Genetic model organisms in the study of N-glycans

Biochimie 83 (2001) 703−712 © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908401012974/FLA

Genetic model organisms in the study of N-glycans Friedrich Altmanna, Gustáv Fabinia, Horst Ahornb, Iain B.H. Wilsona* a

Institut für Chemie der Universität für Bodenkultur, Muthgasse 18, 1190 Vienna, Austria Boehringer Ingelheim Austria GmbH, Dr. Boehringer-Gasse 5-11, 1121 Vienna, Austria

b

(Received 20 April 2001; accepted 19 June 2001) Abstract — Recently the genomic sequences of three multicellular eukaryotes, Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana, have been elucidated. A number of cDNAs encoding glycosyltransferases demonstrated to have a role in N-linked glycosylation have already been cloned from these organisms, e.g., GlcNAc transferases and α1,3-fucosyltransferases. However, many more homologues of glycosyltransferases and other glycan modifying enzymes have been predicted by analysis of the genome sequences, but the predictions of full length open reading frames appear to be particularly poor in Caenorhabditis. The use of these organisms as models in glycobiology may be hampered since they all have N-linked glycosylation repertoires unlike those of mammals. Arabidopsis and Drosophila have glycosylation similar to that of other plants or insects, while our new data from MALDI-TOF analysis of PNGase A-released neutral N-glycans of Caenorhabditis indicate that there exists a range of pauci- and oligomannosidic structures, with up to four fucose residues and up to two O-methyl groups. With all these three ‘genetic model organisms’, however, much more work is required for a full understanding of their glycobiology. © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. N-glycans / glycosyltransferases / Arabidopsis / Caenorhabditis / Drosophila

1. Introduction Even 2 years ago, a general glycobiology text had very little to say with regard to the glycobiology of Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana [1]; also last year, a review on Drosophila glycans indicated the relative dearth of structural information [2]. Whereas, these organisms have become, due to their relatively low generation times in comparison to other multicellular eukaryotes and the ease with which they can be genetically manipulated, important models for genomic and developmental research, biochemical and structural analyses lagged far behind. As anyone working with these organisms knows, acquiring enough material for anything other than extracting nucleic acid requires some significant effort. Certainly the most significant advance in our knowledge of these species has come about due to the essentially complete (but not 100%) genomic DNA sequences that are now available [3–5]. This allows the identification, by homology, of potential genes.

*Correspondence and reprints. E-mail address: [email protected] (I.B.H. Wilson). Abbreviations: Alg, asparagine-linked glycosylation; Dol-P-Glc, dolicholphosphate glucose; Dol-P-Man, dolicholphosphate mannose; EST, expressed sequence tag; HRP, horseradish peroxidase; MALDI-TOF, matrix-assisted laser desorption/ionisation time-of-flight; ORF, open reading frame; PNGase A, peptide-Nglycosidase A from almonds; RNAi, RNA interference.

2. Homology searching Recently, it has become obvious that many glycosyltransferases belong to families that are related by sequence. These families encompass a range of specificities; in some cases transfer of one type of sugar to form different types of linkage is mediated by members of the same family. For example, the members of the eukaryotic sialyltransferase family catalyse all known sialyltransferase-mediated reactions to a range of acceptor substrates forming either α2,3, α2,6 or α2,8 linkages but all using CMP-sialic acid donors [6]. In other cases, members of one glycosyltransferase family transfer different sugars, but form the same type of linkage with a variety of acceptors, as is the case with the β1,3galactosyltransferase family [7], which includes an β1,3N-acetylgalactosaminyltransferase (formerly designated β3Gal-T3) [8] and fringe-type β1,3-N-acetylglucosaminyltransferases [9]. The β1,6-N-acetylglucosaminyltransferase (core 2/large I) family also includes the xylosyltransferase responsible for forming the proteoglycan core. The easiest way to use information from homology searching of the genome databases is to perform RT-PCR based on the extent of the predicted open reading frame (for the predicted number of homologues for each organism, see table I). Experience so far with Arabidopsis and Drosophila glycosyltransferases in this laboratory would suggest that these predictions are reasonably satisfactory, certainly at the 3’-end of the reading frame; sometimes

704 there are also sufficient ESTs from the same, or related, organism(s) in order to aid delineation of the ORF. Occasionally, there are problems with the internal intronexon boundaries (shifting of the splice site or its complete absence). On the other hand, it seems that the predictions of Caenorhabditis ORFs can be highly unsatisfactory. Recently, in a sample of 1000 genes, it was found that 27% of worm reading frames were not properly identified by gene-finding programs [10]. Our own searches validate this: in one case (cosmid H43I07), the Alg5p (Dol-P-Glc synthetase) homologue, as annotated in the databases, is rather long. Indeed, the final 300 amino acids of the predicted ORF are related to the murine RNA polymerase 1-1 40 kDa subunit. In another case, two α1,3fucosyltransferases are predicted to be present on the same polypeptide of ca. 1600 amino acids encoded by cosmid T05A7: more probably, though, these are encoded by two genes 10 kb apart. Showing up the limitations of the ‘computer cloning’ approach is the fact that homologies tend to be strong only in the middle of a given protein sequence; thus, in the absence of sufficient ESTs from the worm, cDNA library screening may prove still necessary. Homology searching can also lead to some puzzling results: for instance the identification of three sialyltransferase homologues in Arabidopsis. To date, there has been no validated report of sialic acid being present in any plant tissue, yet RT-PCR indicates that these homologues are transcribed (I.B.H.W., unpublished data). Since plants have a CMP-3-deoxy-D-manno-2-octulosonate (CMPKdo) synthetase which has some homology to CMP-Sia synthetases [11], thus indicating it is possible that related enzymes can handle Kdo and sialic acid, it may be that these are Kdo-transferases which form, for example, the Kdo-GalA linkages found in rhamnogalacturonan II [12]. In Drosophila, a single sialyltransferase homologue (of unknown function) is predicted and a corresponding cDNA sequence has been deposited in Genbank [13]. 3. Genetic manipulations One of the major objectives in studying ‘genetic model organisms’ is to modulate gene expression by mutation or interference and then, by examining the phenotype, uncover a function for a gene or a process. Whereas the lack of biochemical knowledge is a handicap in the study of Caenorhabditis, Drosophila and Arabidopsis, these organisms are susceptible to genetic manipulation. Genetic methods involve traditional epistatic genetic interactions to study genetic pathways and preparation of either lossor gain-of-function mutants by random mutagenesis (e.g., ethyl methanesulphonate, γ-ray), insertional mutagenesis (P-elements, T-DNA) and transgenesis. P-element and other mutant lines of Drosophila (whose locations are mapped in Flybase; www.flybase.org) [14] and T-DNA lines of Arabidopsis (for which PCR screening is necessary) [15, 16] are publicly available.

Altmann et al. The use of systematic functional genomics has been unbelievably accelerated by the accidental discovery of rapid gene disruption by introduction of homologous double-stranded (ds) RNA in C. elegans, an effect also called RNA interference (RNAi) [17]. The exciting story began a decade ago by observation of a co-suppression in petunia plants [18], followed by so-called quelling in the mould Neurospora crassa [19] and finally the RNAi effect in the nematode C. elegans [17]. In the last 2 years RNAi has also been applied to other species such as Arabidopsis [20], Drosophila [21], Xenopus laevis [22], Trypanosoma brucei [23], and mouse [24]. This technique, which reflects an ancient defence mechanism to foreign mobile DNA elements (in C. elegans and Drosophila) or to RNAs (in form of viruses in plants), now can be widely used to switch off the genes in order to approach their biological function, in the case of fully sequenced genetic model organisms, even without laborious ‘fishing and cloning’ of the genes of interest. The power of RNAi for large scale functional genomics has been recently demonstrated by the systematic ‘knocking-out’ of individual genes comprising a total of one-third of the C. elegans genome (chromosomes I and III) [25, 26]. However, there are great differences in susceptibility of the genes to a RNAi effect. Phenotypic changes are better seen for evolutionary well conserved genes than for species specific ones. For instance, neuronal genes have been shown to be very often resistant. To overcome these obstacles one may use heritable gene silencing, which can be achieved in vivo by expressing hairpin dsRNA from inverted repeat genes under the control of an inducible promoter [27]. The advantages involve the gene specific inactivation at a given time and location (stage specific tissue inactivation), easy maintenance of the manipulated organisms and, more importantly, the possibility of production of large amounts for biochemical assays and analyses (e.g., of aberrations of glycan structure and glycosyltransferase activities). Recent reports confirming this trend describe RNAi inactivation in Drosophila of PIPE, a heparan sulphate 2-Osulphotransferase homologue, and of heparan sulphate 6-O-sulphotransferase [28, 29]. Combining glycobiology with the RNAi technique opens new ways how to elucidate the complex structure, biosynthesis and precise function of various types of glycoconjugates. 4. N-glycans in Arabidopsis Arabidopsis has genes predicted to encode the major enzymes involved in the biosynthesis of the lipid-linked Glc3Man9GlcNAc2 N-glycan precursor (i.e., probable homologues of the yeast transferases Alg1p-Alg12p and mammalian Dol-P-Man synthetase subunits DPM1-3), as well as various oligosaccharyltransferase subunits (Wbp1p/OST48, Stt3p, Ost1p/ribophorin, Ost2p/DAD1), N-acetylglucosaminyltransferases I and II, glucosidases

Genetic model organisms in the study of N-glycans

705

Table I. Eukaryotic processing glycoenzyme families: Estimated numbers of homologues based on BLAST searching of the Caenorhabditis elegans (C.e.), Drosophila melanogaster (D.m.) and Arabidopsis thaliana (A.t.) genomes. Family α1,3/4 fucosyltransferase α1,2/6-fucosyltransferase Protein GalNAc transferase α1,3-galactosyltransferase α1,4-galactosyltransferase α1,6-galactosyltransferaseb β1,3-galactosyltransferase β1,4-galactosyltransferase β1,2-GlcNAc transferase β1,3-GlcNAc transferasec β1,4-GlcNAc transferase β1,6-GlcNAc transferase Protein GlcNAc transferase Glucosidasea Protein glucosyltransferase Collagen glucosyltransferase Glucuronyltransferase GlcA C5 epimerase Mannosidasea Protein mannosyltransferase α2,3/6/8 sialyltransferase Sulphotransferase

β1,2-xylosyltransferase Protein xylosyltransferase

Examples of linkage -(Fucα1,3)GlcNAc- (core) Galβ1,3/4(Fucα1,4/3)Gal (Lewis) -GlcNAcβ1,4(Fucα1,6)GlcNAcFucα1,2Gal (H-type) Fucα1,2Gal (xyloglucan-type) GalNAc-O-Ser Galα1,3Gal Gal/GlcNAcα1,4Gal (Pk-type) Galα1,4Gal (lgtC-type)c Gal/Manα1,2/6-Man Gal/GalNAcβ1,3GlcNAc Galβ1,3GalNAc (core 1) GlcNAcβ1,3Fuc (fringe) Gal β1,4GlcNAc GlcNAc-TI GlcNAc-TII i/LARGE GlcNAcβ1,3Gal GlcNAcTIII GlcNAcTIV/VI GlcNAcβ1,6Gal(NAc) (core 2/I) GlcNAcTV GlcNAc-O-Ser glucosidase I glucosidase II GlcMan9GlcNAc2 Gal-Hyp GlcT/Lys-hydroxylase 3 proteoglycan/HNK-1 type EXT GlcNAc/GlcA type glucuronic/iduronic epimerase mannosidase I mannosidase II Man-O-Ser Siaα2,3/6/8Gal heparan/uronyl sulphate HS2ST heparan sulphate HS3ST heparan sulphate HS6ST N-deacetylase/N-sulphoT HNK-1/GalNAc/chondroitin -(Xylβ1,2)Manβ1,4Xylβ1,O-ser

}

C.e.

Presence in D.m.

A.t.

– 5 1 1 – 9 – – – – 12

1 3 1 – – 14 – 2 – – 7

2 1 – – 10 – – 5 23 5 18

12

16

13

3 3 1 3 – – 20 1 1 1 2 2 1 8 2 1 7 2 – – 1 2 1 1 – – 1

3 1 1 4 1 2 3 – 1 1 1 1 1 4 3 1 6 2 2 1 2 2 1 1 3 – 1

– 1 1 – 5 – 10 – 1 2 1 1 – 2 20 – 3 1 – 3 – – – – – 1 –

Symbols: –, no detected homologue; a excluding putative lysosomal glycosidases; b based on searching with fenugreek galactomannan α1,6-galactosyltransferase which displays homology to Schizosaccharomyces α1,2-galactosyltransferase and Saccharomyces Mnn10p α1,2-mannosyltransferase; c the C-terminus of the human LARGE protein is homologous to human i-β1,3-GlcNActransferase involved in polyLacNAc biosynthesis and some homologues in fly and worm, while the N-terminus of LARGE is homologous to four predicted Arabidopsis proteins with homology in turn to the Neisseria lgtC α1,4-galactosyltransferase and nineteen other Arabidopsis proteins, but not to the Pk blood group-type α1,4-galactosyltransferase.

and α-mannosidases. In some cases, cDNAs encoding these enzymes have already been isolated: the Alg7p homologue [30], GlcNAc-TI [31,32] and GlcNAc-TII from Arabidopsis [33]. A number of mutants relevant to these earlier stages of N-glycosylation have been isolated:

a deficiency in GDP-Man synthesis (the cyt1 mutant), which presumably affects the synthesis of the dolichollinked precursor and so of N-glycans, is associated with a five-fold reduction in cellulose content of Arabidopsis cell walls [34]; a T-DNA insertion line deficient in glucosidase

706 I (gcs1) which is defective in seed development [35]; and the cgl mutant lacking GlcNAc-TI activity and, thereby, complex N-glycans, but which has no apparent defect in viability [36]. Core α1,3-fucosylation of N-glycans is a modification that probably occurs in all plant species and the relevant core α1,3-fucosyltransferase (FucT-C3) was purified in this laboratory from mung beans and its cDNA was cloned [37]. Now, two cDNAs encoding core α1,3fucosyltransferase homologues in Arabidopsis have been identified: one encodes a protein which was verified to have the predicted enzymatic activity, while the other was only isolated in a form with a splice site shift resulting in a premature stop codon [38]. Another form of N-glycan fucosylation, α1,4-fucosylation of the antennae, results in the presence of Lewis a epitopes in plants; however, Arabidopsis is relatively unique in that it, or at least its leaf tissue, apparently lacks N-glycans with this structure [39]. Indeed, a third Arabidopsis α1,3-fucosyltransferase homologue, similar to a tomato Lewis-type α1,3/4fucosyltransferase, has not yet proven to be active [38]. Similar to the core α1,3-fucosyltransferase, the cDNA for core β1,2-xylosyltransferase was only isolated after the soybean enzyme was purified [40] and some of its peptides sequenced. In this case, regions of homology were detected in the Arabidopsis genome (and ESTs) and it was possible to use RT-PCR to clone a cDNA, whose recombinant gene product was found to have xylosyltransferase activity [41]. This xylosyltransferase is apparently a unique enzyme, with clear homologues only found by analysis of ESTs from other plants. 5. N-glycans in Drosophila At a biochemical level the biosynthesis of N-glycans has rarely been studied in fly, although in Drosophila evidence for the synthesis of the Glc3Man9GlcNAc2 dolichol-linked precursor for N-glycans in a cell line has been previously presented [42]. The fly genome, however, encodes the ‘expected’ ER mannosyl- and glucosyltransferases and homologues of the mammalian Dol-P-Man synthetase subunits DPM1 and DPM3. One cloned fly mannosyltransferase cDNA encodes an Alg3p homologue, designated Not56, whose corresponding l(2)not gene contains another gene within its intron [43]. The fly also has various oligosaccharyltransferase subunit homologues, one of whose cDNAs (encoding OST48/WBP1) has also been cloned [44]. Fly homologues of ER/Golgi glucosidases, mannosidases, UDP-Glc:glycoprotein glucosyltransferase and GlcNAc-transferases I and II can also be found. Of these enzymes, cDNAs have been cloned that encode: a glucosyltransferase of the type involved in glycoprotein folding [45]; GlcNAc-TI [46]; one mannosidase I (mas-1) [47]; and one mannosidase II from Drosophila [48], shown to be localised to the Golgi in both cells

Altmann et al. and embryos [49]. BLAST searching indeed suggests the presence of other mannosidase I and II homologues, whose presence is a possible explanation for the ability of MAS-1 null flies to still process oligomannosidic oligosaccharides [50]. There are also homologues, with unknown functions, of N-acetylglucosaminyltransferases III and IV. None of the β1,4-galactosyltransferase homologues identified in this laboratory have yet proven to be active. Interestingly, antibodies raised against a highly glycosylated plant enzyme, horseradish peroxidase (HRP), are useful reagents in the study of the fly neural system. Indeed, all neurons and the male reproductive tissue of Drosophila and grasshoppers can be stained using this antibody [51–53]. In theory, the binding of anti-HRP to these neuronal cells, apparently dependent on the carbohydrate, may be due to α1,3-linked fucose, β1,2-linked xylose or both. By comparison to the glycans of other insects (e.g. bee venom glycoproteins [54, 55]), it would appear that the former type of linkage is most likely, while β1,2-linked xylose has not been found on the glycans of any other insect. Indeed, our most recent investigations would indicate that the N-glycome of adult Drosophila contains no β1,2-linked xylose, but instead, similarly to bee venom glycoproteins, a small quantity of doubly core fucosylated glycans [56]. Previously, only oligomannosidic and core α1,6-fucosylated glycans had been described in Drosophila [57]. Moreover, we cloned a requisite core α1,3-fucosyltransferase cDNA by homology to plant core α1,3-fucosyltransferases and proved the activity of the recombinant enzyme [56]. Three relevant mutants are nac [58, 59], TM3 [51, 58] and Brd15 [60]. The core α1,6-fucosylation is presumably catalysed by the protein encoded by the single fly FUT8 homologue (figure 1). 6. N-glycans in Caenorhabditis As with Drosophila, anti-HRP reactivity has also been reported in Caenorhabditis; specifically 10% of the worm’s neurons can be stained using this reagent [61]. More recently, the results of Western blotting suggested that both the putative anti-fucose and anti-xylose fractions (the latter being a fraction that does not bind immobilised bee venom phospholipase and thus only by subtraction deemed to be ‘xylose-specific’) of an antiserum were found to recognise Caenorhabditis glycoproteins [62]. The only other nematode whose glycans have been described is Haemonchus contortus, a parasite of sheep and cattle. The presence of up to three core fucose residues (two on the proximal GlcNAc and one on the ‘internal’ GlcNAc of the chitobiose core), but no xylose, was detected by FAB-MS of its permethylated N-glycans [63, 64]. The core α1,3-fucose on these glycans has been claimed to be an epitope for IgE from Haemonchusinfected sheep [62].

Genetic model organisms in the study of N-glycans

707

Figure 1. MALDI-TOF mass spectrum of underivatised, reducing N-glycans from C. elegans. The spectrum was acquired on a Dynamo (ThermoBioAnalysis, Santa Fé, USA) in the linear mode. Except for background peaks at ca. 1209 and 1340 Da, the peaks represent N-glycans of C. elegans. While pauci- and oligomannosidic N-glycans could be clearly identified, the linear instrument could not resolve the variously fucosylated and methylated complex-type N-glycans that appear to be typical of C. elegans (see table II and figure 2).

Since no data on the structures of Caenorhabditis N-glycans have been published, we decided to analyse its N-glycome. The experimental approach was similar to that employed by us in the studies on the N-glycomes of a range of plant foods relevant to allergy and of flies [56, 65]. C. elegans (strain N2) were grown at 20 °C with rotary shaking in a liquid S complete medium containing E. coli OP50 resuspended to a final density 20 g/L. After media clearance (ca. 5 days), the worms were sedimented at 4 °C overnight, washed twice with 0.1 M NaCl by centrifugation at 100 g and cleaned by flotation on 30% (w/v) sucrose in water. The worms were then washed twice and resuspended in 0.1 M NaCl, shock-frozen in liquid nitrogen and stored at –80 °C until used. Subsequently, 2 g of frozen worms were thrown into 20 mL stirred boiling water. After cooling, formic acid (to attain a final concentration of 5% (v/v)) and 0.6 mg pepsin were added. Proteolysis was allowed to proceed overnight at 37 °C. The glycopeptides were then purified by serial chromatography on Dowex AG50W×2 and Sephadex G25; the glycans were then released by PNGase A (which unlike PNGase F can remove core α1,3-fucosylated glycans) in 50 mM ammonium acetate, pH 5, and then subject to chromatography on Dowex AG50W×2 ionexchange and Lichroprep reversed-phase resins. Admittedly, this procedure has not been refined for charged glycans; thus, we can only state with certainty that we have prepared the nematode’s neutral N-glycans. The purified glycans were analysed by MALDI-TOF MS in their underivatised state, as well as by RP-HPLC after tagging by pyridylamination. The N-glycans were subject to various degradative procedures and then re-analysed by MS, in the case of jack bean α-mannosidase and 0.5 M

trifluoroacetic acid (1 h, 80 °C), or by RP-HPLC of the unfractionated mixture, in the case of α-mannosidase, bovine kidney α-fucosidase and endoglycosidase H. These techniques were essentially employed as previously described [56, 65]. However, high resolution MALDITOF spectra were also acquired on Elite STR instrument (PerSeptive Biosystems) in the reflectron mode. In order to minimise the confusing influence of potassium adducts, 10 nmol ammonium sulphate was added to samples before co-crystallisation with HIC-DHB matrix [65]. The monosaccharide composition was analysed by gas chromatography/mass spectrometry of alditol acetates [54]. From our analyses, we can conclude that Caenorhabditis glycoproteins contain four types of N-glycans: 1) Oligomannosidic N-glycans up to Man9GlcNAc2 and even some Glc1Man9GlcNAc2 (see table II for masses). Pyridylaminated glycans which had the elution properties of oligomannose structures on RP-HPLC were sensitive to α-mannosidase and endoglycosidase H and gave the expected products. MALDI-TOF MS of the α-mannosidase digest of the entire glycan pool revealed the persistence of a small amount of Hex6GlcNAc2 (presumably Glc1Man5GlcNAc2) which is the expected limit digestion product of Glc1Man9GlcNAc2 if cleavage of α1,6-linked mannose residue in the corepentasaccharide is sterically hindered. 2) Fucosylated ‘paucimannosidic’ N-glycans: certainly the presence of an α1,6-fucosylated trimannosyl core structure (so-called MMF6) was strongly indicated by a respective peak on RP-HPLC and its sensitivity to, and demonstration of the expected shift behaviour upon, treatment with α-fucosidase and α-mannosidase.

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Altmann et al.

Table II. N-Glycan composition of Caenorhabditis elegans. Isotopic masses (Na+ adducts) and peak area percentages were determined by MALDI-TOF MS. According to GLC-MS, the Hex residues are mannose and dHex residues are fucose. Experimental Mass 933.320 1079.374 1095.366 1136.398 1225.456 1239.459 1241.432 1255.454 1257.429 1269.436 1385.501 1387.476 1399.437 1401.492 1415.510 1417.481 1419.499 1533.549 1547.536 1549.537 1561.517 1563.501 1581.494 1693.556 1695.510 1707.591 1709.572 1743.571 1855.659 1905.630 2067.731

Calculated Mass

Area (%)

Molar composition HexNAc

Hex

dHex

O-Me

933.317 1079.375 1095.370 1136.397 1225.433 1239.449 1241.428 1255.444 1257.423 1269.459 1385.507 1387.486 1399.522 1401.502 1415.517 1417.497 1419.476 1533.544 1547.560 1549.539 1561.575 1563.554 1581.529 1693.617 1695.597 1707.633 1709.612 1743.581 1855.670 1905.634 2067.687

14.1 7.7 5.8 2.8 1.5 2.0 3.3 3.9 7.2 1.5 1.1 2.2 0.9 4.2 1.5 1.8 3.7 1.0 2.2 1.9 1.2 1.6 3.5 1.2 1.0 0.8 1.9 6.1 0.9 10.7 0.9

2 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

3 3 4 3 3 3 4 4 5 4 3 4 3 4 4 5 6 4 4 5 4 5 7 4 5 4 5 8 5 9 10

– 1 – – 2 2 1 1 – 1 3 2 3 2 2 1 – 3 3 2 3 2 – 4 3 4 3 – 4 – –

– – – – – 1 – 1 – 2 1 – 2 1 2 1 – – 1 – 2 1 – 1 – 2 1 – 1 – –

3) Minute amounts of a complex type N-glycan, i.e., the core-pentasaccharide with one additional GlcNAc-residue as judged from the mass (table II). 4) Most remarkably, a range of fucosylated paucimannosidic N-glycans containing three to five mannose residues, up to four fucose residues and even O-methyl groups (table II). Removal of fucose residues from the pool of reducing N-glycans by mild acid hydrolysis revealed, in addition to non-methylated Man3GlcNAc2, the presence of methylated Man3GlcNAc2 suggesting the presence of O-methyl-mannose residues. Monosaccharide analysis of the N-glycan pool indeed indicated the presence of 2-Omethyl fucose and 3- or 4-O-methyl mannose in similar amounts in addition to fucose and mannose. Naturally occurring O-methyl groups, e.g. as 3-O-methyl mannose, have been reported for snails [66]. However, often mass spectrometry is performed after a chemical methylation protocol and in these cases natural methylation would not be detected.

While α-mannosidase treatment of the N-glycan pool led to digestion of Man3GlcNAc2Fuc1 (presumably MMF6) as judged by mass spectrometry (and also HPLC), the N-glycans with two or three fucose residues essentially proved resistant to α-mannosidase regardless of the degree of methylation. Therefore we conclude that some fucoses are directly linked to mannose residues. Our preliminary data can neither prove nor disprove the occurrence of minute amounts of core α1,3-fucose. However, the multiple fucose residues of C. elegans N-glycans are apparently organised in a manner different from that postulated for Haemonchus contortus N-glycans as, e.g., (m/z = 1533.55) would be Man4Fuc3GlcNAc2 α-mannosidase sensitive if the three fucose residues were all linked to the chitobiose core. In fact, preliminary data presented by others [67] corroborate the conclusion that some of the fucose-residues are linked to mannoses. Even though no non-reducing terminal GlcNAc could be found on these structures, the fact that they comprise

Genetic model organisms in the study of N-glycans

709 Data on the glycan structures from worm obviously raise questions as to the biosynthesis of these structures. If we hypothesise that one (at least an α1,6-linked fucose), and perhaps even two (although we have no direct proof of α1,3-fucosylation other than anti-HRP binding), of the fucose residues are attached to the core, we should consider that for all known core α1,3- and α1,6fucosyltransferases, including the recombinant Drosophila core α1,3-fucosyltransferase, the prior action of N-acetylglucosaminyltransferase I (GlcNAc-TI) is a prerequisite for an N-glycan being a substrate. Indeed there are three worm homologues of GlcNAc-TI (gly-12, gly13, gly-14) [68], in addition to an N-acetylglucosaminyltransferase II [69] and an N-acetylglucosaminyltransferase V homologue (designated gly-2), the latter able to rescue Chinese hamster ovary Lec4 mutants [70]. On the other hand, both Caenorhabditis and Drosophila N-glycomes contain only low amounts of glycans with GlcNAc residues on the antennae, but a high percentage of the fucosylated glycans. This apparent contradiction is probably reconcilable due to the presence of Golgi β-hexosaminidase; certainly, such an activity has been found in insect cells [71].

Figure 2. Selected parts of the MALDI-TOF mass spectrum of underivatised, reducing N-glycans from C. elegans. Spectra were obtained on a Voyager Elite STR MALDI-TOF instrument in the reflecton mode. The matrix used was a mixture of 2,5dihydroxybenzoic acid and 1-hydroxyisoquinoline as previously described [65], but containing ammonium sulphate which suppresses potassium adducts. The spectrum was internally recalibrated using the peaks of oligomannosidic N-glycans. Noteworthy are the peaks representing di-O-methyl(1561.52 Da), mono-O-methylFuc3Man4GlcNAc2 (1693.55 Da) and di-O-methylFuc4Man4GlcNAc2 Fuc4Man4GlcNAc2 (1707.59 Da).

maximally five mannose residues (reminiscent of the substrate for GlcNAc transferase I) suggests that these glycans may be regarded as the worm’s equivalent of complex type N-glycans. A full structural investigation of these glycans obviously requires NMR to supplement the HPLC and MS data.

Whereas the Drosophila core α1,3-fucosyltransferase cDNA (one of up to four possible α1,3-fucosyltransferase homologues) has been cloned and expressed, it is not clear whether any α1,3-fucosyltransferase homologue in Caenorhabditis could be responsible for core α1,3fucosylation. Of the five α1,3-fucosyltransferase homologues found in the worm (one each encoded by cosmids KO8F8, K12H6 and F59E12 and two encoded by cosmid T05A7), only the first has yet been proven to have enzymatic activity (after engineering of the cDNA to remove a partial putative intron and associated stop codon) as a Lewis-type enzyme [72]. Homology searching offers no clear clue that any of the others may be more related to the insect/plant core α1,3-fucosyltransferases. On the other hand, simple BLAST searching suggests the presence of a single worm core α1,6-fucosyltransferase, of the mammalian FUT8 type, and of a single H-type α1,2-fucosyltransferase. However, searching for short motifs suggests that Caenorhabditis may have up to 22 potential α1,2-fucosyltransferase genes [73, 74]. It obviously remains to be resolved whether these multiple α1,2and α1,3-fucosyltransferases are responsible for the ‘extra’ fucose residues present on Caenorhabditis N-glycans, participate in the fucosylation of other glycoconjugate structures or are just unexpressed evolutionary relicts. Due to the apparent absence of any sequences for sugar O-methyltransferases, the relevant worm genes remain unidentified. Otherwise, all the normal ER mannosyl- and glucosyltransferases are encoded by the worm genome, including a type of Dol-P-Man synthetase like that of mammals and of Schizosaccharomyces (rather than like that of Saccharomyces or protozoa) [75].

710 7. Perspectives In this article, we have summarised the current status of knowledge about the N-glycosylation of three frequentlyused ‘genetic model’ eukaryotes, Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana and present new data on the N-glycans of the first. A number of cDNAs proven to encode enzymatically-active glycosyltransferases have been cloned from these species and the first neutral N-glycome spectra have been obtained. It is obvious that we are quite some way from a full genetic and biochemical dissection of their glycosylation pathways, even though these are simple in comparison to mammals; also, due to this relative simplicity, there may be less than was hoped to be gleaned from their glycobiology that is directly relevant to the study of mammalian systems. Indeed, the most conserved aspects of N-glycan and glycosylphosphatidylinositol biosynthesis in the endoplasmic reticulum have already been extensively examined in yeast (with advantages in the amounts that can be generated for biochemical work), and findings from yeast have direct relations to some human diseases, e.g., some congenital disorders of glycosylation (CDGs) and paroxysmal nocturnal haemoglobinuria (PNH). Indeed, forms of glycosylation such as found in proteoglycans and O-linked fucose in worm and fly (not mentioned in this review for reasons of space) are probably the most promising areas for those wishing to make direct comparisons; however, other than in the general ‘of what are glycans theoretically capable’ type of question, the study of Caenorhabditis, Drosophila and Arabidopsis will certainly have more value when considering other nematodes, insects or plants of medicinal, biotechnological or agricultural interest. Acknowledgments Work in the authors’ laboratories is funded by grants from the Fonds zur Förderung der wissenschaftlichen Forschung (P13810-GEN) and Neose Technologies, Inc., to I.B.H.W. and the Jubiläumsstiftung der Österreichischen Nationalbank (7080) and EU Fifth Framework Programme (QLK1-1999-00765) to F.A. We also thank Ing. Thomas Gramanitsch, Boehringer Ingelheim, for operating the reflectron MALDI-TOF and Verena Jantsch-Plunger, Institute of Molecular Pathology, for advice on worm rearing.

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