Báo cáo khoa học: Neutral N-glycans of the gastropod Arion lusitanicus potx

Neutral N-glycans of the gastropod Arion lusitanicus Martin Gutternigg, Karin Ahrer, Heidi Grabher-Meier, Sabine Bu¨ rgmayr and Erika Staudacher Department fu ¨ r Chemie, Universita ¨ tfu ¨ r Bodenkultur Wien, Vienna, Austria The neutral N-glycan structures of Arion lusitanicus (gas- tropod) skin, viscera and egg glycoproteins were examined after proteolytic digestion, release of the glycans from the peptides, fluorescent labelling with 2-aminopyridine and fractionation by charge, size and hydrophobicity to obtain pure glycan structures. The positions and linkages of the sugars in the glycan were analysed by two dimensional HPLC (size and hydrophobicity) and MALDI-TOF mass spectrometry before and after digestion with specific exoglycosidases. The most striking feature in the adult tis- sues was the high amount of oligomannosidic and small paucimannosidic glycans terminated with 3-O-methylated mannoses. The truncated structures often contained modi- fications of the inner core by b1,2-linked xylose to the b-mannose residue and/or an a-fucosylation (mainly a1,6-) of the innermost GlcNAc residue. Skin and viscera showed predominantly the same glycans, however, in different amounts. Traces of large structures carrying 3-O-methylated galactoses were also detected. The egg glycans contained mainly ( 75%) oligomannosidic structures and some pau- cimannosidic structures modified by xylose or a1,6-fucose, but in this case no methylation of any monosaccharide was detected. Thus, gastropods seem to be capable of producing many types of structures ranging from those typical in human to structures similar to those found in nematodes, and therefore will be a valuable model to understand the regulation of glycosylation. Furthermore, this opens the way for using this organism as a host for the production of recombinant proteins. The detailed know- ledge on glycosylation also may help to identify targets for pest control. Keywords: Arion lusitanicus; gastropod; glycosylation; N-glycans; snail. Gastropods are intermediate hosts for schistosomes, which are pathogenic to humans and domestic animals. In addition to schistosomiasis, diseases such as fascioliasis, clonorchiasis and paragonimiasis represent only a few of the snail transmitted diseases with worldwide medical and economic impact. Other potential candidates for pest control are those gastropods, mainly slugs, which cause damage to vegetables. The worst case is a complete crop failure but even their eating or moving tracks reduce the commercial value of lettuce. Structural features, which do not occur in higher animals, are valuable candidates as a target for pest control. The most effective way would be inhibition of enzymes that are not typical of mammals and that are responsible for structures important for slug/snail survival or reproduction. This would be a convenient way to reduce the population of these animals without high amounts of conventional chemical pesticides. Analysing the complete set of N-glycan structures of a species gives an overview on its biosynthetic capacity for glycosylation. It is the first step for the identification of glycosylation related target enzymes for inhibition. So far, N-glycan structures derived from the hemocyanins of the snails Helix pomaia, Lymnaea stagnalis, Rapana venosa and the keyhole limpet Megathura crenulata have been published. The Helix pomatia glycans show complex structures containing a common core with an a1,6-linked fucose to the reducing GlcNAc and a b1,2-linked xylose to the b-mannose residue. One or both a-mannose residues may be substituted by GalNAcb1,4GlcNAcb1,2 elements which contain two to four b1,3- or b1,6-linked galactoses with or without 3- or 4-O-methyl groups [1]. Lymnaea stagnalis hemocyanin contains low and high molecular mass biantennary oligosaccharides. They lack the a1,6-linked fucose to the inner GlcNAc residue, but some antennae terminate with an a1,2-linked fucose. Similarly to Helix pomatia, the basic element of the antennae is Galb1,3Gal- NAcb1,4GlcNAc [2,3]. The two N-glycans of the functional unit RvH1-a of Rapana venosa hemocyanin are biantennary nonfucosylated oligosaccharides with 3-O-methylated ter- minal b1,3-linked galactose residues. One of these residues also carries a sulfate group on the a1,6-linked core mannose and a 3-O-methylated GlcNAc residue b1,2-linked to the b-mannose of the core [4]. Megathura crenulata hemocyanin is substituted by a novel type of N-glycan with galactoses directly linked in b1,6-linkage to mannose residues [5]. Recently a core structure terminated with two 3-O-methy- lated mannose residues linked to the major soluble protein of the organic shell matrix of Biomphalaria glabrata was identified [6]. Furthermore some characteristics of a few enzymes which are involved in gastropod glycan biosynthesis have Correspondence to E. Staudacher, Department fu ¨ rChemie, Universita ¨ tfu ¨ r Bodenkultur Wien, Muthgasse 18, A-1190 Vienna, Austria. Fax: + 43 136006 6059, Tel.: + 43 136006 6063, E-mail: erika.staudacher@boku.ac.at Abbreviation: endoglycosidase H, endo-b-N-acetylglucosaminidae H. Enzymes: endo-b-N-acetylglucosaminidae H (EC 3.2.1.96). Note: The abbreviations for the glycan structures are detailed in Figs 2 and 4. (Received 22 December 2003, revised 2 February 2004, accepted 18 February 2004) Eur. J. Biochem. 271, 1348–1356 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04045.x been determined. However, the information gained is restricted in most cases to the enzyme specificity in vitro and some biochemical parameters. Lymnaea stagnalis has been shown to contain the key enzyme for the formation of complex N-glycans, GlcNAc-transferase I, which has been proven to be a prerequisite for the action of GlcNAc- transferase II, fucosyltransferases and xylosyltransferase [7]. This organism has also been shown to contain GlcNAc-transferase II and xylosyltransferase [7], a b1,4- GalNAc-transferase which shows similar characteristics to mammalian b1,4-galactosyltransferase [8], a b1,3-galacto- syltransferase and an a1,2-fucosyltransferase [9,10]. Hybridization experiments using a bovine b1,4-galactosyl- transferase cDNA probe resulted in the isolation of a clone encoding a b1,4-GlcNAc-transferase which is similar to the mammalian galactosyltransferase in acceptor specificity but requires a different nucleotide sugar. It is definitely not involved in the biosynthesis of the chitobiose core of N-glycans [11–13]. The function of this enzyme in vivo is not clear. The prostate glands of these snails also contain a b1,4-glucosyltransferase forming Glcb1,4GlcNAc units [14]. Furthermore an a1,3-fucosyltransferase catalysing the transfer of fucose from GDP-fucose to a Galb1,4Glc- NAc acceptor forming the Lewis X -unit has been found in the connective tissue of Lymnaea stagnalis [10] and an a1,3-fucosyltransferase catalysing the transfer of fucose from GDP-fucose to the asparagine-linked GlcNAc has been found in the albumin and prostate glands of the same snail[15].However,noLewis X -containing structures, core a1,3-fucosylated structures, or glucosylated units have been detected in the glycans of this snail so far. An a1,2- L -galactosyltransferase which seems to be involved in the elongation of the storage polysaccharide of the snail was found in Helix pomatia [16]. Although in vitro this galactosyltransferase catalyses the transfer of a fucose into a1,2-linkage from GDP-fucose to a Galb1,3Gal-O-Me substrate, nothing is known about this ability in vivo. A number of exoglycosidases have been described from gastropodian sources. Some of them are commercially available and widely used as tools in glycomic research. The majority of these enzymes seem to be part of the degrada- tion and recycling processes of the cells and not be involved in the N-glycosylation pathway. In the present study we present for the first time the neutral N-glycan structures of a whole gastropod, the slug Arion lusitanicus, in two developmental stages, to show its capability for N-glycan biosynthesis and processing. Materials and methods Materials Slugs were collected by M. Pintar (Department for Integrative Biology, Institute for Zoology, Universita ¨ tfu ¨ r Bodenkultur Wien, Vienna, Austria) and his students in local gardens and were frozen immediately at )80 °C. Eggs were collected by the authors, lyophilized and kept at )20 °C until use. Sephadex G25 fine and Sephadex G15 were purchased from Amersham Biosciences, and Dowex 50W·2 was from Fluka (Fluka Chemie, Vienna, Austria). Standard pyridy- laminated glycans were prepared in the course of previous studies [17,18]. All other materials purchased were of the highest quality available from Merck or Sigma. Preparation of N-glycans Thawed slugs (10 individuals for each preparation) were washed to remove the extraneous mucous components and dissected into three fractions; the skin and inner organs (viscera) were lyophilized separately, while the intestinal tract was discarded. The dry material (skin, viscera or eggs) was suspended in 200 mL of 50 m M Tris/HCl buffer pH 7.5, homogenized with an IKA Ultra Turrax T25 (IKA-Labortechnik, Janke and Kunkel GmbH, Staufen, Germany) at 15 000 r.p.m. for 2· 20 s and centrifuged at 5000 g for 10 min. The supernatant was adjusted to 80% (w/v) of ammonium sulfate and centrifuged at 27 500 g for 40 min. The precipitate was dialyzed against water, con- centrated on rotary evaporation and made up to 150 m M of Tris/HCl, 1 m M CaCl 2 , pH 7.8. Thermolysin (ICN Biomedicals, Vienna, Austria) was added at a 40 : 1 (w/w) ratio of protein/enzyme and incubated for 20 h at 50 °C. The digest was dialyzed against 2% (v/v) acetic acid andappliedtoacolumnof100mLofDowex50W·2 equilibrated in 2% (v/v) acetic acid. The column was washed with 150 mL of the same solution and the (glyco)peptides were eluted with 0.4 M ammonium acetate, pH 6.0, concentrated and applied onto an Sephadex G25 column (1 · 120 cm) equilibrated in 1% (v/v) of acetic acid. Carbohydrate containing fractions detected by the orcinol- sulfuric acid method according to Winzler [19] were pooled, lyophilized and dissolved in approximately 1 mL citrate- phosphate buffer, pH 5.0. The N-glycans were released by incubation with 0.7 U of peptide:N-glycosidase A (Roche) at 37 °C for 24 h, purified on Sephadex G15, Dowex 50W·2 and Lichroprep RP (Merck) according to [18] and labelled with 2-aminopyridine as described previously [20,21]. Analysis of monosaccharides Monosaccharide analysis was carried out by hydrolysis of the glycans with 4 M trifluoroacetic acid at 100 °C followed by derivatization with 3-methyl-1-phenyl-2-pyrazolin-5-one and separation on reverse-phase HPLC according to Fu and O’Neill [22] or by conversion of the monosaccharides into their corresponding alditol acetates, which were then analysed by gas chromatography/mass spectrometry as described [21]. Separation and analysis of N-glycans Fluorescently labelled oligosaccharides were separated into neutral and negatively charged fractions on an Econo-Pac High Q Cartridge (5 mL, Bio-Rad Laboratories) at a flow rate of 1 mLÆmin )1 . Solvent A was 50 m M Tris/HCl, pH 8.5; solvent B was 1 M NaCl in solvent A. The run was started with 5 min at 100% solvent A followed by a linear gradient of 5% per min to 50% solvent B, continued with 10% per min to 100% solvent B and terminated by 1 min at 100% solvent B. Fluorometric detection was carried out at excitation and emission wavelengths of 320 and 400 nm, respectively. Ó FEBS 2004 Neutral N-glycans of Arion lusitanicus (Eur. J. Biochem. 271) 1349 The neutral fraction was further fractionated by a two dimensional mapping technique starting with separation according to hydrophobicity on an Hypersil ODS column (0.4 · 25cm, 5l, Forschungszentrum Seibersdorf, ARC Seibersdorf research GmbH, Seibersdorf, Austria) [21]. Fluorometric detection was performed at excitation and emission wavelengths of 320 and 400 nm, respectively, and peaks were collected and dried prior to subfractionation by size, in the second dimension. The method was modified from the procedure of Khoo et al. [23] using a Palpak type N column (4.6 · 250 mm, Takara, Japan) at a flow rate of 1 mLÆmin )1 . Solvent A was 75 : 25 (v/v) acetonitrile/ stock solution [3% (w/v) acetic acid-triethylamine buffer at pH 7.3 with 10% (v/v) acetonitrile]. Solvent B was 50 : 50 (v/v) acetonitrile/stock solution. The run was started with 5 min at 10% solvent B followed by a linear gradient of 2.8% per min to 80% solvent B, and terminated by 8 min at 80% solvent B. Fluorimetric detection was performed at excitation and emission wavelengths of 310 and 380 nm, respectively. Columns were calibrated in terms of glucose units with a pyridylaminated partial dextran hydrolysate (3–11 glucose units). Peaks from either size fractionation or reverse-phase chromatography were analysed by MALDI-TOF and subjected to exo- or endoglycosidase digestions. MALDI-TOF MS analysis MALDI-TOF MS was carried out as described previously [24]. The sample (1 lL, 0.2–0.8 pmol) was spotted onto a target and dried, followed by the addition of 0.8 lLof matrix [2% (v/v) 2,5-dihydroxybenzoic acid in water containing 30% (v/v) acetonitrile]. The plate was transferred immediately to a desiccator and vacuum was applied until all solvent had evaporated. Spectra were recorded on a DYNAMO linear MALDI-TOF mass spectrometer (Thermo BioAnalysis, Hemel Hempstead, UK) operated with a dynamic extraction setting of 0.1. External mass calibration was performed with pyridylaminated N-glycan standards derived from bovine fibrin. About 20 individual laser shots were summed. In some cases, on-target digestions with exoglycosidases were carried out using 6-aza-2-thiothymine [0.5% (w/v) in water] as the matrix [25]. Exo- and endoglycosidase digests Endoglycosidase H (recombinant from Escherichia coli, Roche) was used at a concentration of 2 mU in 0.15 M citrate-phosphate buffer, pH 5.0 containing 0.1 M NaCl; a-mannosidase (jack bean, Sigma) at 2 mU in 50 m M sodium acetate, pH 4.5 containing 0.2 m M ZnCl 2 ; a-fuco- sidase (bovine kidney, Sigma) at 2 mU in 50 m M sodium citrate, pH 4.5; a1,2-fucosidase (recombinant, Sigma) at 0.2 mU in 50 m M sodium phosphate pH 5.0; b-galactosi- dase (bovine testis, Roche) at 1.6 mU in 50 m M sodium citrate, pH 5.0 and b-hexosaminidase (bovine kidney, Sigma) at 25 mU in 20 lLof0.1 M sodium citrate, pH 5.0). Incubations were carried out in 20 lL of appro- priate buffer at 37 °Covernight. For chemical release of fucose a1,3-linked to the inner GlcNAc-residue, the dry sample was incubated for 48 h at 0 °Cwith20lL of 48% (v/v) hydrofluoric acid. The acid was then removed under a stream of nitrogen [26]. Results Adult tissues Oligomannosidic structures. The N-glycan pattern of the labelled glycans on reverse-phase chromatography can be divided into four regions, I–IV (Fig. 1). The first region (4–6.8 glucose units) contains mainly oligomannosidic structures (M 5 –M 9 ; abbreviations of glycan structures are giveninFig.2),whichwereconfirmedbytheirelution behaviour on HPLC in comparison with standard glycans, their mass on MALDI-TOF and their sensitivity to a-mannosidase and endoglycosidase H (Table 1 and data not shown). Using MALDI-TOF, moderate digestion with a-mannosidase gave a ladder of structures with masses with a distance of 162.1 mass units, this effect could also be observed on Palpak-HPLC. Endoglycosidase H digest on MALDI-TOF caused a shift by 281 mass units, indicating the loss of a GlcNAc-residue containing the fluorescent group. Using HPLC, just the pyridylaminated GlcNAc- residue is still visible by the detector. Structural isomers of M 7 and M 8 were identified by their elution behaviour on reverse-phase. Methylated oligomannosidic structures. Region II of the reverse-phase pattern (Fig. 1) contained, in the preparations of the adult snails, methylated mannosidic structures with mainly five to seven mannose residues and two or more, often three, methyl groups (abbreviations of glycan structures are given in Fig. 2). Methylated M 4 ,M 8 and M 9 structures were also found, however,in very low amounts (Table 1). All these structures were sensitive to endoglycosidase H (Fig. 3). To confirm the presence of 3-O-methylmannose residues, we performed carbohydrate composition analysis by gas chromatography/mass spectrometry. Incomplete methy- Fig. 1. HPLC analysis of pyridylaminated neutral N-glycans of Arion lusitanicus on a reverse-phase column. (A) Isomaltose standard, 4–14 glucose units, (B) skin, (C) viscera and (D) eggs. Regions I–IV are indicated with arrows. I, oligomannosidic structures; II, methylated oligomannisidic structures; III, a1,6-fucosylated structures; IV, large galactose containing structures. 1350 M. Gutternigg et al.(Eur. J. Biochem. 271) Ó FEBS 2004 lated structures were subjected to an a-mannosidase digest which made it possible to identify the position of the unmethylated mannose in most cases. For example, if the terminal mannose of the a1,3-arm of a M 9 structure was not methylated, three mannoses could be released. If one of the terminal mannoses of the a1,6-arm lacked the methyl group, two mannoses could be released, but we saw in our experiments that the middle arm appeared to be less accessible to the enzyme and so only one mannose was cleaved in this case. Due to their insensitivity to a-mannosidase, the majority of the methylated oligo- mannosidic (M 5 ,M 6 and M 8 ) glycans were determined to be methylated on each terminal mannose. Structures lacking one methyl group were present only in a few percent of the oligomannosidic methylated glycans (<10%), whereas structures lacking two methyl groups were detectable only in trace amounts. If one methyl group was missing, it was in most cases the middle antennae which was unmethylated, whereas, if just one methyl group was present no preferences could be determined. a1,6-fucosylated structures. The third region of the reverse-phase pattern was characterized by structures with an a1,6-fucose linked to the inner GlcNAc (Fig. 1, Table 1). This fucose could be easily removed by a-fucosidase from bovine kidney. A shift of )146.1 mass units on MALDI-TOF and the characteristic shift to earlier elution times on reversed phase chromatography confirmed the loss of a fucose linked a1,6 to the inner core. The main compound was dimethylated Me 2 MMF 6 in skin and viscera (abbreviations of glycan structures are given in Fig. 4). However, in viscera the monomethylated variant MeMMF 6 , and in skin a xylosylated variant Me 2 MMXF 6 were also detected. No a1,6-fucosylated glycans lacking the methyl groups could be determined in adult tissues (Table 1). Fig. 2. Structures of paucimannosidic (four mannose residues or less) and oligomannosidic glycans. The abbreviation system applied herein (according to [18]) names the terminal residues, starting with the residue on the 6-linked antenna and proceeding counter clockwise. Ó FEBS 2004 Neutral N-glycans of Arion lusitanicus (Eur. J. Biochem. 271) 1351 Paucimannosidic structures. Examination of regions I and II of the reverse-phase pattern suggested the presence of small paucimannosidic structures. Therefore a further preparation removing the oligomannosidic structures by digestion with endoglycosidase H prior to fractionation on reverse-phase was performed. Using this strategy in both tissues, small truncated structures were found (Table 1). They contained up to four mannose residues and additional xylose and/or fucose residues linked to the inner core. Similar to the previously described oligo- mannosidic structures, MMX occurred in a nonmeth- ylated, a mono- and a di-3-O-methylated form at the terminal mannose residues. In most of the cases the expected GlcNAc-residue linked to the a1,3-mannose was missing. This ÔGlcNAc IÕ (incor- porated by N-acetylglucosaminyltransferase I in b1,2-link- age to the a1,3-linked mannose) has been proven in other sources to be a prerequisite for the further transfer of core- modifying enzymes (fucosyltransferases and xylosyltrans- ferase). It can be speculated that the snail enzymes do not need this GlcNAc I residue for their action or, more probably, that a Golgi-hexosaminidase removes the Glc- NAc I in an early processing stage of the developing N-glycan as it has been shown previously for insects and nematodes [27,28]. The paucimannosidic glycans eluting in the first two regions on reversed phase HPLC and carrying a fucose residue were subjected to more intensive investigation. The small size of the glycans and successful b-hexosaminidase and/or a-mannosidase digests led to the conclusion that these fucose residues were linked to the core. While a1,6- linked fucose at the inner GlcNAc residue increases elution time drastically on a reverse-phase column [29], glycans with an a1,3-fucoselinkedtothesameGlcNAcelute earlier at the positions found for the paucimannosidic Table 1. Neutral N-glycan profiles of Arion lusitanicus. Wherever the detected traces are less then 0.2% an exact quantitation is not possible. Therefore the amount is considered to be 0.1%. Structure Skin (%) Viscera (%) Eggs (%) Mannosidic structures MU  0.1 1.6 1.5 MM – – 5.1 M 4 1.5 2.5 1.0 M 5 1.0 21.6 19.6 M 6 2.1 8.2 16.9 M 7 1.5 3.7 14.8 M 8 1.5 4.2 14.9 M 9 1.0 5.2 1.5 GlcM 9  0.1 – – Sum 8.8 47.0 75.3 Methylated mannosidic structures MeMU 2.1 1.2 – MeMM  0.1 1.2 – Me 2 MM 26.9 10.1 – Me 1-2 M 4 1.0 1.3 – Me 1-2 M 5 5.3 2.5 – Me 3 M 5 24.1 10.8 – Me 1-2 M 6 0.3 0.6 – Me 3 M 6 6.6 9.4 – Me 1-2 M 7 0.7 2.9 – Me 3 M 7 0.5  0.1 – Me 1-2 M 8  0.1 0.4 – Me 3 M 8 0.8  0.1 – Me 1-2 M 9 0.6  0.1 – Me 3 M 9 –– – Me 2 GlcM 9 0.3 – – Sum 69.6 40.7 – a1,6-fucosylated structures MUF 6 – – 5.0 MMF 6 – – 2.9 MGnF 6 – – 2.3 MGnXF 6 – – 1.8 Sum – – 12.0 Methylated a1,6-fucosylated structures MeMMF 6 – 0.3 – Me 2 MMF 6 8.7 4.4 – Me 2 MMXF 6 1.2 – – Sum 9.9 4.7 – Other paucimannosidic structures MUX – – 1.2 MMX 0.4 0.7 11.5 M 4 X  0.1 – – MMXF 3 1.7 0.4 – GnGnXF 3 0.7  0.1 – Sum 2.9 1.2 12.7 Other methylated paucimannosidic structures MeMMX 0.4 2.2 – Me 2 MMX 3.5 1.2 – MeM 4 X 0.6 0.8 – Sum 4.5 4.2 – Complex type structures with methylated galactoses Sum 3.9 2.0 – Fig. 3. MALDI-TOF MS spectra of pyridylaminated oligosaccharides from region II. Before (A) and after (B) digest with endoglycosidase H. Structures labelled with an asterisk were not cleaved by endoglycosi- dase H. 1352 M. Gutternigg et al.(Eur. J. Biochem. 271) Ó FEBS 2004 glycans under study. The investigated fucose residues could only be cleaved by HF-treatment of the glycans and not by the usual amounts of commercially available fucosidases (Fig. 5), which confirmed the occurrence of a low amount of a1,3-fucosylation of the core in both adult tissues. Complex structures. Less than 4% of the structures of adult tissues contained various larger N-glycans with a number of galactose residues terminated with methyl groups, eluting in region IV of the reversed phase pattern (Fig. 1). The linkage of the methyl groups was identified by gas chromatography/mass spectrometry to be 3-O- methylation. In contrast to [4], we could not observe a removal of the methylated galactose by bovine testis galactosidase, therefore subsequent exoglycosidase diges- tions were not possible. Due to the low amount and the heterogeneity of the fractions a detailed analysis of those glycans was omitted. From our data we suspect that the structures may be similar to those described by Lommerse Fig. 4. Structures of paucimannosidic glycans with or without core fucosylation or xylosyla- tion. The abbreviation system applied herein (according to [18]) names the terminal resi- dues, starting with the residue on the 6-linked antenna and proceeding counter clockwise. In the case of the core fucose, which occurs in more than one type of linkage, the linkage is depicted as a superscript. Fig. 5. HPLC analysis of pyridylaminated MMXF 3 on a reverse-phase column. (A) Isomaltose standard, 3–11 glucose units, (B) MMXF 3 , (C) MMXF 3 after incubation with a-fucosidase from bovine kidney and (D) MMXF 3 after incubation with hydrofluoric acid. Ó FEBS 2004 Neutral N-glycans of Arion lusitanicus (Eur. J. Biochem. 271) 1353 et al.[1]forHelix pomatia a D -hemocyanin, where one or both antennae of biantennary xylosylated glycans termin- ate with a varying number of methylated galactose residues. Eggs The egg glycans differed from those derived from adult tissues (Table 1). While in preparations of adult slugs the unmethylated, oligomannosidic structures were restricted to 8.8% and 47% in skin and viscera, respectively, in the eggs  75% of the total N-glycans were oligomannosidic struc- tures, dominated by M 5 –M 8 glycans. The remaining 25% of structures were equally divided into MMX and a series of a1,6-fucosylated small glycans, some of them carrying the GlcNAc I. The most striking result, however, was the complete absence of methylated structures in eggs. No oligomannosidic or paucimannosidic structures were sub- stituted by methyl groups. Discussion In order to obtain information about the N-glycan biosyn- thesis capacity/capability of the gastropod Arion lusitanicus we performed a protein preparation of whole animals (except the digestion system) separated into viscera and skin fractions. The tissues were homogenized and the proteins were precipitated and digested with thermolysin. After purifica- tion of the (glyco)peptides by ion exchange chromatography and gelfiltration, the N-glycans were released by PNGase A in order to ensure that a1,3-fucosylated structures were also released [30]. The oligosaccharides were labelled with 2-aminopyridine and separated by anion exchange chro- matography into neutral and negatively charged fractions. To obtain individual structures the neutral fraction was further fractionated on reverse-phase HPLC and the collected peaks were subfractionated on a Palpak column. Aliquots of all fractions were analysed by MALDI-TOF mass spectrometry. Further information was gained by gas- chromatography/mass spectrometry of the alditol acetates. Elution behaviour on both HPLC systems compared with standard oligosaccharides, in combination with the mass information from MALDI-TOF, led to the conclusions about the structure which were confirmed by digestion with specific exoglycosidases. For relative quantitation of the structures see Table 1. In the course of our work we found that the percentages of structures vary slightly with the area where the slugs had been collected (due to nutritional conditions), the age (size) of the individuals and their physiological status (carrying eggs or not). However, skin and viscera preparations contained the same spectrum of N-glycans. Therefore it can be ruled out that unusual structures are due to food or environmental contaminants. The most obvious structural feature of these slug adult tissues is the high degree of structures with terminal 3-O- methylated mannose residues (>80% in skin and  50% in viscera) and traces of structures with 3-O-methylated galactoses (Table 1). Methylated sugars were first described in the early 1970s in the polysaccharides of procaryotes, lower eucaryotes, algae and fungi with soil habitat. In gastropod hemocyanin 3-O-methylated mannose and 3-O-methylated galactose were found in 1977 [31]. Since that time a number of methylated sugars have been found in polysaccharides from plants and procaryotes. In molluscs 3-O-methylated mannose and/or 3-O-methylated galactose were found in some hemocyanins [32], 6-O-methylation of mannose was found in the giant clam Hippopus hippoppus [33] and 3-O-methyl galactose and 3-O-methyl GlcNAc in Rapana venosa [4]. In nematodes 2-O-methylated fucose was found in Toxocara [34] and Caenorhabditis elegans [35]. The high degree of methylation and its occurrence on so many different structures in Arion lusitanicus leads to the assumption that methylation is an important regulating event in this organism. The enzyme(s) responsible appear to be very active and widely distributed along the modifying oligosaccharide steps during the biochemical pathway of the N-glycans. As this modification, as far as we know now, is restricted to lower animals it may be an interesting target for pest control. However, the slugs also contain another set of N-glycans, the occurrence of which seems to be highly regulated. The traces of Me 2 GlcM 9 may be a relic of the early events of glycan processing or play an important role in folding or assembly of a special protein, as has been speculated recently for GlcM 9 of Antheraea pernyi and Bombyx mori arylphorin [36]. Besides the usual set of oligomannosidic structures, Arion lusitanicus tends to accumulate short antennary chains similar to plants, insects and C. elegans, lacking the GlcNAc I residue which has been shown to be neces- sary for the action of a number of modifying enzymes (core-fucosyltransferases, xylosyltransferase and GlcNAc- transferases II–V) [7,37]. A highly active Golgi-located b-N-acetylhexosaminidase has been suggested, which removes the GlcNAc residue from the Mana1,3-antenna after fucosylation and xylosylation; such an enzyme has already been described in insects and C. elegans [27,28]. Due to the small size of the glycans, heterogeneity is mainly caused by modification of the core. A remarkable amount of xylose linked b1,2 to the b-mannose and/or fucosylation of the reducing GlcNAc, was detected. Mainly a1,6-linked fucose was observed. a1,3-linked fucose, like that typical for plants, occurred only in trace amounts. A corresponding a1,3-fucosyltransferase has been detected in Lymnaea stagnalis [15], but here it is the first time that one of its products has been found in a snail. It can be speculated that this structural feature is limited to some very specialized cells and does not occur randomly in the organism. There was no evidence for the presence of difucosylation of the inner GlcNAc-residue found in lepidopteran insects [29] and squid rhodopsin [38], or of difucosylation in combination with a core xylose as is present in Schistosoma japonicum eggs [39]. Terminal fucosylation such as the a1,2- fucosylation seen in another gastropodian source (Lymnaea stagnalis)[3]orLe X determinants were also not found. Arion lusitanicus contains an enormous potential for generating a large set of structural elements commonly found in eukaryotic N-glycosylation: they sialylate [40], they carry a1,6-linked as well as a1,3-linked fucose as shown for some insects, nematodes and trematodes, and b1,2-linked xylose, as found in plants and trematodes, and they are able to methylate terminal sugars (mannose and galactose) as 1354 M. Gutternigg et al.(Eur. J. Biochem. 271) Ó FEBS 2004 found in nematodes. Thus they combine structural features from mammals, plants, insects, nematodes and trematodes. This is the first known complete system where it is possible to investigate the regulation of N-glycan modification in its fullest variety. An understanding of this complex system, i.e. why a distinct structure occurs on a certain protein, will improve our knowledge on the rules of glycan modification and help to optimize the production of recombinant glycoproteins. In addition, the snail system itself may be useful for the production of a large variety of glycoproteins. For example it may present the first opportunity to produce some structures similar to those in pathogenic nematodes or trematodes. Proteins modified in the snail system could for instance be used for the elucidation of the immune response to those nonmammalian structures. Furthermore the snail- produced glycans may be a safe way to stimulate and improve the human immune response to recognize and fight against those pathogenic nematodes and trematodes. Acknowledgements This project was financed by the Austrian Fonds zur wissenschaftlichen Forschung Project number P13928-BIO. We want to thank Dr Manfred Pintar (Department for Integrative Biology, Institute for Zoology, Universita ¨ tfu ¨ r Bodenkultur, Wien) for identification and classification of the slugs, Daniel Kolarich and Dr Friedrich Altmann for support on the MALDI-TOF and Dr Iain Wilson for reading the manuscript. The technical help of Thomas Dalik, Susanna Eglseer and Denise Kerner is highly appreciated. References 1. Lommerse, J.P.M., Thomas-Oates, J.E., Gielens, C., Pre ´ aux, G., Kamerling, J.P. & Vliegenthart, J.F.G. (1997) Primary structure of 21 novel monoantennary and diantennary N-linked carbohydrate chains from a- D -hemocyanin of Helix pomatia. Eur. J. Biochem. 249, 195–222. 2. Van Kuik, J.A., Sijbesma, R.P., Kamerling, J.P., Vliegenthart, J.F.G. & Wood, E.J. (1986) Primary structure of a low-molecular- mass N-linked oligosaccharide from hemocyanin of Lymnaea stagnalis.3-O-methyl- D -mannose as a constituent of the xylose- containing core structure in an animal glycoprotein. Eur. J. Bio- chem. 160, 621–625. 3. Van Kuik, J.A., Sijbesma, R.P., Kamerling, J.P., Vliegenthart, J.F.G. & Wood, E.J. (1987) Primary structure determination of seven novel N-linked carbohydrate chains derived from hemocyanin of Lymnaea stagnalis.3-O-methyl- D -galactose and N-acetyl- D -galactosamine as constituents of xylose-containing N-linked oligosaccharides in an animal glycoprotein. Eur. J. Bio- chem. 169, 399–411. 4. Dolashka-Angelova, P., Beck, A., Dolashki, A., Beltramini, M., Stevanovic, S., Salvato, B. & Voelter, W. (2003) Characterization of the carbohydrate moieties of the functional unit RvH1-a of Rapana venosa haemocyanin using HPLC/electrospray ionization MS and glycosidase digestion. Biochem. J. 374, 185–192. 5. Kurokawa, T., Wuhrer, M., Lochnit, G., Geyer, H., Markl, J. & Geyer, R. (2002) Hemocyanin from the keyhole limpet Megathura crenulata (KLH) carries a novel type of N-glycans with Gal(b1- 6)Man-motifs. Eur. J. Biochem. 269, 5459–5473. 6. Marxen, J.C., Nimtz, M., Becker, W. & Mann, K. (2003) The major soluble 19.6 kDa protein of the organic shell matrix of the freshwater snail Biomphalaria glabrata is an N-glycosylated dermatopontin. Biochim. Biophys. Acta. 1650, 92–98. 7. Mulder, H., Dideberg, F., Schachter, H., Spronk, B.A., De Jong- Brink, M., Kamerling, J.P. & Vliegenthart, J.F.G. (1995) In the biosynthesis of N-glycans in connective tissue of the snail Lymnaea stagnalis of incorporation GlcNAc by b2GlcNAc-transferase I is an essential prerequisite for the action of bGlcNAc-transferase II and b2Xyl-transferase. Eur. J. Biochem. 232, 272–283. 8. Mulder, H., Spronk, B.A., Schachter, H., Neeleman, A.P., Van den Eijneden, D.H., De Jong-Brink, M., Kamerling, J.P. & Vlie- genthart, J.F.G. (1995) Identification of a novel UDP-Gal- NAc:GlcNAcb-Rb1-4 N-acetylgalactosaminyltransferase from the albumen gland and connective tissue of the snail Lymnaea stagnalis. Eur. J. Biochem. 227, 175–185. 9. Mulder, H., Schachter, H., De Jong-Brink, M., Van der Ven, J.G.M., Kamerling, J.P. & Vliegenthart, J.F.G. (1991) Identifica- tion of a novel UDP-Gal:GalNAcb1-4GlcNAc-R b1-3-galacto- syltransferase in the connective tissue of the snail Lymnaea stagnalis. Eur. J. Biochem. 201, 459–465. 10. Mulder, H., Schachter, H., Thomas, J.R., Halkes, K.M., Kamerling, J.P. & Vliegenthart, J.F.G. (1996) Identification of a GDP-Fuc:Galb1-3GalNAc-R (Fuc to Gal) a1-2 fucosyltransf- erase and a GDP-Fuc: Galb1-4GlcNAc(FuctoGlcNAc)a1-3 fucosyltransferase in connective tissue of the snail Lymnaea stagnalis. Glycoconjugate J. 13, 107–113. 11. Bakker, H., Agterberg, M., Van Tetering, A., Koeleman, C.A.M., Van den Eijnden, D.H. & Van Die, I. (1994) A Lymnaea stagnalis gene, with sequence similarity to that of mammalian b1-4- galactosyltransferases, encodes a novel UDP-GlcNAc:Glc- NAcbRb1-4-N-acetylglucosaminyltransferase. J. Biol. Chem. 269, 30326–30333. 12. Bakker, H., Schoenmakers, P.S., Koeleman, C.A.M., Joziasse, D.H., Van Die, I. & Van den Eijnden, D.H. (1997) The substrate specificity of the snail Lymnea stagnalis UDP-GlcNAc:GlcNAcb- Rb4-N-acetylglucosaminyltransferase reveals a novel variant pathway of complex-type oligosaccaride synthesis. Glycobiology 7, 539–548. 13. Bakker, H., Van Tetering, A., Agterberg, M., Smit, A.B., Van den Eijnden, D.H. & Van Die, I. (1997) Deletion of two exons from the Lymnaea stagnalis b1-4-N-acetylglucosaminyltransferase gene elevates the kinetic efficiency of the encoded enzyme for both UDP-sugar donor and acceptor substrates. J. Biol. Chem. 272, 18580–18585. 14. Van Die, I., Cummings, R.D., Van Tetering, A., Hokke, C.H., Koeleman, C.A.M. & Van den Eijnden, D.H. (2000) Identification of a novel UDP-Glc:GlcNAcb1-4-glucosyltransferae in Lymnea stagnalis that may be involved in the synthesis of complex type oligosaccharide chains. Glycobiology 10, 263–271. 15. Van Tetering, A., Schiphorst, W.E.C.M., Van den Eijnden, D.H. & Van Die, I. (1999) Characterization of a core a1-3-fuc- osyltransferase from the snail Lymnaea stagnalis that is involved in the synthesis of complex-type N-glycans. FEBS Lett. 461, 311–314. 16. Lu ¨ ttge,H.,Heidelberg,T.,Stangier,K.,Thiem,J.&Bretting,H. (1997) The specificity of an a(1-2)- L -galactosyltransferase from albumen glands of the snail Helix pomatia. Carbohydr. Res. 297, 281–288. 17. Staudacher, E. & Ma ¨ rz, L. (1998) Strict order of (Fuc to Asn- linked GlcNAc) fucosyltransferases forming core-difucosylated structures. Glycoconjugate J. 15, 355–360. 18. Wilson, I.B.H., Zeleny, R., Kolarich, D., Staudacher, E., Stroop, C.J.M., Kamerling, J.P. & Altmann, F. (2001) Analysis of Asn- linked glycans from vegetable foodstuffs: widespread occurrence of Lewis a, core a1,3-linked fucose and xylose substitutions. Glycobiology 11, 261–274. 19. Winzler, R.J. (1955) Determination of serum glycoproteins. Methods Biochem. Anal. 11, 279–311. Ó FEBS 2004 Neutral N-glycans of Arion lusitanicus (Eur. J. Biochem. 271) 1355 20. Hase, S., Ibuki, T. & Ikenaka, T. (1984) Reexamination of the pyridylamination used for fluorescence labelling of oligosacchar- ides and its application to glycoproteins. J. Biochem. 95, 197–203. 21. Kubelka, V., Altmann, F., Staudacher, E., Tretter, V., Ma ¨ rz, L., Ha ˚ rd, K., Kamerling, J.P. & Vliegenthart, J.F.G. (1993) Primary structures of the N-linked carbohydrate chains from honeybee venom phospholipase A 2 . Eur. J. Biochem. 213, 1193–1204. 22. Fu, D. & O’Neill, R.A. (1995) Monosaccharide composition analysis of oligosaccharides and glycoproteins by high-perform- ance liquid chromatography. Anal. Biochem. 227, 377–384. 23. Khoo, K H., Huang, H H. & Lee, K M. (2001) Characteristic structural features of schistosome cercarial N-glycans: expression of Lewis X and core xylosylation. Glycobiology 11, 149–164. 24. Kolarich, D. & Altmann, F. (2000) N-Glycan analysis by matrix- assisted laser desorption/ionization mass spectrometry of electro- phoretically separated nonmammalian proteins: application to peanut allergen Ara h 1 and olive pollen allergen Ole e 1. Anal. Biochem. 285, 64–75. 25. Geyer, H., Schmitt, S., Wuhrer, M. & Geyer, R. (1999) Structural analysis of glycoconjugates by on-target enzymatic digestion and MALDI-TOF-MS. Anal. Chem. 71, 476–482. 26. Haslam, S.M., Coles, G.C., Morris, H.R. & Dell, A. (2000) Structural characterization of the N-glycans of Dictyocaulus vivi- parous: discovery of the Lewis X structure in a nematode. Glyco- biology 10, 223–229. 27. Altmann, F., Schwihla, H., Staudacher, E., Glo ¨ ssl,J.&Ma ¨ rz, L. (1995) Insect cells contain an unusual, membrane-bound b-N- acetylglucosaminidase probably involved in the processing of protein N-glycans. J. Biol. Chem. 270, 17344–17349. 28. Zhang, W., Cao, P., Chen, S., Spence, A.M., Zhu, S., Staudacher, E. & Schachter, H. (2003) Synthesis of paucimannose N-glycans by Caenorhabditis elegans requires prior actions of UDP-N-acetyl- D -glucosamine:a-3- D -mannosideb1,2-N-acetylglucosaminyl transferase I, a3,6-mannosidase II and a specific membrane-bound b-N-acetylglucosaminidase. Biochem. J. 372, 53–64. 29. Staudacher, E., Kubelka, V. & Ma ¨ rz,L.(1992)DistinctN-glycan fucosylation potentials of three lepidopteran cell lines. Eur. J. Biochem. 207, 987–993. 30. Altmann, F., Schweiszer, S. & Weber, C. (1995) Kinetic com- parison of peptide-N-glycosidases F and A reveals several differ- ences in substrate specificity. Glycoconjugate J. 12, 84–93. 31. Hall, R.L., Wood, E.J., Kamerling, J.P., Gerwig, G.J. & Vlie- genthart, J.F.G. (1977) 3-O-methyl sugars as constituents of glycoproteins. Identification of 3-O-methylgalactose and 3-O-methylmannose in pulmonate gastropod haemocyanins. Biochem. J. 165, 173–176. 32. Kamerling, J.P. & Vliegenthart, J.F.G. (1997) Hemocyanins. In Glycoproteins II (Montreuil, J., Vliegenthart, J.F.G. & Schachter, H., eds), pp. 123–161. Elsevier, Amsterdam, Lausanne, New York, Oxford, Shannon, Singapore, Tokyo. 33. Puanglarp, N., Oxley, D., Currie, G.J., Bacic, A., Craik, D.J. & Yellowlees, D. (1995) Structure of the N-linked oligosaccharides from tridacnin, a lectin found in the haemolymph of the giant clam Hippopus hippopus. Eur. J. Biochem. 232, 873–880. 34. Khoo, K H., Maizels, R.M., Page, A.P., Taylor, G.W., Rendell, N.B. & Dell, A. (1991) Characterization of nematode glycoproteins: the major O-glycans of Toxocara excretory- secretory antigens are O-methylated trisaccharides. Glycobiology 1, 163–171. 35. Guerardel, Y., Balanino, L., Maes, E., Leroy, Y., Coddeville, B., Oriol, R. & Strecker, G. (2001) The nematode Caenorhabditis elegans synthesizes unusual O-linked glycans: identification of glucose-substituted mucin-type O-glycans and short chondroitin- like oligosaccharides. Biochem. J. 357, 167–182. 36. Kim, S., Hwang, S.K., Dwek, R.A., Rudd, P.M., Ahn, Y.H., Kim, E H., Cheong, C., Kim, S.I., Park, N.S. & Lee, S.M. (2003) Structural determination of the N-glycans of a lepidopteran arylphorin reveals the presence of a monoglucosylated oligo- saccharide in the storage protein. Glycobiology 13, 147–157. 37. Schachter, H. (1995) The Ôyellow brick roadÕ of branched complex N-glycans. Glycobiology 1, 453–461. 38. Takahashi, N., Masuda, K., Hiraki, K., Yoshihara, K., Huang, H.H.,Khoo,K.H.&Kato,K.(2002)N-Glycanstructuresof squid rhodopsin. Eur. J. Biochem. 270, 2627–2632. 39. Khoo, K H., Chatterjee, D., Caulfield, J.P., Morris, H.R. & Dell, A. (1997) Structural mapping of the glycans from egg glycopro- teins of Schistosoma mansoni and Schistosoma japonicum: identi- fication of novel core structures and terminal sequences. Glycobiology 7, 663–677. 40. Bu ¨ rgmayr, S., Grabher-Meier, H. & Staudacher, E. (2001) Sialic acids in gastropods. FEBS Lett. 508, 95–98. 1356 M. Gutternigg et al.(Eur. J. Biochem. 271) Ó FEBS 2004 . not involved in the biosynthesis of the chitobiose core of N-glycans [11–13]. The function of this enzyme in vivo is not clear. The prostate glands of these snails. contain another set of N-glycans, the occurrence of which seems to be highly regulated. The traces of Me 2 GlcM 9 may be a relic of the early events of glycan