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The modification of α1,6-linked fucose residues attached to the proximal (reducing-terminal) core N-acetylglucosamine residue of N-glycans by β1,4-linked galactose (“GalFuc” epitope) is a feature of a number of invertebrate species including the model nematode Caenorhabditis elegans. A pre-requisite for both core α1,6-fucosylation and β1,4-galactosylation is the presence of a nonreducing terminal N-acetylglucosamine; however, this residue is normally absent from the final glycan structure in invertebrates due to the action of specific hexosaminidases. Previously, we have identified two hexosaminidases (HEX-2 and HEX-3) in C. elegans, which process N-glycans. In the present study, we have prepared a hex-2;hex-3 double mutant, which possesses a radically altered N-glycomic profile. Whereas in the double mutant core α1,3-fucosylation of the proximal N-acetylglucosamine was abolished, the degree of galactosylation of core α1,6-fucose increased, and a novel Galα1,2Fucα1,3 moiety attached to the distal core N-acetylglucosamine residue was detected. Both galactosylated fucose moieties were also found in two parasitic nematodes, Ascaris suum and Oesophagostomum dentatum. As core modifications of N-glycans are known targets for fungal nematotoxic lectins, the sensitivity of the C. elegans double hexosaminidase mutant was assessed. Although this mutant displayed hypersensitivity to the GalFuc-binding lectin CGL2 and the N-acetylglucosamine-binding lectin XCL, the mutant was resistant to CCL2, which binds core α1,3-fucose. Thus, the use of C. elegans mutants aids the identification of novel N-glycan modifications and the definition of in vivo specificities of nematotoxic lectins with potential as anthelmintic agents.
During evolution, glycosylation has accumulated an increasing number of biological functions. Thereby, it is generally considered that the glycans of “lower” organisms are less complex and are less essential for viability than those of vertebrates. This assumption is based on the results of knocking out the N-acetylglucosaminyltransferase I (GlcNAc-TI) gene in various organisms: whereas in mice the ability of this enzyme to commence synthesis of hybrid and classical complex N-glycans is essential (1, 2), ablating the homologous gene in Arabidopsis, Drosophila, or Caenorhabditis results in milder phenotypes in the laboratory, such as lowered stress resistance or behavioral changes (3–5). However, recent studies indicate that this view should be modified and that the glycans of some lower organisms are relatively complex while presenting targets in “warfare” between species. For instance, the N-glycome of Caenorhabditis elegans features very unusual glycans, including the presence of galactosylated core α1,6-fucose residues (6, 7). These glycans are bound by both endogenous carbohydrate-binding proteins (galectins LEC-6 and LEC-10 (8, 9)) and, in laboratory experiments, by a fungal lectin with nematotoxic activity (CGL2 (10)). A range of other fungal lectins also exist that target other glycans presumed, by analogy to C. elegans, to be present in fungivorous nematodes (11).
Previous studies have indicated that modification by galactosylated fucose (12) depends on the prior action of N-acetylglucosaminyltransferase I. However, paradoxically the final paucimannosidic structures of many glycans in nematodes, as in other lower organisms, lack the N-acetylglucosamine residue transferred by this enzyme. Therefore, based on data from insect cells (13), the concept of a Golgi hexosaminidase in these organisms was hypothesized and results on the Drosophila and Spodoptera fdl genes have supported the presence of such an enzyme in insects (14, 15).
Five years ago, some of us discovered that a similar hexosaminidase also operates in C. elegans. Indeed, two genes were identified that encoded enzymes possessing the relevant specific hexosaminidase activity (16). These two genes, designated hex-2 and hex-3, have distinct cellular expression profiles and although a mutation in the hex-2 gene resulted in some shift in the N-glycome and an apparent abolition in N-glycan-specific hexosaminidase activity, the paucimannosidic structures such as Man3GlcNAc2Fuc0–1 were still dominant (16). These data suggested that hex-2 was not alone in determining the processing of N-glycans in C. elegans; therefore, a double hex-2;hex-3 mutant has now been prepared and the impact on the N-glycome was assessed. The observed changes in the N-glycome could be correlated to altered sensitivity to nematotoxic lectins. Furthermore, N-glycans similar to those in the double mutant were also found in two parasitic nematode species indicating a wider distribution of certain glycomic features, such as galactosylation of fucose residues, than previously thought.
The C. elegans single mutants hex-2 (tm2530) and hex-3 (tm2725) were obtained from the National Bioresource Project for the Experimental Animal Nematode C. elegans, Tokyo Women's Medical University, Japan; N2 wild type was obtained from the Caenorhabditis Genetics Centre, University of Minnesota, MN. All C. elegans strains were cultured under standard conditions at 20 °C (17). Double mutants were generated by standard crossings. Genotypes were followed using the primer pairs below.
Primers were designed to amplify DNA fragments with distinguishable lengths: as the deletion within the hex-2 gene is 467 bp long, PCR using the hex-2-specific primers (5′-CAGAAAATGCAAAACCAAT-3′ and 5′-GCCTTCAAATGAGCCAGC-3′) was predicted to generate DNA fragments of either 1324 bp (wild type) or 857 bp (mutation), whereas the deletion within the hex-3 gene is 146 bp long and so PCR using the hex-3-specific primers (5′-AAATACCCGAAACTCTCTCC-3′ and 5′-TCTTTAATCAATTCCCACGC-3′) was predicted to generate 401-bp (wild type) or 255-bp (mutation) DNA fragments. 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 of 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 of genomic DNA from worm lysates, 10 μl of GoTaq® Green Master Mix (Promega), and 8.6 μl of sterile water. PCR fragments were electrophoresed in a 1.5% (w/v) agarose gel mixed with 0.02% (v/v) of Gel-Red (Biotium, USA) and visualized on a UV transilluminator.
Worms were homogenized in lysis buffer (20 mm MES, pH 7.0, 1% (w/v) Triton X-100, 0.01% (v/v) Sigma protease inhibitor mixture) and incubated on ice for 10 min followed by 10 min of sonication. The crude extract was cooled down on ice for another 10 min prior to centrifugation at 10,000 × g for 10 min at 4 °C. The supernatant was transferred to a fresh Eppendorf tube, mixed with an equal volume of 2× reducing SDS buffer, and denatured at 95 °C for 5 min. The remaining debris within the supernatant was removed by a brief centrifugation prior to SDS-PAGE, using 12.5% gels and subsequent transfer onto a nitrocellulose membrane using a semi-dry blotting apparatus (25 V for 40 min). The quality of transfer was assessed by Ponceau staining prior to blocking for 1 h in TTBS/BSA (Tris-buffered saline containing 0.05% (w/v) Tween 20 and 0.5% (w/v) bovine serum albumin). The membrane was then first incubated with rabbit anti-horseradish peroxidase antibody (anti-HRP; Sigma, 1:10,000 diluted in blocking buffer) for 1 h and then with a goat anti-rabbit secondary antibody (Vector Laboratories, alkaline phosphatase conjugated, 1:2,000 diluted in blocking buffer) for another hour. After washing with TTBS, SigmaFASTTM 5-bromo-4-chloro-3-indolyl phosphate was used as substrate for chromogenic detection.
C. elegans were grown in liquid culture with Escherichia coli OP50 in standard S complete medium, harvested after cultivation at room temperature for 4–6 days, and purified by sucrose density centrifugation. Ascaris suum was obtained from pig intestines at a slaughterhouse and was the kind gift of Dr. Günter Lochnit, Universität Gießen. The monospecific strain of Oesophagostomum dentatum, OD-Hann (18), was routinely maintained by infection of parasite-free pigs kept at the animal facilities of the Institute of Parasitology, University of Veterinary Medicine Vienna. Adult females and males were isolated from the intestines of pigs during the patent stage of infection according to Slotved et al. (19) and differentiated as described (20).
N-Glycans were released from worm peptic peptides using peptide:N-glycosidase F (capable of removing most eukaryotic N-glycans other than those carrying α1,3-fucose on the reducing terminal GlcNAc), followed by peptide:N-glycosidase A (which is capable of removing core α1,3-fucosylated N-glycans), according to the procedures described previously (21). The N-glycome of the mutant was profiled by MALDI-TOF MS (Ultraflex I, Bruker Daltonics, Germany) in positive mode. Free glycans were labeled with 2-aminopyridine prior to fractionation by normal phase HPLC (NP-HPLC) and reversed-phase HPLC (RP-HPLC). All the HPLC peaks were collected and examined by MALDI-TOF MS, using either 2,5-dihydroxybenzoic acid or 6-aza-2-thiothymine as matrices; predicted glycan species were subject to fragmentation by MS/MS (post-source decay) and the spectra were analyzed manually.
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) 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) 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. For the glycans prepared from A. suum and O. dentatum, an Ascentis® Express RP-Amide column (Sigma) was used with the same buffers as for RP-HPLC; a gradient of buffer D up to 35% over 35 min was applied at a flow rate of 0.8 ml/min.
To isolate and purify the unique glycan structure Hex4HexNAc3Fuc2-PA (m/z 1646), a two-dimensional HPLC approach was employed. Thereby, a portion of the pyridylaminated glycans was first separated on a normal phase column, collected, lyophilized, analyzed by MALDI-TOF MS, and then re-injected onto the reversed phase column.
In general, a 1-μl aliquot of a HPLC fraction was mixed with 0.2 μl of exoglycosidase and 0.8 μl of 50 mm ammonium acetate solution, pH 5.0; after an overnight incubation at 37 °C, a 0.5-μl aliquot of the mixture was analyzed by MALDI-TOF MS. Exoglycosidases employed were: α-galactosidase from green coffee bean (Sigma, 11 milliunits), β-galactosidase from Aspergillus oryzae (Sigma, 27 milliunits) repurified by ion-exchange chromatography (22), α-l-fucosidase from bovine kidney (Sigma, 10 milliunits), β-N-acetylglucosaminidase from jack bean (Sigma, 6.25 milliunits), recombinant Xanthomonas manihotis α1–2/3-mannosidase (New England Biolabs, 6.4 units), and recombinant X. manihotis α1–6-mannosidase (New England Biolabs, 8 units). For removal of α1,3-linked fucose, glycan samples were dried in a SpeedVac and then incubated with 3 μl of 48% (v/v) hydrofluoric acid (HF)2 on ice for 24 h. The HF was allowed to evaporate overnight.
For monosaccharide linkage analysis, PA-labeled glycans (~0.5 nmol) were permethylated, hydrolyzed, reduced, and peracetylated prior to analysis of the obtained partially methylated alditol acetates by capillary GLC/MS using the instrumentation and microtechniques described elsewhere (23, 24).
Single L4 hermaphrodites were cultivated at 20 °C and transferred onto fresh NGM plates every 24 h until no more eggs were laid. Laid eggs were counted and after 24 h the fraction of hatched progeny was assessed.
A C. elegans liquid toxicity assay was performed as previously described (25) to compare the effect of the fungal lectins CCL2, CGL2, TAP1, and XCL on the wild-type N2 strain and the hex-2;hex-3 double mutant. In brief, ~30 L1 larvae were added to 100 μl of bacterial suspension (final A600 = 2) in sterile PBS in 96-well plates. For the bacterial suspension, lectin-expressing E. coli were mixed at different percentages with vector control transformed E. coli. The percentage of individuals reaching each developmental stage was quantified after 48 h of incubation at 20 °C; each treatment was performed in quintuplicate.
In our previous studies on C. elegans hexosaminidases we identified two enzymes (HEX-2 and HEX-3) that specifically remove the GlcNAc residue transferred by GlcNAc-TI and so are putatively involved in the formation of paucimannosidic N-glycans in vivo (16). In single mutants, especially in the hex-2 mutant, however, only a partial shift in the N-glycomic profile away from paucimannosidic glycans was observed. Also, the use of GFP-promoter constructs suggested that different hexosaminidases have distinct expression patterns and that no hexosaminidase was ubiquitously expressed. Therefore, we hypothesized that multiple hexosaminidases are involved in N-glycan processing in the nematode in vivo and so a double hexosaminidase mutant (hex-2;hex-3) was prepared by crossing the relevant single mutants. Progress of crossing was assessed by use of specific primers; PCR of the genomic DNA of the final double mutant indicated that both alleles of both genes contained the expected deletions (Fig. 1A). There was no obvious effect on the reproductive capability of the double mutant; the brood size of the hex-2:hex-3 double mutant was only slightly reduced (189 ± 10) as compared with the N2 wild type (233 ± 6). No obvious abnormalities were observed under the microscope. An initial biochemical screening of this new strain by Western blotting with an anti-carbohydrate antibody (anti-horseradish peroxidase) that recognizes core α1,3-fucose residues in wild-type worms (26) indicated that the epitope was now absent (Fig. 1B); these data were the first indication of a glycomic shift caused by knocking out both the hex-2 and hex-3 genes.
The N-glycans of the hex-2;hex-3 double mutant were released using PNGase F prior to PNGase A. The mass spectrum of the glycans released with PNGase F indicated a rather simple N-glycome (Table 1 and Fig. 2), in terms of the complexity of the spectrum, as compared with wild-type worms. Consistent with the lack of anti-HRP staining (see above), only trace amounts of one glycan (m/z 1281; 7 glucose units on RP-HPLC) carrying two fucoses on the proximal GlcNAc could be released with PNGase A (data not shown). Noticeable also is the reduction in the relative peak heights corresponding to oligomannosidic glycans; this may either be due to the lower number of the processed structures (and so relatively higher concentrations of such species) or unknown intracellular effects on glycan processing.
The vast majority of the PNGase F-released glycans had putative compositions indicating the presence of three or four N-acetylhexosamine residues (Hex3–4HexNAc3–4Fuc0–2), a result suggestive of a lack of hexosaminidase activity during the biosynthesis of these structures. These glycans were pyridylaminated and subject to NP-HPLC and RP-HPLC either singly or in succession. As with the MALDI-TOF MS data, the NP- and RP-HPLC chromatograms indicated the presence of four or five major N-glycan structures (Fig. 2, C and D). Each individual fraction was subject to MALDI-TOF MS and MS/MS to gain data regarding the composition and structure.
Depicted in Fig. 3 are the MS/MS spectra corresponding to the four major N-glycan compositions. Particularly dominant in these spectra are fragments of m/z 203.5–204.1 probably derived from a nonreducing terminal GlcNAc; one isomer of the species with m/z 1338 contains core fucose as indicated by a fragment of m/z 445.0 (Fig. 3B), whereas all the other major fucose-containing structures are capped with hexose as indicated by fragments of m/z 607.4–607.8 (Fig. 3, C–E). This fragment has been previously observed when analyzing pyridylaminated glycans of a planaria (27) and the C. elegans hex-2 mutant (16). Four minor glycan components (m/z 1395, two isomers of 1484 and 1541) also share a selection of these features (supplemental Fig. S1). Another indication, in all these spectra, for the presence of antennal GlcNAc, is a fragment of m/z ~1030, which corresponds to Hex2HexNAc3-PA; this common element in N-glycans of the hex-2;hex-3 double mutant is a demonstration of the effective elimination of processing hexosaminidase activity.
A particular focus of our analyses was on the glycan with m/z 1646. First, MS/MS (Fig. 3E) of this glycan indicated a serial loss of Hex, dHex, Hex, and dHex resulting in the aforementioned core Hex2HexNAc3-PA (m/z 1030); thereby, no deoxyhexose (fucose) residue was predicted to be terminal. The glycan was then isolated by two-dimensional HPLC (NP-HPLC followed by RP-HPLC) and was indeed found to be α-fucosidase resistant (Fig. 4B). On the other hand, Aspergillus β-galactosidase, known to be β1,4-specific (22), and, in part, coffee bean α-galactosidase each caused loss of one hexose residue (Fig. 4, C and E), whereas a combination of β1,4-galactosidase and bovine α-fucosidase resulted in loss of up to two residues (one hexose and one deoxyhexose; Fig. 4D). The corresponding MS/MS spectra indicate that β1,4-galactosidase digestion results in a loss of the m/z 607.4 fragment upon MS/MS and appearance of a fragment of m/z 445.7 (Fig. 4H); subsequent digestion with the fucosidase results in appearance of the unmodified core GlcNAc-PA fragment (m/z 299.7; Fig. 4I). This indicates that the same “GalFuc” epitope as found in planaria or mollusks is also present in the double mutant.
The location of the putatively α-linked galactose and second fucose was initially less clear. As mentioned above the Hex4HexNAc3Fuc2 glycan was partially sensitive (approximately two-thirds) to α-galactosidase as indicated by MS (Fig. 4E) and RP-HPLC (data not shown); incubation with hydrogen fluoride, known to remove core and Lewis-type α1,3-linked fucose as well as phosphodiesters (28, 29), removed one fucose and one hexose at once (loss of 308 mass units, Fig. 4F). However, treatment with either α-galactosidase or hydrogen fluoride did not remove the aforementioned GalFuc epitope evidenced by the continued presence of the fragment of m/z 608 (Fig. 4, J and K). Further serial exoglycosidase sequencing of the hydrogen fluoride-treated material with β-galactosidase, α-fucosidase, and β-hexosaminidase resulted in degradation down to Hex2HexNAc2-PA (m/z 827; supplemental Fig. S2).
Thus, under consideration of all the MS/MS and digestion data, we hypothesized that a different galactofucosyl moiety was present on either the core β-mannose or distal (second) core GlcNAc residues. MS/MS after perdeuteromethylation yielded ambiguous data. Therefore, the PA-glycan was subjected to GC-MS linkage analysis, which indicated the presence of terminal galactose, terminal and disubstituted GlcNAc, 2- and 3-substituted mannose, as well as 2- and 4-substituted fucose (Table 2 and supplemental Fig. S3).
Consistent with the digestion and MS/MS data, no terminal fucose was detected by GC-MS; indeed, the presence of 4-substituted fucose verifies the occurrence of the classical GalFuc epitope with β1,4-galactose as a capping residue, whereas the 2-substituted fucose and the 3,4-disubstituted GlcNAc reveal the modification of the distal GlcNAc with an HF-sensitive Galα1,2Fucα1,3 motif. Furthermore, the substitution pattern of the mannose residues indicates that only an α1,3-antenna is present. The putative structure offers an explanation for the incomplete nature of the combined β-galactosidase/α-fucosidase digestion (Fig. 4D): the modification of the distal GlcNAc sterically hinders the action of the fucosidase unless the Galα1,2Fuc moiety is first removed by HF (supplemental Fig. S2). Interestingly, a glycan of m/z 1322 (Hex2HexNAc3Fuc2) lacking any galactose residues on its fucose moieties loses (in part) two fucose residues when treated with α-fucosidase. The MS/MS of the products suggests that the proximal α1,6-fucose is completely removed, whereas the distal α1,3-fucose is partially sensitive to the enzyme; on the other hand, the α1,3-fucose is completely HF-sensitive (supplemental Fig. S4).
As summarized in Scheme 1, we propose a structure of GlcNAcβ1,2Manα1,3Manβ1,4(Galα1,2Fucα1,3)GlcNAcβ1,4(Galβ1,4Fucα1,6)GlcNAc-PA for the fourthmost major glycan with m/z 1646 in the double mutant; this is probably also the definitive structure of the glycan with the same mass previously found in the hex-2 single mutant (16). We assume that the novel Galα1,2Fuc moiety is also present on a number of glycans with compositions of Hex3–5HexNAc3Fuc2–3Me0–1 (m/z 1484, 1792, 1806, and 1808; see also below) and that the previous detection of a hexosyl modification of fucose on the distal GlcNAc by Reinhold and colleagues (6) can be explained by our data showing α-galactosylation.
A detailed examination of both the complete spectra of pyridylaminated hex-2;hex-3 N-glycans as well as the individual fractions revealed four other modifications; three of these are extended GalFuc motifs and the fourth is a phosphorylcholine-substituted HexNAc2. Example MS/MS spectra of glycans carrying these modifications are shown in Fig. 5, which reveal fragments of m/z 769, 753, 767, and 571. The first of the GalFuc modifications (Fig. 5, A and C) is, on the basis of the removal of two hexoses by Aspergillus β1,4-specific galactosidase, predicted to be a Galβ1,4Galβ1,4Fucα1,6 motif as found in keyhole limpet hemocyanin; removal of the galactose residues results in loss of the m/z 769.9 fragment and appearance of one of m/z 445.6 (Fig. 5, B and D). These digestion data, therefore, complement the previous MSn data of Hanneman (6). The second GalFuc variant (Fig. 5E) is predicted to be capped with a deoxyhexose as shown by the presence of the m/z 753.7 fragment when performing MS/MS of the Hex4HexNAc3Fuc3 glycan, which renders the m/z 1792 structure β-galactosidase-insensitive (see Fig. 5B).
The third modified form of the GalFuc, present in three glycans (m/z 1660, 1674, and 1806; Fig. 5, F–H), contains a residue with a mass of 160, which could correspond to methylfucose, and results in fragments of m/z 767.2–768.2. Some of these glycans (m/z 1792, 1806, and 1808) with aforementioned modified forms of Galβ1,4Fucα1,6 on the proximal GlcNAc probably also carry the Galα1,2Fucα1,3 motif on the distal GlcNAc as judged by their HF sensitivity (loss of 308 mass units; data not shown). The glycan with m/z 1674 is also proposed, based on the MS/MS data to contain a methylated mannose residue (Fig. 5G).
Finally, of various putatively phosphorylcholine-modified glycans, MS/MS of the species with m/z 1763 (Hex3HexNAc5PC1; Fig. 5I) reveals a HexNAc2PC motif. Previous studies on C. elegans have only shown the modification of a single terminal GlcNAc with phosphorylcholine and this is probably the first direct observation of a HexNAc2PC fragment, by MS/MS, in an N-glycan; it is uncertain whether the HexNAc2 in this case is LacdiNAc, as in Trichinella (30), or a chitobiose unit, as in filarial nematodes (31).
As discussed above, the glycans of lower organisms are often products resulting from the specific removal of the GlcNAc residue attached to the α1,3-linked mannose. Thereby, N-glycans with a composition of Hex3HexNAc3 are actually the products of Golgi hexosaminidase action on Hex3HexNAc4 (GnGn according to the nomenclature of Schachter (32)) to remove that GlcNAc transferred by GlcNAc-TI, thus yielding so-called GnM, which has a later RP-HPLC elution time than MGn (33). To assess which isomer of Hex3HexNAc3 (m/z 1192) is present in the hex-2;hex-3 double mutant, the relevant fraction was examined in comparison to a partial jack bean hexosaminidase digestion of GnGn, which yields MGn, MM, and GnM. As expected from the function of HEX-2 and HEX-3, the isomer of Hex3HexNAc3 isolated from the double mutant indeed corresponded to MGn (7 glucose units; Fig. 2D and supplemental Fig. S5). On the other hand, the glycan with the composition Hex3HexNAc4 (m/z 1395, 10 glucose units; Fig. 2D and supplemental Fig. S5) co-elutes with GnGn, indicating the presence of some nonextended biantennary glycans, whereas the very minor Hex3HexNAc2 glycan (m/z 989) is indeed MM as indicated by its sensitivity to both α1,2/3- and α1,6-specific mannosidases (data not shown). In addition to the aforementioned glycans, only low amounts of oligomannosidic structures were detected in preparations of N-glycans from hex-2;hex-3 worms.
To date, the GalFuc epitope has, in the Nematoda, only been structurally shown in C. elegans (6, 10, 16). On the other hand, the modification of the distal core GlcNAc by unsubstituted α1,3-fucose has been shown in the parasite Hemonchus contortus (34). Therefore, our question was whether both types of GalFuc moieties are indeed expressed in parasitic nematodes, specifically in two porcine parasites A. suum and O. dendatum. We have previously examined the N-glycans of A. suum by ESI-MS (35), but it appeared that, in comparison to later MALDI-TOF MS data, some in-source fragmentation occurred that overestimated the occurrence of N-glycans with lower molecular masses; in contrast, the glycans of O. dendatum have not been examined other than by lectin blots (36, 37). HPLC fractionation on an amide-capped RP column and MALDI-TOF MS was performed (Fig. 6) and strikingly some glycans with the same m/z values as in the C. elegans double mutant were detected. We focused on fractions of glycans released by PNGase F from A. suum (fraction 17) and O. dendatum (fraction 22), which co-elute with the m/z 1646 glycan present in the mutant (23.5 min; ~13 glucose units). As shown by rechromatography, fraction 17 from A. suum consists of three subfractions, the most dominant of which co-elutes with the purified glycan from the C. elegans mutant (Fig. 6A).
Fractions 17 and 22 indeed contained a number of species and some of these glycans were sensitive to β1,4-galactosidase and/or hydrogen fluoride (Fig. 6B) indicating the presence of galactose, α1,3-linked fucose, and phosphorylcholine modifications. This conclusion is also confirmed by the MS/MS data. The fragment of m/z 369.2 upon MS/MS of the HF-sensitive O. dendatum glycan of m/z 1503 is consistent with a terminal PC-HexNAc (Fig. 7A). MS/MS of a number of other glycans resulted in the appearance of fragments of m/z 607.7–608.2 (Fig. 7, B–F). For instance, the galactosidase-sensitive O. dendatum glycan with a composition of Hex4HexNAc2Fuc1 (m/z 1297; Fig. 7B) is predicted to possess a GalFuc modification of the reducing-terminal GlcNAc residue. Another example is the HF- and galactosidase-sensitive O. dendatum glycan of m/z 1443 (Hex4HexNAc2Fuc2; Fig. 7C), which not only has an m/z 608 fragment but also shows fragments of 1281, 1135, 973, and 827 (sequential loss of hexose and deoxyhexose residues resulting in Hex2HexNAc2); here, we assume that the second fucose is linked to the distal GlcNAc as in C. elegans. Furthermore, the MS/MS spectra for the m/z 1646 species from both parasitic nematodes were similar to that from the hex-2;hex-3 mutant in displaying two serial losses of 308 mass units, suggestive of the presence of two GalFuc moieties (Fig. 7, D and F). Therefore, we conclude that late-eluting Hex3–4HexNAc2–3Fuc2 glycans from A. suum and O. dendatum contain galactosylated fucose on the proximal (reducing terminal) GlcNAc as well as unsubstituted or substituted α1,3-fucose probably attached to the distal core GlcNAc.
Due to the glycomic shift observed in the hex-2;hex-3 mutant (increased GalFuc epitope expression, increased terminal GlcNAc, and lack of core α1,3-fucose), it was predicted that the sensitivity of this strain to nematotoxic fungal lectins would be altered. In total, the sensitivity to four lectins (11) was tested: CCL2 and CGL2 from Coprinopsis cinerea targeting, respectively, core α1,3-fucose and GalFuc epitopes, TAP1 from Sordaria macrospora binding to T-antigen (Galβ1,3GalNAc) and XCL from Xerocomus chrysenteron, which binds both T-antigen and terminal GlcNAc. L1 larvae of wild-type and double mutant C. elegans were fed with mixed populations of E. coli with a certain percentage of the bacteria expressing recombinant forms of the lectins; the percentage of larvae reaching L4 was determined. Consistent with the increased degree of modification by GalFuc and terminal GlcNAc, Fig. 8 shows that the sensitivity to CGL2 and XCL are significantly increased in the hex-2;hex-3 double mutant, whereas sensitivity to CCL2 was abolished due to the lack of core α1,3-fucose on the proximal GlcNAc; only small differences in the nematoxicity were observed with TAP1.
An examination of the developmental profile of the arrested worm population indicated no development to adults of either wild-type or mutant worms in the presence of a concentration of >5% of TAP1-expressing E. coli, whereas wild-type worms showed a lower rate of developmental arrest across the concentration range (5–50%; see supplemental Fig. S6). Thereby, the effect of XCL on the double mutant is in keeping with the glycomic changes; the reasons for the higher toxicity of TAP1 are unknown.
As shown by this and other studies, hexosaminidases play an important role in molding N-glycomes of lower animals and plants. Whereas in plants, hexosaminidases may rather have a role in random removal of nonreducing terminal N-acetylglucosamine residues on N-linked oligosaccharides (16), in insects the processing fused lobes (fdl) hexosaminidase present in the secretory pathway is highly specific for the residue transferred by N-acetylglucosaminyltransferase I (14). In C. elegans, we also assume that the ”processing“ hexosaminidases are present in the secretory pathway. Based on in vitro data (16), two candidate hexosaminidases (HEX-2 and HEX-3) were identified that have a similar specificity to that encoded by the Drosophila fdl gene. As hex-2 and hex-3 single knock-outs did not result in an absence of paucimannosidic glycans (16), we have now constructed a double mutant. The prediction that these two hexosaminidases are involved in N-glycan biosynthesis was confirmed, as the double knock-out is lacking almost all paucimannosidic glycans. Only traces of Hex2–3HexNAc2Fuc0–2 remain, whereas the vast majority of N-glycans present nonreducing terminal GlcNAc residues; the data suggest that this GlcNAc residue is bound to the α1,3-mannose.
However, the glycans in the double mutant are not just marked by the presence of the residue transferred by GlcNAc-TI: most of the N-glycans detected carry the so-called GalFuc epitope. Furthermore, a significant proportion has a proposed composition of Hex4HexNAc3Fuc2. The fragmentation pattern as well as the digestions with exoglycosidases and hydrofluoric acid indicated that this and some other glycans in the hex-2;hex-3 mutant are unusual in carrying two GalFuc motifs: one is the Galβ1,4Fucα1,6 motif decorating the proximal (reducing-terminal) GlcNAc present in a number of invertebrates as diverse as planaria and molluscs (27, 38–40), the other is proposed to be a Galα1,2Fucα1,3 moiety on the distal (second) core GlcNAc residue. This is a novel glycan modification, although its presence (but not the sequence of the residues) could be surmised from MSn data on wild-type glycans (6). Although α-galactosidase did not completely remove the galactose residue from this motif, the GC-MS data indicating the 2-substitution of a fucose residue and the sensitivity of this second GalFuc motif to hydrofluoric acid are evidence enough for the unusual nature of this modification. Furthermore, the proximal GalFuc motif can also be further modified by fucose, methylfucose, or galactose; the latter modification is known from keyhole limpet hemocyanin, whereas methylhexose (not found in the present study) is present in planaria (27, 39).
As previously shown by us (12), the modification of glycans by galactosylation of the core α1,6-fucose is most efficient when a nonreducing terminal GlcNAc is present; thus, the increase in the presence of this GalFuc modification in the double hexosaminidase mutant is in accordance with the properties of the relevant enzymes GALT-1 and FUT-8 (12, 41). The enzymatic origin of the second GalFuc modification is certainly less clear; it can be assumed that one of the five α1,3-fucosyltransferase homologues in C. elegans (26) is responsible for transfer of fucose to the distal core GlcNAc. On the other hand, there are no clear α-galactosyltransferase homologues in C. elegans; perhaps the only α-galactosyltransferase activity known to date in nematode extracts, specifically in H. contortus, uses GalNAc and not fucose as an acceptor (42). Perhaps coincidentally, the N-glycans modified by fucose on the distal core GlcNAc lack α1,6-mannose residues; thus, a mannosidase activity is either a pre-requisite for modification of the distal GlcNAc or this modification is a ”GO“ signal for a mannosidase.
Another biosynthetic conclusion from the glycomic data is that the occurrence of α1,3-fucose on the proximal reducing-terminal GlcNAc is inhibited when a nonreducing terminal GlcNAc is present. This is in accordance with the enzymatic properties of the relevant core α1,3-fucosyltransferase FUT-1 (26). Therefore, it was expected that no, or only a low amount of, difucosylated glycans would be found in the double mutant (as shown by the almost complete lack of glycans displaying MS/MS fragments of m/z 591) and that anti-HRP staining would be abolished (as indeed observed).
Based on the increased occurrence of the GalFuc modification of the proximal GlcNAc, an increased sensitivity to the GalFuc-binding fungal lectin CGL2 could be predicted. Indeed, this was the case; on the other hand, the double mutant was insensitive to CCL2, recently shown to be a core α1,3-fucose binding lectin (25).
The dual specificity (GlcNAc- and T-antigen-binding) lectin XCL was also investigated, considering the large increase in nonreducing terminal GlcNAc in the hex-2;hex-3 mutant: in comparison to the homologous lectin TAP1, for which no major change in susceptibility of wild-type versus mutant worms was observed, a shift toward higher sensitivity of the mutant is seen with XCL. The reason for the somewhat higher overall toxicity of TAP1, particularly obvious when examining the dose dependence, is unknown. There may be an unidentified glycan target interacting with the second binding site in this lectin. TAP1 is homologous to XCL but lacks this second binding site according to glycan array data (11); thus, the higher sensitivity of the mutant to XCL can be assigned to its interaction with nonreducing terminal GlcNAc.
There are further fungal lectins that target nematodes; other than defensive lectins, it is also claimed that some nematophagous fungi use lectins to trap their nematode prey (43). The specificities of such trapping lectins as well as other uncharacterized lectins displaying toxicity toward nematode parasites of plants (44), may potentially encompass some of the glycans found in this study.
A further aspect of our data is the demonstration of GalFuc epitopes, probably on both the distal and proximal GlcNAc residues, in parasitic nematodes. Previously, the only nematode proven to contain the Galβ1,4Fucα1,6 motif was C. elegans (6, 10, 16). On the other hand, modification of the distal GlcNAc with α1,3-fucose alone was previously shown in the parasite H. contortus, but GalFuc epitopes were not detected in that organism (34). Therefore, the discovery of GalFuc in both A. suum and O. dendatum is the first indication that GalFuc is present in parasitic organisms.
It can probably be presumed that other parasites may also carry these glycan decorations and so this opens up, considering the lectin sensitivity data, a new avenue for potential anthelmintic therapy. This study is also the first to present actual structural data on glycans of O. dendatum; previously our glycomic knowledge was restricted to lectin binding studies (36, 37). Ongoing experiments indicate that other N-glycans of this species carry multiple fucose residues as well as phosphorylcholine.3 Furthermore, the present data indicate a higher degree of complexity of glycans from A. suum as compared with our previous LC/ESI-MS study (35).
A final consideration is that we can reveal further complexities in the N-glycosylation of nematode species. The glycans of the “simple” worm continue to present analytical challenges; hereby, the use of double mutants is an invaluable approach to increase the expression of glycans normally of low abundance. It is also clear that blocking the processing by hexosaminidases in C. elegans does not result so much in elongation of the antennae, but an accumulation of core modifications including galactosylation events, which in higher animals are associated with the outer branches. The “daily” biological significance of these glycan decorations to C. elegans is unclear; however, challenging C. elegans in a laboratory setting with lectin toxins (which in nature are rather part of the fungal arsenal against fungivorous nematodes) indicates the importance of the fine structure of glycans and the relative levels of their expression in the interactions between organisms.
We thank Dr. H. Geyer for help with the GC-MS data, Peter Kysel for help preparing Ascaris glycans, Dr. Shohei Mitani from the Tokyo Women's Medical University for the original hex-2 and hex-3 single mutants, Dr. Harry Schachter for the triple GlcNAc-TI knock-out, and the Caenorhabditis Genomics Centre for the VC378 fut-1 and N2 strains.
*This work was supported by Austrian Fonds zur Förderung der wissenschaftlichen Forschung Grant P21946 (to K. P.), European Union Euroglycoarrays Initial Training Network Grant PITN-GA-2008–215536, Swiss National Science Foundation Grant 31003A-130671, and the ETH Zürich.
This article contains supplemental Figs. S1–S6.
3K. Paschinger, unpublished data.
2The abbreviations used are: