bean α-mannosidase or β-galactosidase, but only the latter resulted in loss of a hexose residue (C and D). Further digestion of the β-galactosidase product using α1,2/3-mannosidase resulted in removal of the “lower arm” mannose and formation of a final product with m/z 665.3.

Among other fractions, we examined the α-galactosidase and hydrofluoric acid sensitivity of glycans of m/z 1135 (Hex3HexNAc2Fuc1-PA) and 1163 (Hex3HexNAc2Fuc1Me2-PA) as well as two isomers of m/z 1311 (Hex4HexNAc2Fuc1Me1-PA) separated in the two fractions of 4.8 and 5.5 g.u.; the earlier fraction also contains an m/z 989 structure, which is a putative reducing-terminal epimer of standard Man3GlcNAc2 (epimerization of up to 10% of the reducing-terminal GlcNAc to ManNAc has been previously reported (36)). Whereas the m/z 1311 glycan (Hex4HexNAc2Fuc1Me1-PA) of 5.5 g.u. was sensitive to α-galactosidase, the isobaric structure eluting at 4.8 g.u. was resistant to this treatment (Fig. 6B and 6E). On the other hand, all four fucosylated structures in the two fractions were sensitive to hydrofluoric acid as indicated by the incomplete loss of 146 or 160 Da (fucose or methylfucose; Fig. 6C and 6F). Notably, the Hex3HexNAc2Fuc1Me2-PA glycan (m/z 1163) appears to be modified with methyl groups on both the fucose and the α1,3-mannose residues. This is compatible with the MS/MS spectrum indicating an m/z 841 fragment (putatively Man2GlcNAc2Me1-PA; Fig. 2G). Due to their similar fragmentation and digestion properties, it is concluded that the basic structures of the Hex3–4HexNAc2Fuc1 and Hex3–4HexNAc2Fuc1Me1 glycans are the same. Indeed, the fragmentation patterns of the isobaric hydrofluoric acid digestion products of m/z 1297 and m/z 1311 were highly similar suggestive of a common basic structure (supplemental Fig. S4C and S4D).

Fig. 6.
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Fig. 6.

Structural characterization of two isomeric N-glycans by α-galactosidase digestion and HF treatment. Two different RP-HPLC fractions containing Hex4HexNAc2Fuc1Me1-PA with m/z 1311.6 (4.8 g.u. and 5.5 g.u.; A and D) were treated with α-galactosidase (B and E); complete loss of one hexose was observed only for the late eluting fraction, resulting in a product of m/z 1149.5. Hydrofluoric acid treatment resulted in partial removal of either fucose or methylfucose (FMe) residues from Hex3–4HexNAc2Fuc1Me0–2-PA (C and F); thus, the fucose is not α1,3-linked (which would be fully removed by this treatment) and not α1,6-linked (which would be resistant), but the degree of release is compatible with the proposed Fucα1,2 linkage.

NMR Analysis Indicative of Bisecting Galactose

As the data from chemical and enzymatic digestions were indicative for an unusual location of galactose residues, which blocked the action of α-mannosidase, we sought further confirmation for our model of bisecting galactose being a feature of the mannosidase-resistant glycans of C. elegans. Because of the relatively low amount of glycan material available, we considered use of a nondestructive method of glycan analysis and so turned to NMR to yield further insights. To aid definition of the novel features of the putatively bisected glycans from the C. elegans, 1H NMR and homonuclear 2D NMR spectra of “classical” Man3GlcNAc2-PA and a pool of mannosidase-resistant paucihexosidic glycans from the triple mutant were compared (Fig. 7). Data on related N-glycan structures were used for comparative analysis of the interglycosidic bonds (37⇓–39).

Fig. 7.
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Fig. 7.

1H NMR and TOCSY of a standard trimannosyl N-glycan and the pool of bisected N-glycans. Separated signals in the 1H NMR spectra of the 7.2 g.u. fraction containing Man3GlcNAc2-PA (A) and of the 4.0–5.5 g.u. fractions (B, lacking the glycan of 4.8 g.u.) from the triple mutant are indicated according to the numbering shown on the structures. In the TOCSY spectra, separated cross peaks of the involved nuclei in according spin systems are marked. The chemical shifts of both compounds are listed in Table II.

For the pool of putatively bisected glycans, the structural variants led to broadening or doubling of some proton NMR signals; certainly, the modification of the core β-d-Manp (residue 3; see Fig. 7), as compared with standard Man3GlcNAc2-PA, can be surmised by the alterations in a number of the chemical shifts for this residue (e.g. 3.77, 3.80 and 3.85 ppm rather than 3.82, 3.69 and 3.77 ppm for the H-3, H-4 and H-6b protons; see Table II). The strong shift for H-4 can be taken as confirmation for a bisecting residue on C-4 of the core β-mannose and so shares a trend observed with data on a bisected mammalian glycan (40), whereas the data are not compatible with a C-2 modification. The presence of β-d-Galp (residue 4′) and some α-d-Galp (residue C) linked to mannose as well as some α-l-Fucp linked to galactose is shown by relevant chemical shifts typical for such residues (4.6–4.3 (38, 40), 5.5–5.1 (41), and 5.3–5.2 ppm (42) respectively for the anomeric H-1); for the latter, it is noteworthy that older NMR data was interpreted as showing α1,2-fucosylation of mannose (43), but our data overall indicate the presence of a bisecting Fucα1,2Gal motif. Indeed, the proton signals of the β-d-Galp 4′ show the most pronounced shift variations, which may be caused by the fucose or methylfucose bound in substoichiometric amounts to this unit, whereas the α-galactose is assumed to be linked to position 2 of the α-d-Manp (residue 4). In conclusion, the NMR data support the presence of 3,4,6-trisubstituted bisected core β-mannose as well as the presence of galactose and fucose residues.

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Table II 1H NMR chemical shifts (parts per million) of the mannosidase-resistant N-glycan pool. The nomenclature for the numbering of the saccharide units is according to that of Halbeek et al. (37). Structures and spectra are shown in Figure 7; numbers in brackets refer to the corresponding chemical shifts for the Man3GlcNAc2 structure. Significant differences in the chemical shifts (0.08–0.12 ppm) can be observed for H-3, H-4, and H-6b of the β-mannose 3 and the H-1 and H-2 of the α1,3-linked mannose 4. nd not determined
LC-MS Analyses Confirm the Fucα1,2Galβ1,4 Modification

As a final analytical method, purified N-glycans were also applied to LC-MS/MS in negative ion mode (Fig. 8). All MS/MS spectra were dominated by an ion resulting from 2,4A cross-ring cleavage of penultimate GlcNAc, which are diagnostic for N-glycans. In the case of the singly-charged precursors, we observed Y3 ions indicative of loss of one antennal hexose ([M-H]− ions such as m/z 825, 985 or 987 corresponding to the positive mode MALDI-TOF MS/MS [M+H]+ fragment ions at m/z 827, 987 or 989 shown in Fig. 2) and B ions resulting from loss of GlcNAc-PA (e.g. m/z 688 in Fig. 8A and m/z 850 in Fig. 8B) together with 2,4A cross-ring cleavage of the penultimate GlcNAc suggested that the reducing-terminal GlcNAc-PA was not modified. Lower intensity MS2 ions, though, were more informative in terms of the linkage of the bisecting galactose and of the fucose residues.

Fig. 8.
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Fig. 8.

Detection of fucose-substituted and nonsubstituted bisecting Gal by LC-MS/MS. LC-MS/MS spectra of N-glycans from the 4.0–5.5 g.u. fut-1;fut-6;fut-8 pool: A, a paucihexosidic N-glycan containing a bisecting galactose (Hex3HexNAc2-PA, [M-H]− of m/z 987), B, a Man3GlcNAc2 N-glycan with a bisecting Gal (Hex4HexNAc2-PA, [M-H]− of m/z 1150), C, an N-glycan in which a methylated Fuc is linked to the bisecting Gal (Hex3HexNAc2Fuc1Me1-PA, [M-H]− of m/z 1147), (D) an bisected Man3GlcNAc2 N-glycan with a methylated fucose substitution of the bisecting Gal (Hex4HexNAc2Fuc1Me1-PA, [M-H] of m/z 1310) and E, a bisected Man3GlcNAc2 N-glycan in which Fuc is linked to the bisecting Gal (Hex4HexNAc2Fuc1-PA, [M-2H]2− of m/z 647). Schematic representations explaining the Domon and Costello nomenclature of indicative glycosidic or cross-ring cleavages are shown in the panels on the right.

To confirm the linkage of bisecting Gal, a glycan with composition of Hex3HexNAc2-PA ([M-H]− ions at m/z 987) was analyzed (Fig. 8A). Fragment ions at m/z 627 (Z3α/Z3β) suggest bi-substitution by hexose residues of the β-Man. In comparison to a typical Man3GlcNAc2 N-glycan, “D ions” (m/z 323 and 305, mass of 6-antenna plus β-Man (44)) were absent indicating that one of the hexoses should be α1,3-linked Man. This was further confirmed by the presence of Z3α/Z3β-CH2O ions at m/z 597, which indicated a C-3 and C-4 bi-substitution of the β-Man (Fig. 8A, right panel); this is similar to the Zi/Zi-CH2O fragmentation pattern of a Lewis type structure (45). In addition, fragment ions at m/z 443 were concluded to result from 0,2A cleavage of β-Man, which would be absent if the C-2 of the core β-mannose is substituted; together with the β-galactosidase sensitivity and NMR data, the MS/MS results are compatible with this glycan carrying a bisecting β1,4-linked Gal. Furthermore, the MS/MS spectrum of a glycan with a 6-antenna (composition of Hex4HexNAc2-PA, [M-H]− ions at m/z 1150; Fig. 8B) shows triple Z ions (Z3α/Z3β/Z3γ) at m/z 609 indicative that the β-Man is tri-substituted with two Man and one Gal. D ions at m/z 485 and 323 (with or without bisecting Gal) were also consistent with the presence of tri-substituted β-Man. Thus, this structure was assigned as Man3GlcNAc2 modified with a bisecting Gal.

To confirm that fucose could modify the bisecting Gal, a glycan with composition of Hex3HexNAc2Fuc1Me1-PA was analyzed (m/z 1147; Fig. 8C). The 2,4A cleavage of the penultimate GlcNAc (ion at m/z 705) and B4 ions at m/z 848 suggested that the fucose was on the nonreducing end of the N-glycan; the Y3β ions at m/z 985 and B2α/C2α (m/z 321/339) are compatible with the presence of one terminal hexose and one terminal Me-Fuc-Hex motif (322 Da). The Z3α/Z3β-CH2O ions at m/z 597 indicate, as discussed for Fig. 8A, the C3 and C4 bi-substitution of the β-Man (i.e. by α1,3-linked mannose and a methylfucose-capped β1,4-linked galactose). The 0,2A cleavage of β-Man (0,2A3α ions at m/z 603) and dominant fragment ions at m/z 423 (0,2A3α-H2O/Y3β) suggest that the methylated Fuc is linked to the bisecting Gal. A further variant is exemplified by a structure with an additional α1,6-linked Man (Hex4HexNAc2Fuc1Me1-PA, m/z 1310; Fig. 8D) displaying an 0,2A3-H2O ion at m/z 747 and an 0,2A3/Y3γ ion at m/z 603. As for the linkage between Fuc and the bisecting Gal, MS/MS of a doubly charged ion (Hex4HexNAc2Fuc1Me1-PA, [M-2H]2− ions at m/z 647; Fig. 8E) resulted in fragment ions at m/z 205 (which could be because of either 0,4AGal, 1,3AGal, or 2,4AGal cleavage) but not at m/z 265 (0,2AGal); this is suggestive of a 1,3A cleavage of bisecting Gal, which would correlate with Fuc substituting C-2 of the bisecting Gal. Fragmentation ions at both m/z 205 and m/z 247 (Fuc1Gal1-C2H4O2) are diagnostic for an H type structure (45). Thus, consistent with the partial sensitivity toward hydrofluoric acid, we conclude that the bisecting Gal is modified with α1,2-linked Fuc.

Galactose as a Component of Paucihexosidic N-glycans in Wild-type C. elegans

Considering the results with the triple mutant, we sought for glycans of the same RP-HPLC retention time (4.0–5.5 g.u.) as the mannosidase-resistant forms in other strains. As part of an earlier study, extensive 2D-HPLC fractionation (normal phase followed by reversed phase) had been performed on the pmk-1 strain, which is defective in a p38 MAP kinase homolog and is hypersensitive to some fungal galectins (46), but in glycosylation terms is “pseudowild-type”. We found one 2D-HPLC fraction containing a glycan with the composition Hex5HexNAc2-PA (m/z 1313), which did not co-elute with standard Man5GlcNAc2-PA (7.2 g.u.) on a standard RP-HPLC column, but instead eluted at 4.6 g.u., i.e. within the elution range for the mannosidase-resistant glycans from the triple mutant. The MS/MS spectrum of this structure was also different as compared with that of the standard Man5GlcNAc2 (i.e. the dominance of the m/z 827 fragment in the latter was not apparent; supplemental Fig. S4E and S4F).

Considering the hexosidases available and data indicative for the presence of galactose on C. elegans N-glycans, we attempted both α-mannosidase and α- or β-galactosidase treatments of this glycan. Indeed, the Hex5HexNAc2-PA from the pmk-1 strain was resistant to α-mannosidase, but sensitive to the recombinant fungal β1,4-galactosidase (Fig. 9). After this treatment, a further hexose could then be removed with jack bean α-mannosidase, but further digestion was still not possible. We observed that α-galactosidase could remove one hexose and that a final hexose is cleaved by an α1,2/3-specific mannosidase. Thus, the Hex5HexNAc2-PA glycan was concluded to have the composition Gal2Man3GlcNAc2; the digestion data is compatible with a bisecting position for β-galactose and α-galactosylation of the α1,3-mannose residue. Furthermore, in the wild-type N2 embryo N-glycome, an Hex4HexNAc2 glycan (4.2 g.u.) displayed a similar α-mannosidase resistance, but β-galactosidase sensitivity, as for the co-eluting m/z 1151 glycan from the triple mutant (data not shown).

Fig. 9.
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Fig. 9.

Sequential enzymatic digestions of an Hex5HexNAc2 isoform. A structure (m/z 1313.5 as [M+H]+) produced by the pmk-1 strain was isolated on HPLC via a two-dimensional approach (NP-HPLC followed by RP-HPLC); this glycan elutes unusually early (4.6 g.u.) on the reversed phase column (A). Jack bean α-mannosidase digestion resulted in no loss of mannose (B), whereas β-galactosidase removed one hexose residue (C). Mannosidase digestion of the β-galactosidase product resulted in additional loss of one hexose (D) yielding a glycan (m/z 989.3), which was further trimmed down to Hex2HexNAc2-PA with green coffee bean α-galactosidase (E). Finally, the Hex2HexNAc2-PA was digested by α1,2/3-mannosidase (F) to yield the core trisaccharide structure (m/z 665.2).

DISCUSSION

Studies over the past 15 years have revealed a huge diversity in the N-glycome of the “simple” multicellular nematode Caenorhabditis elegans (3, 14). Some of the features are shared with other parasitic and nonparasitic nematode species (18⇓–20, 47, 48). Examples include trifucosylation of the core chitobiose (including fucosylation of the distal core GlcNAc), galactosylation of core fucose residues and the antennal modification with phosphorylcholine. On the other hand, although antennal α1,3-fucosylation (Lewis and Lewis-type epitopes) is known from some parasitic nematode species (49), this feature is lacking from C. elegans. However, a fourth fucose residue is apparent in a subset of N-glycans of C. elegans (13, 15, 26⇓⇓⇓–30) and structural models have suggested that this is attached either directly or indirectly (via galactose) to α-mannose residues of the trimannosylchitobiosyl core (15, 26, 50, 51). However, tetrafucosylation of a trimannosylchitobiosyl core is a structural proposition contradicting not only the known specificity of the enzyme (the FUT-6 α1,3-fucosyltransferase), which modifies the distal core GlcNAc (17), but also GC-MS data indicating that a portion of the core β-mannose residues is 3,4,6-trisubstituted and that 2-substituted galactose is also present in wild-type C. elegans (27).

Mutant C. elegans strains have been highly valuable in verifying glycobiosynthetic pathways in this organism: in our laboratory we have analyzed single fucosyltransferase mutants (13), double fucosyltransferase mutants (17), a Golgi mannosidase II mutant (52), and hexosaminidase single and double mutants (18, 53). Also, the N-glycomes of worm strains with other glycosyltransferase, fucosylation and methylation defects have been analyzed (28, 51, 54, 55). Very often one observes not just the loss of N-glycans but the apparent gain of others.

In order to simplify the N-glycome, we constructed a triple mutant featuring deletions in the genes known to encode fucosyltransferases modifying the core chitobiose unit of C. elegans N-glycans. Indeed, as predicted the N-glycans of the triple mutant contained maximally one fucose residue. Focusing on a range of HPLC-enriched N-glycans, which possess up to five hexose residues, but which were α-mannosidase resistant (hence “paucihexosidic”), we reveal, using chemical and enzymatic treatments in conjunction with off-line MALDI-TOF MS and on-line LC-MS as well as NMR, that the fucose residue on such glycans is α1,2-linked via a bisecting β1,4-galactose to the core β-mannose. Thereby, some of these glycans either lack an α1,6-mannose or carry an α-galactose on the α1,3-mannose; variants of these glycans are also methylated.

Preliminary data, as well as the aforementioned “old” GC-MS data (27), lead us to believe that the antennal fucose is also significantly present on core fucosylated glycans in the wild-type. Furthermore, we have previously shown that the “GalFuc” epitope can also carry a fucose residue (18), which yields a fifth attachment point for fucose on C. elegans N-glycans; nevertheless, only maximally four fucose residues have ever been detected on oligosaccharides of this organism. The difficulty in digesting these bisected N-glycans with glycosidases may be explained by predicted 3D-conformations suggesting that the bisecting modification “folds back” onto the core of the glycan (supplemental Fig. S5).

Thus, after some fifteen years of work on C. elegans glycans by various laboratories, we offer a reinterpretation of a number of previously-published studies regarding the N-glycome of this organism, which may have also repercussions for the understanding of data regarding glycan-binding nematotoxic proteins. As a double hexosaminidase worm mutant, which apparently lacks bisecting galactose (18), is completely or partially resistant to tectonin and MpL (51, 56), it would be attractive to propose that the binding sites on N-glycan antennae for these nematotoxins are indeed the methylated or nonmethylated forms of the α1,2-fucose attached to the bisecting galactose. Armed with the exact structure of C. elegans N-glycans, the binding specificities of such proteins can be reassessed; furthermore, such glycomic knowledge is a prerequisite to fine-tune approaches to understand the biological significance of the seemingly endless variation of the C. elegans glycome in order to make this otherwise well-understood organism into a system truly suitable for examination of glycobiological paradigms.

Acknowledgments

The authors would also like to thank Maria Strobl for purification of N-glycans.

Footnotes

  • Author contributions: S.Y., V.J., I.B.W., and K.P. designed research; S.Y., L.B., C.J., and A.T. performed research; M.D., N.K., and V.J. contributed new reagents or analytic tools; S.Y., L.B., C.J., I.B.W., and K.P. analyzed data; I.B.W. and K.P. wrote the paper; S.Y. prepared figures; K.P. MS interpretation.

  • ↵* This work was funded in part by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (FWF) (grants P21946 to K.P. and P23922 to I.B.H.W.); A.T. was recipient of an EMBO Short Term Fellowship (ASTF-377.00-2008), NGK was supported by Swedish Research Council (2013-5895 and 2010-5322) and CJ was supported by the Knut and Alice Wallenberg Foundation. The fut-1, fut-6 and fut-8 strains used were provided by the Caenorhabditis Genetics Centre (CGC), which is funded by the NIH Office of Research Infrastructure Programmes (P40 OD010440). The LTQ mass spectrometer was obtained by a grant from the Swedish Research Council (342-2004-4434).

  • ↵Embedded Image This article contains supplemental Figs. S1 to S5.

  • ↵1 The abbreviations used are:

    MALDI-TOF
    matrix-assisted laser desorption-ionisation time-of-flight
    NMR
    nuclear magnetic resonance
    NP-HPLC
    normal-phase HPLC
    PA
    pyridylamino
    PC
    phosphorylcholine
    RP-HPLC
    reversed-phase HPLC
    TOCSY
    total correlation spectroscopy.

  • Received March 10, 2015.
  • Revision received April 22, 2015.
  • © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

REFERENCES

  1. 1.↵
    1. Sommer R. J.,
    2. Streit A.
    (2011) Comparative genetics and genomics of nematodes: genome structure, development, and lifestyle. Annu. Rev. Genet. 45, 1–20
    OpenUrlCrossRefPubMed
  2. 2.↵
    1. Blaxter M.
    (2011) Nematodes: the worm and its relatives. PLoS Biol. 9, e1001050
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. Paschinger K.,
    2. Gutternigg M.,
    3. Rendić D.,
    4. Wilson I. B. H.
    (2008) The N-glycosylation pattern of Caenorhabditis elegans. Carbohydr. Res. 343, 2041–2049
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Schachter H.
    (2009) Paucimannose N-glycans in Caenorhabditis elegans and Drosophila melanogaster. Carbohydr. Res. 344, 1391–1396
    OpenUrlCrossRefPubMed
  5. 5.↵
    1. Yazdanbakhsh M.,
    2. Kremsner P. G.,
    3. van Ree R.
    (2002) Allergy, parasites, and the hygiene hypothesis. Science 296, 490–494
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Maizels R. M.
    (2005) Infections and allergy - helminths, hygiene and host immune regulation. Curr. Opin. Immunol. 17, 656–661
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. Rook G. A.
    (2009) Review series on helminths, immune modulation and the hygiene hypothesis: the broader implications of the hygiene hypothesis. Immunology 126, 3–11
    OpenUrlCrossRefPubMed
  8. 8.↵
    1. Summers R. W.,
    2. Elliott D. E.,
    3. Urban J. F. Jr..,
    4. Thompson R.,
    5. Weinstock J. V.
    (2005) Trichuris suis therapy in Crohn's disease. Gut 54, 87–90
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Ebner F.,
    2. Hepworth M. R.,
    3. Rausch S.,
    4. Janek K.,
    5. Niewienda A.,
    6. Kuhl A.,
    7. Henklein P.,
    8. Lucius R.,
    9. Hamelmann E.,
    10. Hartmann S.
    (2014) Therapeutic potential of larval excretory/secretory proteins of the pig whipworm Trichuris suis in allergic disease. Allergy 69, 1489–1497
    OpenUrlCrossRefPubMed
  10. 10.↵
    1. Tawill S.,
    2. Le Goff L.,
    3. Ali F.,
    4. Blaxter M.,
    5. Allen J. E.
    (2004) Both free-living and parasitic nematodes induce a characteristic Th2 response that is dependent on the presence of intact glycans. Infect. Immun. 72, 398–407
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. Harnett W.
    (2014) Secretory products of helminth parasites as immunomodulators. Mol. Biochem. Parasitol 195, 130–136
    OpenUrlCrossRefPubMed
  12. 12.↵
    1. Newton S. E.,
    2. Meeusen E. N.
    (2003) Progress and new technologies for developing vaccines against gastrointestinal nematode parasites of sheep. Parasite Immunol 25, 283–296
    OpenUrlCrossRefPubMed
  13. 13.↵
    1. Paschinger K.,
    2. Rendić D.,
    3. Lochnit G.,
    4. Jantsch V.,
    5. Wilson I. B. H.
    (2004) Molecular basis of anti-horseradish peroxidase staining in Caenorhabditis elegans. J. Biol. Chem. 279, 49588–49598
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    1. Haslam S. M.,
    2. Dell A.
    (2003) Hallmarks of Caenorhabditis elegans N-glycosylation: complexity and controversy. Biochimie 85, 25–32
    OpenUrlCrossRefPubMed
  15. 15.↵
    1. Hanneman A. J.,
    2. Rosa J. C.,
    3. Ashline D.,
    4. Reinhold V.
    (2006) Isomer and glycomer complexities of core GlcNAcs in Caenorhabditis elegans. Glycobiology 16, 874–890
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    1. Paschinger K.,
    2. Staudacher E.,
    3. Stemmer U.,
    4. Fabini G.,
    5. Wilson I. B. H.
    (2005) Fucosyltransferase substrate specificity and the order of fucosylation in invertebrates. Glycobiology 15, 463–474
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    1. Yan S.,
    2. Serna S.,
    3. Reichardt N. C.,
    4. Paschinger K.,
    5. Wilson I. B. H.
    (2013) Array-assisted Characterization of a Fucosyltransferase Required for the Biosynthesis of Complex Core Modifications of Nematode N-Glycans. J. Biol. Chem. 288, 21015–21028
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Yan S.,
    2. Bleuler-Martinez S.,
    3. Plaza Gutierrez D. F.,
    4. Künzler M.,
    5. Aebi M.,
    6. Joachim A.,
    7. Razzazi-Fazeli E.,
    8. Jantsch V.,
    9. Geyer R.,
    10. Wilson I. B. H.,
    11. Paschinger K.
    (2012) Galactosylated fucose epitopes in nematodes: increased expression in a Caenorhabditis mutant associated with altered lectin sensitivity and occurrence in parasitic species. J. Biol. Chem. 287, 28276–28290
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Yan S.,
    2. Wilson I. B. H.,
    3. Paschinger K.
    (2015) Comparison of RP-HPLC modes to analyse the N-glycome of the free-living nematode Pristionchus pacificus Electrophoresis 36, 1314–1329
    OpenUrlCrossRefPubMed
  20. 20.↵
    1. Paschinger K.,
    2. Wilson I. B. H.
    (2015) Two types of galactosylated fucose motifs are present on N-glycans of Haemonchus contortus. Glycobiology, 25, 585–590
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    1. Brenner S.
    (1974) The genetics of Caenorhabditis elegans. Genetics 77, 71–94
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    1. Fay D.
    (2006) Genetic mapping and manipulation: chapter 7–Making compound mutants. WormBook 1–4
  23. 23.↵
    1. Paschinger K.,
    2. Hykollari A.,
    3. Razzazi-Fazeli E.,
    4. Greenwell P.,
    5. Leitsch D.,
    6. Walochnik J.,
    7. Wilson I. B. H.
    (2012) The N-glycans of Trichomonas vaginalis contain variable core and antennal modifications. Glycobiology 22, 300–313
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    1. Dragosits M.,
    2. Pflugl S.,
    3. Kurz S.,
    4. Razzazi-Fazeli E.,
    5. Wilson I. B. H.,
    6. Rendić D.
    (2014) Recombinant Aspergillus β-galactosidases as a robust glycomic and biotechnological tool. Appl. Microbiol. Biotechnol. 98, 3553–3567
    OpenUrlCrossRef
  25. 25.↵
    1. Domon B.,
    2. Costello C. E.
    (1988) A Systematic Nomenclature for Carbohydrate Fragmentations in Fab-Ms Ms Spectra of Glycoconjugates. Glycoconjugate J. 5, 397–409
    OpenUrlCrossRef
  26. 26.↵
    1. Altmann F.,
    2. Fabini G.,
    3. Ahorn H.,
    4. Wilson I. B. H.
    (2001) Genetic model organisms in the study of N-glycans. Biochimie 83, 703–712
    OpenUrlCrossRefPubMed
  27. 27.↵
    1. Haslam S. M.,
    2. Gems D.,
    3. Morris H. R.,
    4. Dell A.
    (2002) The glycomes of Caenorhabditis elegans and other model organisms. Biochem. Soc. Symp. 69, 117–134
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Zhu S.,
    2. Hanneman A.,
    3. Reinhold V.,
    4. Spence A.,
    5. Schachter H.
    (2004) Caenorhabditis elegans triple null mutant lacking UDP-N-acetyl-D-glucosamine:α-3-D-mannoside β1,2 N-acetylglucosaminyltransferase I. Biochem. J. 382, 995–1001
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    1. Cipollo J. F.,
    2. Awad A. M.,
    3. Costello C. E.,
    4. Hirschberg C. B.
    (2004) srf-3, a mutant of Caenorhabditis elegans, resistant to bacterial infection and to biofilm binding, is deficient in glycoconjugates. J. Biol. Chem. 279, 52893–52903
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    1. Takeuchi T.,
    2. Hayama K.,
    3. Hirabayashi J.,
    4. Kasai K.
    (2008) Caenorhabditis elegans N-glycans containing a Gal-Fuc disaccharide unit linked to the innermost GlcNAc residue are recognized by C. elegans galectin LEC-6. Glycobiology 18, 882–890
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Tomiya N.,
    2. Lee Y. C.,
    3. Yoshida T.,
    4. Wada Y.,
    5. Awaya J.,
    6. Kurono M.,
    7. Takahashi N.
    (1991) Calculated two-dimensional sugar map of pyridylaminated oligosaccharides: Elucidation of the jack bean α-mannosidase digestion pathway of Man9GlcNAc2. Anal. Biochem. 193, 90–100
    OpenUrlCrossRefPubMed
  32. 32.↵
    1. Chen Y. J.,
    2. Wing D. R.,
    3. Guile G. R.,
    4. Dwek R. A.,
    5. Harvey D. J.,
    6. Zamze S.
    (1998) Neutral N-glycans in adult rat brain tissue - Complete characterisation reveals fucosylated hybrid and complex structures. Eur. J. Biochem. 251, 691–703
    OpenUrlPubMed
  33. 33.↵
    1. Hykollari A.,
    2. Balog C. I.,
    3. Rendic D.,
    4. Braulke T.,
    5. Wilson I. B. H.,
    6. Paschinger K.
    (2013) Mass spectrometric analysis of neutral and anionic N-glycans from a Dictyostelium discoideum model for human congenital disorder of glycosylation CDG IL. J. Proteome Res. 12, 1173–1187
    OpenUrlCrossRefPubMed
  34. 34.↵
    1. Wilson I. B. H.,
    2. Altmann F.
    (1998) Structural analysis of N-glycans from allergenic grass, ragweed and tree pollens: Core α1,3-linked fucose and xylose present in all pollens examined. Glycoconjugate J. 15, 1055–1070
    OpenUrlCrossRefPubMed
  35. 35.↵
    1. Zheng Q.,
    2. Van Die I.,
    3. Cummings R. D.
    (2008) A novel α1,2-fucosyltransferase (CE2FT-2) in Caenorhabditis elegans generates H-type 3 glycan structures. Glycobiology 18, 290–302
    OpenUrlAbstract/FREE Full Text
  36. 36.↵
    1. Hase S.,
    2. Ibuki T.,
    3. Ikenaka T.
    (1984) Reexamination of the pyridylamination used for fluorescence labelling of oligosaccharides and its application to glycoproteins. J. Biochem. (Tokyo) 95, 197–203
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    1. Van Halbeek H.,
    2. Dorland L.,
    3. Veldink G. A.,
    4. Vliegenthart J. F.,
    5. Strecker G.,
    6. Michalski J. C.,
    7. Montreuil J.,
    8. Hull W. E.
    (1980) A 500 MHz 1H NMR study of urinary oligosaccharides from patients with mannosidosis. FEBS Lett. 121, 71–77
    OpenUrlCrossRefPubMed
  38. 38.↵
    1. Sato H.,
    2. Fukae K.,
    3. Kajihara Y.
    (2008) 2D selective-TOCSY-DQFCOSY and HSQC-TOCSY NMR experiments for assignment of a homogeneous asparagine-linked triantennary complex type undecasaccharide. Carbohydr. Res. 343, 1333–1345
    OpenUrlCrossRefPubMed
  39. 39.↵
    1. Grass J.,
    2. Pabst M.,
    3. Kolarich D.,
    4. Pöltl G.,
    5. Leonard R.,
    6. Brecker L.,
    7. Altmann F.
    (2011) Discovery and structural characterization of fucosylated oligomannosidic N-glycans in mushrooms. J. Biol. Chem. 286, 5977–5984
    OpenUrlAbstract/FREE Full Text
  40. 40.↵
    1. Gallego R. G.,
    2. Blanco J. L.,
    3. Thijssen-van Zuylen C. W.,
    4. Gotfredsen C. H.,
    5. Voshol H.,
    6. Duus J. O.,
    7. Schachner M.,
    8. Vliegenthart J. F. G.
    (2001) Epitope diversity of N-glycans from bovine peripheral myelin glycoprotein P0 revealed by mass spectrometry and nano probe magic angle spinning 1H NMR spectroscopy. J. Biol. Chem. 276, 30834–30844
    OpenUrlAbstract/FREE Full Text
  41. 41.↵
    1. Ohashi T.,
    2. Ikeda Y.,
    3. Tanaka N.,
    4. Nakakita S.,
    5. Natsuka S.,
    6. Giga-Hama Y.,
    7. Takegawa K.
    (2009) The och1 mutant of Schizosaccharomyces pombe produces galactosylated core structures of N-linked oligosaccharides. Biosci. Biotechnol. Biochem. 73, 407–414
    OpenUrlCrossRefPubMed
  42. 42.↵
    1. van Leeuwen S. S.,
    2. Schoemaker R. J.,
    3. Gerwig G. J.,
    4. van Leusen-van Kan E. J.,
    5. Dijkhuizen L.,
    6. Kamerling J. P.
    (2014) Rapid milk group classification by 1H NMR analysis of Le and H epitopes in human milk oligosaccharide donor samples. Glycobiology 24, 728–739
    OpenUrlAbstract/FREE Full Text
  43. 43.↵
    1. Cipollo J. F.,
    2. Costello C. E.,
    3. Hirschberg C. B.
    (2002) The fine structure of Caenorhabditis elegans N-glycans. J. Biol. Chem. 277, 49143–49157
    OpenUrlAbstract/FREE Full Text
  44. 44.↵
    1. Harvey D. J.
    (2005) Fragmentation of negative ions from carbohydrates: part 3. Fragmentation of hybrid and complex N-linked glycans. J. Am. Soc. Mass Spectrom. 16, 647–659
    OpenUrlCrossRefPubMed
  45. 45.↵
    1. Karlsson N. G.,
    2. Schulz B. L.,
    3. Packer N. H.
    (2004) Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn. J. Am. Soc. Mass Spectrom. 15, 659–672
    OpenUrlCrossRefPubMed
  46. 46.↵
    1. Butschi A.,
    2. Titz A.,
    3. Wälti M.,
    4. Olieric V.,
    5. Paschinger K.,
    6. Nöbauer K.,
    7. Guo X.,
    8. Seeberger P. H.,
    9. Wilson I. B. H.,
    10. Aebi M.,
    11. Hengartner M.,
    12. Künzler M.
    (2010) Caenorhabditis elegans N-glycan core β-galactoside confers sensitivity towards nematotoxic fungal galectin CGL2. PLOS Pathogens 6, e1000717
    OpenUrlCrossRefPubMed
  47. 47.↵
    1. Haslam S. M.,
    2. Coles G. C.,
    3. Munn E. A.,
    4. Smith T. S.,
    5. Smith H. F.,
    6. Morris H. R.,
    7. Dell A.
    (1996) Haemonchus contortus glycoproteins contain N-linked oligosaccharides with novel highly fucosylated core structures. J. Biol. Chem. 271, 30561–30570
    OpenUrlAbstract/FREE Full Text
  48. 48.↵
    1. Haslam S. M.,
    2. Houston K. M.,
    3. Harnett W.,
    4. Reason A. J.,
    5. Morris H. R.,
    6. Dell A.
    (1999) Structural studies of N-glycans of filarial parasites. Conservation of phosphorylcholine-substituted glycans among species and discovery of novel chito-oligomers. J. Biol. Chem. 274, 20953–20960
    OpenUrlAbstract/FREE Full Text
  49. 49.↵
    1. Haslam S. M.,
    2. Coles G. C.,
    3. Morris H. R.,
    4. Dell A.
    (2000) Structural characterisation of the N-glycans of Dictyocaulus viviparus: discovery of the Lewisx structure in a nematode. Glycobiology 10, 223–229
    OpenUrlAbstract/FREE Full Text
  50. 50.↵
    1. Roberts B.,
    2. Antonopoulos A.,
    3. Haslam S. M.,
    4. Dicker A. J.,
    5. McNeilly T. N.,
    6. Johnston S. L.,
    7. Dell A.,
    8. Knox D. P.,
    9. Britton C.
    (2013) Novel expression of Haemonchus contortus vaccine candidate aminopeptidase H11 using the free-living nematode Caenorhabditis elegans. Vet. Res. 44, 111
    OpenUrlCrossRefPubMed
  51. 51.↵
    1. Wohlschlager T.,
    2. Butschi A.,
    3. Grassi P.,
    4. Sutov G.,
    5. Gauss R.,
    6. Hauck D.,
    7. Schmieder S. S.,
    8. Knobel M.,
    9. Titz A.,
    10. Dell A.,
    11. Haslam S. M.,
    12. Hengartner M. O.,
    13. Aebi M.,
    14. Kunzler M.
    (2014) Methylated glycans as conserved targets of animal and fungal innate defense. Proc. Natl. Acad. Sci. U.S.A. 111, E2787–E2796
    OpenUrlAbstract/FREE Full Text
  52. 52.↵
    1. Paschinger K.,
    2. Hackl M.,
    3. Gutternigg M.,
    4. Kretschmer-Lubich D.,
    5. Stemmer U.,
    6. Jantsch V.,
    7. Lochnit G.,
    8. Wilson I. B. H.
    (2006) A deletion in the Golgi α-mannosidase II gene of Caenorhabditis elegans results in unexpected non-wild type N-glycan structures. J. Biol. Chem. 281, 28265–28277
    OpenUrlAbstract/FREE Full Text
  53. 53.↵
    1. Gutternigg M.,
    2. Kretschmer-Lubich D.,
    3. Paschinger K.,
    4. Rendić D.,
    5. Hader J.,
    6. Geier P.,
    7. Ranftl R.,
    8. Jantsch V.,
    9. Lochnit G.,
    10. Wilson I. B. H.
    (2007) Biosynthesis of truncated N-linked oligosaccharides results from non-orthologous hexosaminidase-mediated mechanisms in nematodes, plants and insects. J. Biol. Chem. 282, 27825–27840
    OpenUrlAbstract/FREE Full Text
  54. 54.↵
    1. Griffitts J. S.,
    2. Haslam S. M.,
    3. Yang T.,
    4. Garczynski S. F.,
    5. Mulloy B.,
    6. Morris H.,
    7. Cremer P. S.,
    8. Dell A.,
    9. Adang M. J.,
    10. Aroian R. V.
    (2005) Glycolipids as receptors for Bacillus thuringiensis crystal toxin. Science 307, 922–925
    OpenUrlAbstract/FREE Full Text
  55. 55.↵
    1. Struwe W. B.,
    2. Reinhold V. N.
    (2012) The Conserved Oligomeric Golgi (COG) Complex is Required for Fucosylation of N-Glycans in C. elegans. Glycobiology 22, 863–875
    OpenUrlAbstract/FREE Full Text
  56. 56.↵
    1. Žurga S.,
    2. Pohleven J.,
    3. Renko M.,
    4. Bleuler-Martinez S.,
    5. Sosnowski P.,
    6. Turk D.,
    7. Künzler M.,
    8. Kos J.,
    9. Sabotič J.
    (2014) A novel β-trefoil lectin from the parasol mushroom (Macrolepiota procera) is nematotoxic. FEBS J. 281, 3489–3506
    OpenUrlCrossRefPubMed
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Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans
Shi Yan, Lothar Brecker, Chunsheng Jin, Alexander Titz, Martin Dragosits, Niclas G. Karlsson, Verena Jantsch, Iain B. H. Wilson, Katharina Paschinger
Molecular & Cellular Proteomics August 1, 2015, First published on May 22, 2015, 14 (8) 2111-2125; DOI: 10.1074/mcp.M115.049817

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Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans
Shi Yan, Lothar Brecker, Chunsheng Jin, Alexander Titz, Martin Dragosits, Niclas G. Karlsson, Verena Jantsch, Iain B. H. Wilson, Katharina Paschinger
Molecular & Cellular Proteomics August 1, 2015, First published on May 22, 2015, 14 (8) 2111-2125; DOI: 10.1074/mcp.M115.049817
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Molecular & Cellular Proteomics: 14 (8)
Molecular & Cellular Proteomics
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