Glycobiology vol. 10 no. 3 pp. 263–271, 2000

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Identification of a novel UDP-Glc:GlcNAc β1→4-glucosyltransferase in Lymnaeastagnalis that may be involved in the synthesis of complex-type oligosaccharide chains

Irma van Die1, Richard D.Cummings2, Angelique vanTetering, Cornelis H.Hokke, Carolien A.M.Koeleman andDirk H.van den Eijnden

Department of Medical Chemistry, Vrije Universiteit, Van derBoechorststraat 7, 1081BT Amsterdam, the Netherlands and 2Department ofBiochemistry and Molecular Biology, University of Oklahoma HealthSciences Center, Oklahoma City, OK 73190, USA

Received on June 28, 1999; revised on August 12, 1999; accepted on August30, 1999

Several studies suggest, that the snail Lymnaea stagnaliscontains glycoproteins whose oligosaccharide side chainshave structural features not commonly found in mamma-lian glycoproteins. In this study, prostate glands of L.stag-nalis were incubated in media containing either [3H]-mannose, [3H]-glucosamine, or [3H]-galactose, and themetabolically radiolabeled protein-bound oligosaccharideswere analyzed. The newly synthesized diantennary-likecomplex-type asparagine-linked chains contained a consid-erable amount of glucose, next to mannose, GlcNAc, fucose,galactose, and traces of GalNAc. Since glucose has not beenfound before as a constituent of diantennary N-linkedglycans as far as we know, we assayed the prostate gland ofL.stagnalis for a potential glucosyltransferase activityinvolved in the biosynthesis of such structures. We reporthere, that the prostate gland of L.stagnalis contains aβ1→4-glucosyltransferase activity that transfers glucosefrom UDP-glucose to acceptor substrates carrying aterminal N-acetylglucosamine. The enzyme preferssubstrates carrying a terminal GlcNAc that is β6 linked toa Gal or a GalNAc, structures occurring in O-linkedglycans, or a GlcNAc that is β2 linked to mannose, as ispresent in N-linked glycans. Based on combined structuraland enzymatic data, we propose that the novel β1→4-gluco-syltransferase present in the prostate gland may beinvolved in the biosynthesis of Glcβ1→4GlcNAc units incomplex-type glycans, in particular in N-linked dianten-nary glycans.

Key words: glycoprotein biosynthesis/glycosyltransferase/N-glycan/glycosylation

Introduction

Most of the complex-type N-linked oligosaccharides inmammalian glycoproteins are based on Galβ1→4GlcNAc

(lacNAc) units, that serve as backbone structural elements. Ininvertebrates, occurrence of lacNAc units is only occasionallyreported, i.e., in Schistosoma mansoni (Srivatsan et al., 1992b;Van Dam et al., 1994). Instead, a variant of the lacNAc unit inwhich the Gal residue is replaced by a GalNAc, resulting in aGalNAcβ1→4GlcNAc (lacdiNAc) unit, is found in anincreasing number of invertebrates (reviewed in Van denEijnden et al., 1995, 1997). The characterization of an UDP-GlcNAc:GlcNAc β4-N-acetylglucosaminyltransferase fromthe prostate gland of the snail Lymnaea stagnalis suggests thatin this snail a third backbone synthesizing enzyme is presentthat may be involved in the biosynthesis of chitobiose units incomplex-type glycans (Bakker et al., 1994, 1997).

The backbone structural element is part of the terminaloligosaccharide that often defines the function of the glycan.Regulation of the biosynthesis of these backbone structuralelements, as well as the terminating steps, are thereforeexpected to be critically important for modulation of the finaloligosaccharide structures on individual glycoproteins. Wetherefore are interested in the variations in the backbone struc-ture occurring in different species, and their regulation ofexpression.

In this study we examined the newly synthesized glycans inthe prostate gland of Lymnaea stagnalis and present data thatsuggest that Glc is present in diantennary N-glycans. This isremarkable in that Glc has not been reported in complex-typeN-glycans previously. These results led us to assay the prostategland for a potential glucosyltransferase activity involved inthe biosynthesis of such structures. This resulted in the detec-tion and characterisation of a novel enzyme, an UDP-Glc:GlcNAcβ-R β1→4-glucosyltransferase (β4-GlcT), thatmay be involved in the synthesis of a fourth variant backbonestructural unit in complex-type glycans.

Results

SDS-PAGE and fluorography of radiolabeled prostate glandsof Lymnaea stagnalis

Prostate glands were dissected from L.stagnalis and metaboli-cally radiolabeled in vitro with either [2-3H]mannose,[6-3H]glucosamine, or [6-3H]galactose to radiolabel glyco-proteins synthesized by the glands, and allow analyses of thestructures of the glycoprotein glycans. To assess incorporationof radiolabel in the glycoproteins, equal amounts of radio-labeled prostate gland were homogenized and analyzed bySDS-PAGE and fluorography. Many glycoproteins wereradiolabeled by the [6-3H]galactose precursor, indicating thepresence of galactose and/or glucose (Reitman et al., 1982;1To whom correspondence should be addressed

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Cummings and Kornfeld, 1984) in the glycoproteins (Figure1). Also numerous glycoproteins were labeled with[6-3H]glucosamine precursor, that can be incorporated inGlcNAc, GalNAc, and sialic acid residues (Cummings et al.,1983). A much weaker radiolabeling was found using

[2-3H]mannose precursor, indicating the relatively limitedpresence of Man and Fuc residues in the radiolabeled glyco-proteins (Kornfeld et al., 1978).

Con A Sepharose column chromatography of glycopeptides

Glycopeptides were prepared by pronase digestion from meta-bolically labeled prostate glands, and subjected to Con ASepharose column chromatography (Figure 2). Unboundglycopeptides were designated as the CAI fraction. Boundmaterial was eluted first with 10 mM α-methylglucoside andsubsequently with 100 mM α-methylmannoside, and desig-nated as CA2 and CA3, respectively (Figure 2A). It has beenshown previously that glycopeptides that do not bind to Con Ausually consist of O-linked oligosaccharides, complex-typebisected diantennary, tri- and tetraantennary N-linked glycans(CAI fraction), whereas Con A interacts with relatively highaffinity with many of the complex-type diantennary N-linkedoligosaccharides (CA2 fraction) and with very high affinitywith high mannose- and/or hybrid-type N-linked glycans (CA3fraction) (Ogata et al., 1975; Krusius et al., 1976; Cummingsand Kornfeld, 1982; Merkle and Cummings, 1987). The CA2fraction was subsequently passed over a Bio Gel P6 column(Figure 2B). This resulted in two peaks, CA2-1 and CA2-2,the latter of which showed the sized of a small diantennarycomplex N-glycan and was chosen for further analysis. Thecompositions of both the radiolabeled Con A fractions, as wellas the unlabeled CA2-2 fraction, were determined by HPLC(Table I). The results show that the CA2-2 glycopeptidescontain, next to mannose, fucose, GlcNAc, and Gal, which areexpected to be found in diantennary N-linked oligosaccha-rides, a relatively high amount of Glc, the presence of whichwas not expected.

Enzymatic digestion of the ConA-II fraction, radiolabeled with[3H]-Gal

To investigate the nature of the oligosaccharides that containGlc, glycopeptides in the CA2-2 fraction, labeled with

Fig. 1. Fluorograph of SDS-PAGE of metabolically radiolabeled glycoproteinsof prostate glands of L.stagnalis. Glands were incubated in vitro in mediacontaining 1 mCi/ml of either [3H] glucosamine, [3H]galactose, or[3H]mannose as described in Materials and methods. The radiolabeledprostates were homogenized and equal amounts of each homogenate wereanalyzed by SDS-PAGE and fluorography. Lane 1, [3H] glucosamine-labeledglycoproteins; lane 2, [3H]mannose-labeled glycoproteins; lane 3,[3H]galactose-labeled glycoproteins. The positions of molecular weightstandards are indicated.

Fig. 2. Chromatography of [3H]galactose labeled glycopeptides on Con A-Sepharose (A) and Bio Gel P-6 (B). (A) [3H]Galactose-labeled prostate glands weresolubilized and treated with pronase to generate glycopeptides. The glycopeptides were applied on Con A-Sepharose. Bound glycopeptides were elutedsequentially with 10 mM α-methylglucoside and 100 mM α-methylmannoside. The radioactive glycopeptide fractions were pooled and designated as fraction CA-1 (unbound, fraction 1–8), CA-2 (fraction 20–35) and CA-3 (fraction 40–47). Fraction CA-2 was applied subsequently to a column of Bio Gel P6, and 4-mlfractions were collected. Radioactive fractions were pooled and designated as pool CA2-1 (fractions 27–34) and pool CA2-2 (fractions 50–68).

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[3H]-Gal, were treated with different exoglycosidases. Norelease of [3H]-monosaccharides could be detected by treat-ment of the CA2-2 fraction with α-amylase, indicating that the[3H]-Glc is not part of glycogen, known to bind with relatively

high affinity to Con A as well. In contrast, glycopeptides offractions CA2-1 and CA3 appeared sensitive to α-amylasetreatment. Treatment of the CA2-2 glycopeptides with α-glucosidase, β-glucosidase, β-hexosaminidase, α-galactosidase,or β-galactosidase did not result in release of radiolabeledmaterial, suggesting that the radiolabeled glucose/galactose isnot accessible to digestion. To establish that the CA2-2 glyco-peptides contain diantennary N-linked glycans, they weredefucosylated and part of this material digested with Endo F,known to act specifically on diantennary N-glycans. Equalamounts of radioactivity from both the defucosylated and thedefucosylated/Endo F treated material were analyzed byHPAEC. The results (Figure 3) show that the endo F treatedmaterial eluted as one peak on HPAEC, whereas the glycopep-tides that were not treated with Endo F eluted over a broadrange of the column, due to peptide heterogeneity. Theseresults suggest that the Glc containing CA2-2 oligosaccharidesare mainly present as diantennary N-linked glycans that areinaccessible to digestion with common glycosidases.

Glucosyltransferase activity in the L.stagnalis prostate gland

Since the structural data described above suggest the presenceof glucose in diantennary N-glycans of the prostate gland ofL.stagnalis, we have assayed these glands for a potentialglucosyltransferase (GlcT) activity involved in the biosyn-thesis of such structures. A GlcT activity was detected, usingGlcNAcβ-S-pNP as the acceptor. Optimal assay conditionswere determined by varying the pH (pH 5–8) and the concen-tration of Triton X-100 and Mn2+. Highest enzyme activity wasmeasured in 100 mM cacodylate at pH 7.6, containing 20 mMMn2+ and 0.5% Triton X-100. The GlcT activity measured inthe prostate gland was relatively low compared to theGlcNAcT and GalNAcT activities using the same acceptorsubstrate, and appeared to be tissue-specific, since the activitywas hardly detectable in the albumen gland (Table II).

Fig. 3. HPAEC analysis of [3H]galactose-labeled glycopeptides released byEndo F treatment. An aliquot of [3H]galactose-labeled glycopeptides wasdefucosylated. Half of this material was treated with Endo F. Equal amounts(in dpm) of nontreated and Endo F treated sample were analyzed by HPAEC.In both cases, 90% of the radioactivity was recovered from the column. (A)Elution pattern of nontreated [3H]galactose-labeled glycopeptides; (B) elutionpattern of Endo F treated [3H]galactose-labeled glycopeptides.

Table I. Composition of radiolabeled and unlabeled ConA glycopeptide fractions. Fractions were hydrolyzed and analyzed by HPAEC as described in Materials

and methods

ConA fraction % Monosaccharides in glycopeptides obtained after radiolabeling with:

[3H]-glucosamine [3H]-Mannose [3H]-Galactose

GlcNAc GalNAc Fuc Man Gal Glc

CAI 60 40 93 7 95 5

CA2-2 93 7 38 62 20 80

CA3 100 — 12 88 5 95

Table II. Glycosyltransferase activities in Triton-X100 extracts of the prostateand albumen gland of L.stagnalis

Standard assays were carried out as described in Materials and methods, usingUDP-Glc, UDP-GlcNAc, and UDP-GalNAc as sugar-donors, and GlcNAcβ-S-pNP as acceptor substrate

Source Activity (pmol·min–1·mg–1 protein)

GlcT GlcNAcT GalNAcT

Prostate gland 15 135 650

Albumen gland <1 15 1750

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Acceptor substrate specificity of the GlcT

GlcT assays were performed using a number of oligosaccha-rides and a glycopeptide as potential acceptor substrates (TableIII). The results show that some substrates carrying a terminalGlcNAc in β-configuration can serve as an acceptor for theGlcT, whereas GlcNAc in α-anomeric configuration, orGalNAc, Gal, or Glc in β-anomeric configuration, were noteffective as acceptor. The oligosaccharides 10 and 11, repre-senting partial structures of O-linked glycans, as well as 12 and13, representing partial diantennary N-glycans, appeared to begood acceptors. Also the glycopeptide ag-GP-F4 served as anacceptor, although it was slightly less effective in comparisonwith the oligosaccharide substrates. HPAEC analysis revealedthat the radioactive monosaccharides released by acid hydrol-ysis of the glucosylated product obtained with glycopeptide12, could be identified as glucose (results not shown), indi-cating that the glycosyltransferase activity involved is a trueGlcT. The Glcβ1→4GlcNAc structure that was obtained couldnot be cleaved by β-glucosidase, indicating that the enzymeused cannot act on this substrate.

In contrast, oligosaccharides 8 and 9 were not effective asacceptor substrates. These results show that terminal GlcNAcresidues that are β1→6 linked to Gal/GalNAc or β1→2 linkedto Man, are effective as substrates, whereas GlcNAc residuesthat are β1→3 or β1→4 linked are not effective.

The acceptor specificity of the GlcT differs from that of β4-GalNAcT, that was shown to act on all substrates with terminalβ-linked GlcNAc, although chitobiose was less preferred(Bakker et al., 1997b). The GlcT resembles the recombinantβ4GlcNAcT except for the preference for diantennary N-glycans (Bakker et al., 1997a).

Product characterization

The product formed by incubation of UDP-Glc with a prostategland extract as the enzyme source and GlcNAcβ-O-pNP as

acceptor substrate was analyzed by 1D- and 2D-1H-NMRspectroscopy to determine the glycosidic linkage between theGlc and the GlcNAc residue. The 1D-1H-NMR spectrum of theproduct is shown in Figure 4. 2D-TOCSY spectra with mixingtimes of 20–100 ms were recorded of the acceptor substrateGlcNAcβ-O-pNP as well as the product, affording completeassignment of the protons of each GlcNAc or Glc residue (seeTable IV). Compared to GlcNAcβ-O-pNP, the GlcNAc H-3,H-4, and H-5 signals of the product show downfield shifts of∆δ 0.15, 0.26, and 0.14, respectively. These shifts are essen-tially the same as the shifts of ∆δ 0.17, 0.25, and 0.13, respec-tively, observed for GlcNAc H-3, H-4, and H-5 whencomparing GlcNAcβ-OMe (data not shown) and Glcβ1–4GlcNAcβ-OMe (Hokke et al., 1993). Together with thecoupling constant of the Glc H-1 signal (J1,2 8.3 Hz), these datashow that the Glc residue is attached to GlcNAcβ-O-pNP in aβ1–4-linkage.

Discussion

In studies aimed at the structural characterization of protein-linked glycans of Lymnaea stagnalis, evidence was obtainedthat glucose is present in a specific subfraction of complex-type N-linked glycans in the prostate gland (CA2-2), knowntypically to contain diantennary glycans. Due to the lowamount of material obtained, and the inaccessibility of theglycopeptides to degradation with common glycosidases, a fullstructural characterization of the glycan structure has not beenperformed. Analysis of the monosaccharide composition ofthis CA2-2 fraction by HPLC revealed next to Glc, the pres-ence of Man, GlcNAc, Fuc, and Gal, all compounds generallyfound in diantennary N-glycans. As Glc never has been foundin complex-type N-linked glycans previously, we set out toassay glucosyltransferase activity in prostate extracts, andfound a glucosyltransferase activity toward β-linked GlcNAc

Fig. 4. Structural-reporter-group regions of the 1H-NMR spectrum recorded at 27°C and 600 MHz of Glcβ1–4GlcNAcβ-O-pNP, obtained by incubation ofL.stagnalis prostate extract, UDP-Glc and the acceptor substrate GlcNAcβ-O-pNP.

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as an acceptor compound. Product characterization by NMRshowed that the enzyme could be identified as a UDP-Glc:GlcNAc β1→4-glucosyltransferase. The enzyme highlyprefers a terminal GlcNAc that is β6 linked to a Gal orGalNAc, structures occurring in O-linked glycans, or aGlcNAc that is β2-linked to mannose, as is present in N-linkedglycans. Based on combined structural and enzymologicaldata, we propose that the β4-GlcT present in prostate glands isinvolved in the biosynthesis of Glcβ1→4GlcNAc units incomplex-type glycans, in particular in diantennary N-glycans.It can, however, not be excluded that this enzyme is alsoinvolved in the biosynthesis of Glcβ1→4GlcNAc units in O-linked glycans.

Several β4-glucosyltransferases have been described in liter-ature previously, none of them, however, involved in complex-type glycosylation of proteins. A Bacillus coagulans β4-gluco-syltransferase has been reported that catalyzes the conversionof GlcNAc-PP-undecaprenol into Glcβ1→4GlcNAc-PP-unde-caprenol, and thus can use a terminal GlcNAc as an acceptor,like the L.stagnalis enzyme. Other bacterial β4-glucosyltrans-ferases have been described, for example a Neisseria meningi-tidis enzyme involved in the biosynthesis of lipo-

oligosaccharide (LOS) (Kahler et al., 1996) and a cellulosesynthase from Acetobacter xylinum (Aloni et al., 1983). A ratliver β-glucosyltransferase was recently purified and charac-terized, and the cDNA encoding the human enzyme has beencloned (Ichikawa et al., 1996; Paul et al., 1996). This enzymecatalyzes the transfer of glucose from UDP-Glc to ceramide,the first glycosylation step of glycosphingolipid biosynthesis.

Until now, the only GlcT that has been found to act on N-linked glycans is an α-glucosyltransferase, which is postulatedto participate in the quality control mechanism of glycoproteinfolding and catalyzes the glucosylation of protein-linked, highmannose-type oligosaccharides (Trombetta et al., 1989; Sousaand Parodi, 1995).

Little is known about the structure of invertebrate glycans.The N-linked glycans present on L.stagnalis hemocyanin havebeen shown to resemble mammalian glycans in many aspects,but also unusual modifications have been shown to occur (VanKuik et al., 1986, 1987). Several L.stagnalis glycosyltrans-ferases have been characterized, among them enzymes relatedto mammalian enzymes (Mulder et al., 1991, 1995a, 1995b,1996; Bakker et al., 1994). The backbone structure of the N-glycans found on hemocyanin was shown to consist of aLacdiNAc unit (Van Kuik et al., 1986, 1987), similarly as hasbeen found in several other invertebrate glycans (Nyame et al.,1989; Srivatsan et al., 1992a; Kubelka et al., 1993, 1995;Hollander et al., 1993; Reason et al., 1994). In accordance withthese data, a high β4-GalNAcT activity was demonstrated inthe connective tissue of L.stagnalis, the site of synthesis ofhemocyanin (Mulder et al., 1995b).

Based on the size of the diantennary glycan fraction, it isexpected that extension of the GlcNAc2Man3 core will be veryshort, possibly consisting of a terminal GlcNAc, or a backbonestructure, i.e., Xβ1→4GlcNAc.

Table III. Acceptor specificity of the GlcT activity from L.stagnalis andcomparison to other prostate gland glycosyltransferases

The GlcT and GalNAcT activities are measured in prostate gland extract. TheGlcNAcT activity represents recombinant β4-GlcNAcT purified from insectcells (Bakker et al., 1997a). 100% GlcT activity corresponds to an enzymeactivity of 15 pmol·min–1·mg–1 protein. Values for the GalNAcT andGlcNAcT activities are taken from (Bakker et al., 1997b) and (Bakker et al.,1997a), respectively.aR = O-(CH2)8-COOCH3.

Table IV. 1H-NMR chemical shifts of the acceptor substrate GlcNAcβ-O-PNPand its glucosylated product Glcβ1–4GlcNAcβ-O-PNP, obtained byincubation with L.stagnalis prostate extract

*Given with only two decimals because of spectral overlap.

GlcNAcβ-O-PNP Glcβ1–4GlcNAcβ-O-PNP

GlcNAc H-1 5.325 5.346

H-2 4.034 4.077

H-3 3.698 3.85*

H-4 3.582 3.84*

H-5 3.698 3.84*

H-6 3.963 4.026

H-6' 3.801 3.895

NAc 2.018 2.015

Glc H-1 — 4.574

H-2 — 3.339

H-3 — 3.527

H-4 — 3.427

H-5 — 3.510

H-6 — 3.925

H-6' — 3.743

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Since little GalNAc could be detected in the CA2-2 glyco-peptide fraction, LacdiNAc units are not expected to occurfrequently as backbone on diantennary glycans in the prostategland. As also low amounts of Gal were found, and GalTactivity towards GlcNAc is hardly detectable (unpublishedresults), the occurrence of LacNAc units in the CA2-2 glyco-peptides is unlikely. The β4-GlcNAcT that was cloned fromthe prostate gland did not act efficiently on diantennary glycanacceptors, but rather preferred terminal GlcNAc that is β6-linked to Gal or GalNAc, structures occurring in O-linkedglycans (Bakker et al., 1994, 1997). It is therefore unlikely thatthis enzyme plays a role in the synthesis of diantennary N-linked glycans. The acceptor specificity of the β4-GlcT charac-terized in this study indicates that this enzyme is able tosynthesize Glcβ1→4GlcNAc units in N-linked diantennaryglycans in vitro. It might be possible, therefore, that part of theprostate diantennary glycans carry novel Glcβ1→4GlcNAcbackbone structural units, a supposition that is supported bythe observation that Glc is part of the diantennary glycopeptidefraction.

Interestingly, comparison of the activities in the prostategland of glycosyltransferases that are able to synthesize abackbone structure (i.e., Xβ1→4GlcNAcβ-) revealed that aβ4-GalNAcT was present as the major backbone-synthaseactivity, in addition to lower β4GlcNAcT and β4GlcT activi-ties. This poses the question why hardly any GalNAc can bedetected in the N-linked diantennary glycans. Preliminaryacceptor specificity studies of the prostate β4-GalNAcTshowed that the enzyme acts efficiently on diantennary glycansin vitro, similarly as has been shown for several other inverte-brate β4-GalNAcTs (Neeleman et al., 1994; Mulder et al.,1995b; Van Die et al., 1996). This suggests that next to therelative activity and acceptor specificity of the glycosyltrans-ferase, other parameters may play a role in regulation of glyco-protein synthesis. It might be that specific protein-motifs areinvolved. Alternatively, as the L.stagnalis prostate gland iscomposed of several different cell-types, the proteinscontaining the diantennary glycans might be expressed in aspecific cell-type only, concomitantly with the β4-GlcT and/orβ4-GlcNAcT, whereas expression of β4-GalNAcT is limitedto another cell-type.

The results described in this manuscript indicate that theprostate gland of L.stagnalis contains a novel β4-GlcT, poten-tially involved in the synthesis of unusual diantennary glycans.The data suggest that the regulation of biosynthesis ofcomplex-type glycans in the L.stagnalis prostate gland iscomplex. Apparently, despite the presence of several glycosyl-transferases that are able to act on biantennary glycans in vitro,only specific structures are formed. This seems to be due partlyto a restricted acceptor specificity as found for the β4-GlcNAcT and β4-GlcT, but other, yet unknown, factors aresupposed to play a role as well. This complex regulation mightappear to be similar to the mammalian system, in whichseveral different UDP-Gal:GlcNAc β4-GalTs with yet notfully known properties, in some cell types in addition to aβ4GalNAcT, are supposed to be involved in the synthesis ofbackbone structures in mammalian protein-linked complex-type glycans (Schachter and Roseman, 1980; Do et al., 1997;Schwientek et al., 1998; Ujita et al., 1998; Van Die et al.,1999; Van den Nieuwenhof et al., 1999). Detailed analysis ofthe Lymnaea stagnalis protein-linked glycans, in combination

with enzymatic studies and comparison to the mammalian situ-ation, might give more insight in the factors that are relevantfor the regulation of glycoprotein synthesis in general.

Materials and methods

Animals and materials

Specimens of L.stagnalis were bred under standard laboratoryconditions. Acceptor compounds (see Table III) 1, 2, 3, and 5and GlcNAcβ-OMe were purchased from Sigma Chemical Co.(St. Louis, MO), 8, 9, and 11 from Toronto Research Chemi-cals (Toronto, Ont), 10 from Dr. A.Veyrières (Université ParisSud, Orsay, France), 12 from Dr. O.Hindsgaul (University ofAlberta, Edmonton, Alberta), and 4, 6, and 7 from Koch-LightLaboratories, England.

Glycopeptide GP-F4 was prepared from asialo-fibrinogen bypronase digestion as described previously (Nemansky and Vanden Eijnden, 1993). GP-F4 was enzymatically degalacto-sylated with jack bean β-galactosidase (0.2 U/µmol terminalGal) in 50 mM sodium acetate pH 4, to yield the agalacto (ag)form.

UDP[3H]Glc (19.7 Ci/mmol) was obtained from Amersham,UDP[3H]Gal (50 Ci/mmol), UDP[3H]GlcNAc (30.4 Ci/mmol),and UDP[3H]GalNAc (24 Ci/mmol) were purchased fromNEN Life Science Products. The sugar nucleotides werediluted with unlabeled UDP-sugars (Sigma) to give the desiredspecific radioactivity. [2-3H]Mannose (17.6 Ci/mmol), [6-3H]glucosamine (29 Ci/mmol), and [6-3H]galactose (29.5 Ci/mmol) were purchased from NEN Life Science Products.Concanavalin A-Sepharose (Con A-Sepharose) was obtainedfrom Pharmacia Biotech Inc.

Metabolic radiolabeling of L.stagnalis prostate glands andSDS-PAGE of radiolabeled glycoproteins

Prostates were dissected from 75 snails. After washing, theseprostates were incubated in vitro with 1 mCi/ml of either[3H]mannose, [3H]glucosamine, or [3H]galactose inDulbecco’s modified Eagle’s medium supplemented with 10%fetal calf serum, 100 U/ml penicillin and 100 µg streptomycin(all from Life Technologies, Inc.), in a final volume of 2 ml for4 h at 37°C, to radiolabel their oligosaccharides. From eachlabeling, one prostate gland was taken, washed twice in PBS,and sonicated in SDS-PAGE buffer. The radiolabeled glyco-proteins were then analyzed by SDS-PAGE on a 10% acryl-amide slab gel (Laemmli, 1970) under reducing conditions.The gel was fixed in an aqueous solution containing 25% (v/v)isopropanol and 10% (v/v) acetic acid and incubated for 40min in “Amplify” (Amersham) at room temperature and visu-alized by fluorography.

Preparation of radiolabeled glycopeptides

From each labeling, 20 radiolabeled prostate glands werewashed two times with PBS, homogenized and after lipidextraction resuspended in 10 ml PBS. The residual protein wasdigested with 100 µg pronase E (Sigma) for 72 h at 37°C.Subsequently, the sample was boiled for 30 min and desaltedby passage over a column (0.75 × 40 cm) of Sephadex G25 inwater containing 7% n-propanol. The radiolabeled glyco-peptides were recovered in the void fractions and freeze-dried.

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Con A column chromatography

Radiolabeled glycopeptides were fractionated on a 3 mlcolumn (0.9 × 5 cm) of Con A-Sepharose at room temperature,as described previously (Cummings and Kornfeld, 1982).Glycopeptides bound at the column were eluted with 10 mMα-methylglucoside followed by elution with 100 mM α-meth-ylmannoside. Fractions of 2 ml were collected from thecolumn and aliquots of each fraction were taken to determinethe radioactivity by liquid scintillation. Radioactive fractionsof unbound (CA1), bound (CA2), and strongly bound (CA3)glycopeptides were pooled and desalted by passage overSephadex as described above. Chromatography of the sampleCA2, radiolabeled with [3H]galactose, on Bio-Gel P6 wasperformed on a 1.6 × 200 cm column equilibrated in 50 mMammonium acetate pH 5.2 and 4 ml fractions were collected.

Analysis of sugar composition in radiolabeledoligosaccharides or glycopeptides

Radiolabeled oligosaccharides or glycopeptides were hydro-lyzed by heating in 2 M TFA at 100°C for 4 h whereafter thehydrolysates were freeze dried. Samples were analyzed byhigh-pH anion exchange chromatography with pulsed ampero-metric detection (HPAEC-PAD). The system consisted of aDionex Bio-LC gradient pump, a CarboPac MA-1 column (4 ×250 mm) and a PAD model-2 detector. For separation of theproducts, isocratic elution was conducted with 0.2 M NaOH ata flow rate of 1 ml/min. Other conditions on chromatographyand detection were as described before (Bakker et al., 1994).

Glycosidase treatments

[3H]Galactose labeled glycopeptides were treated with 10 U ofα-amylase (Bacillus species, Sigma) in 75 µl of 50 mM phos-phate buffer, pH 6.8, for 18 h at 37°C. [3H]Galactose labeledglycopeptides were incubated with either 50 mU α-glucosi-dase in 50 mM citrate buffer at pH 6.0, or 50 mU β-glucosidasein 50 µl 50 mM citrate buffer pH 5.0 for 20 h at 37°C or witheither 60 mU α-galactosidase (Sigma) in 100 µl 50 mM phos-phate buffer pH 6.0, 50 mU jack bean β-galactosidase (Sigma)in 100 µl 50 mM citrate buffer pH 3.5 or 30 mU bovine testesβ-galactosidase (Sigma) in 100 µl 50 mM citrate buffer pH 4.5for 20 h at 25°C. To separate released monosaccharides fromthe glycopeptide fraction after digestion, the samples wereapplied on a column of Con A-Sepharose (1 ml) run in TBS.The columns were washed three times with 1 ml TBS, andeluted with 100 mM α-methylmannoside. The radioactivity ofall fractions was determined by liquid scintillation.

Analysis of Endo F treated [3H]galactose labeledglycopeptides by HPLC

[3H]-Labeled glycopeptides were defucosylated by heating in0.1 N TFA for 75 min at 95°C, freeze dried and dissolved inwater. Half of this sample was incubated with 750 mU endog-lycosidase F (Boehringer) in a final volume of 500 µl 50 mMphosphate buffer, containing 25 mM EDTA, pH 7.0, and 0.4%Nonidet NP40, for 20 h at 37°C. Both the Endo F treated andthe nontreated samples were passed over a Sep-Pak C-18cartridge to remove the Nonidet, and over a 1.6 × 200 cmcolumn of Bio-Gel P4 (200–400 mesh), run in 50 mM ammo-nium acetate pH 5.2. Both samples were analyzed by HPAEC-

PAD, consisting of a Dionex Bio-LC gradient pump, aCarboPac PA100 column (4 × 250 mm) and a PAD model-2detector. Chromatographic conditions and detection were asdescribed previously (Van Die et al., 1996).

Preparation of the enzyme and glycosyltransferase assays

Three grams of L.stagnalis prostate glands were homogenizedin 30 ml 10 mM sodium cacodylate buffer pH 7.6 containing0.25 M sucrose, 10 mM MnCl2, and 0.5% Triton X-100 andincubated under stirring for 1 h at 4°C. The homogenate wassubsequently centrifuged for 1 h at 20,000 × g, and the super-natant recovered as enzyme source. The standard incubationsystem contained in a volume of 50 µl 25 nmol of either UDP-[3H]Glc (2.5 Ci/mol), UDP-[3H]GlcNAc (1 Ci/mol), UDP-[3H]GalNAc (1 Ci/mol) or UDP-[3H]Gal (1 Ci/mol), 0.2 µmolATP (to counteract enzymatic degradation of the nucleotidesugar), 50 nmol of oligosaccharide acceptor, 25 µl of prostateextract and 5 µmol sodium-cacodylate buffer pH 7.6 and1 µmol MnCl2 (for GlcT assays) or 5 µmol sodium-cacodylatebuffer pH 7.2 and 2 µmol MnCl2 (for GalNAcT or GlcNAcTassays). After incubation at 37°C for 60 min the radioactiveproducts from 2 and 10 (Table III) were isolated by chroma-tography of the mixtures on a column (1 ml) of Dowex 1X8(Cl–-form) as described previously (Neeleman et al., 1994),for substrates with a hydrophobic aglycon, on Sep-Pak C-18cartridges (Waters, Milford, MA) as described previously(Palcic et al., 1988) and for the diantennary N-glycans 13 and14 by Con A chromatography (as described under “glycosidasetreatments”). The radioactivity incorporated into the acceptorwas assayed by liquid scintillation. Control assays lacking theacceptor substrate were carried out to correct for incorporationinto endogenous acceptors.

Product characterization

The glucosylated product obtained with substrate 12 wasisolated by Con A-Sepharose chromatography and hydrolyzedby incubation in 2 M TFA at 100°C for 4 h. Subsequently, thehydrolysate was freeze-dried and analyzed by HPAEC asdescribed above.

The product obtained by incubation of UDP-Glc withL.stagnalis prostate extract as enzyme source and GlcNAcβ-O-pNP as acceptor was analyzed by 1H-NMR spectroscopy.Prior to NMR spectroscopic analysis, samples were exchangedtwice in 99.9% 2H2O with intermediate lyophilization. Finally,samples were dissolved in 500 µl 99.95% 2H2O (Merck). 1H-NMR spectra were recorded at 600 MHz on a Bruker AMX2-600 spectrometer (NSR Center, University of Nijmegen, theNetherlands) at a probe temperature of 27°C. Chemical shiftsare expressed in ppm by reference to internal acetone (δ 2.225)(Vliegenthart et al., 1983). The spectra were recorded andprocessed as described previously (Hokke et al., 1998).

Acknowledgments

We thank Wietske E.C.M.Schiphorst for expert technicalassistance in part of the work and Drs. A.Veyrières (UniversitéParis Sud, Orsay, France) and O.Hindsgaul (University ofAlberta, Edmonton, Alberta) for gift of oligosaccharides.

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