Glycobiology vol. 12 no. 3 pp. 217–228, 2002

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Several polylactosamine-modifying glycosyltransferases also use internal GalNAcβ1-4GlcNAc units of synthetic saccharides as acceptors

Hanna Salo1,3, Olli Aitio3, Kristiina Ilves3, Eija Bencomo3, Suvi Toivonen3, Leena Penttilä3, Ritva Niemelä3, Heidi Salminen3, Eckart Grabenhorst4, Risto Renkonen5,6, and Ossi Renkonen2,3,5,7

3Institute of Biotechnology, Laboratory of Glycobiology, FIN-00014 University of Helsinki, Finland; 4Protein Glycosylation, Gesellschaft für Biotechnologische Forschung mbH, D-38124 Braunschweig, Germany; 5Rational Drug Design Program, Biomedicum Helsinki, PB 63, FIN-00014 University of Helsinki, Finland; 6Department of Bacteriology and Immunology, Haartman Institute, FIN-00014 University of Helsinki, Finland; and 7Department of Biological Sciences, Division of General Microbiology, FIN-00014 University of Helsinki, Finland

Received on November 25, 2001; revised on January 7, 2002; accepted on January 13, 2002

The GalNAcβ1-4GlcNAc determinant (LdN) occurs in somehuman and bovine glycoconjugates and also in lower verte-brates and invertebrates. It has been found in unsubstitutedas well as terminally substituted forms at the distal end ofconjugated glycans, but it has not been reported previouslyat truly internal positions of polylactosamine chains. Here,we describe enzyme-assisted conversion of LdNβ1-OR oligo-saccharides into GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-OR.The extension reactions, catalyzed by human serum, weremodeled after analogous β3-GlcNAc transfer processes thatgenerate GlcNAcβ1-3Galβ1-4GlcNAcβ1-OR. The newlysynthesized GlcNAcβ1-3GalNAc linkages were unambiguouslyidentified by nuclear magnetic resonance data, includingthe appropriate long-range correlations in heteronuclearmultiple bond correlation spectra. The novel GlcNAcβ1-3′LdNdeterminant proved to be a functional acceptor for severalmammalian glycosyltransferases, suggesting that humanpolylactosamines may contain internal LdN units in manydistinct forms. The GlcNAcβ1-3′LdN determinant wasunusually resistant toward jackbean β-N-acetylhexosamini-dase; the slow degradation should lead to a convenientmethod for the search of putative internal LdN determinantsin natural polylactosamine chains.

Key words: polylactosamine/glycosyltransferase/synthetic/saccharide/glycoconjugate

Introduction

The GalNAcβ1-4GlcNAc determinant (known also as N,N′-dia-cetyllactosediamine or LacdiNAc; here as LdN) replaces thedistal N-acetyllactosamine (LN) unit in some human andbovine glycoconjugates; it is common also in lower vertebratesand invertebrates (reviewed in van den Eijnden et al., 1997).LdN and its terminally substituted derivatives are immunogenic(Nyame et al., 1999, 2000; van Remoortere et al., 2000, 2001)and sometimes modify the biological activities of cognate glyco-proteins, for example, glycodelins (Seppala et al., 2001), someglycoprotein hormones (Smith and Baenziger, 1988; Fiete et al.,1991), and Protein C of human plasma (Grinnell et al., 1994).

Polylactosamine-type elongation reactions of the LdN determi-nant, generating GlcNAcβ1-3GalNAcβ1-4GlcNAc-OR, have notbeen reported, and the presence of truly internal LdN determinantsin natural polylactosamine chains has not been established either.However, the closely related internal GalNAcβ1-4Glc determinantwith distal polylactosamine extensions has been described inSchistosoma mansoni glycolipids (Wuhrer et al., 2000). Theapparent scarcity of information concerning internal LdN isslightly surprising because the synthesis of the distal LdNdeterminant is not uncommon in the animal kingdom, asdiscussed, and the generation of the GlcNAcβ1-3GalNAclinkage is also well established, though only in the context of O-glycan core 3 biosynthesis (Brockhausen et al., 1985).

Here, we describe enzyme-assisted conversion of unconjugatedGalNAcβ1-4GlcNAcβ1-OR oligosaccharides and methyl glyco-sides into products of GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-ORtype. Pure products of definite structures were obtained fromthese reactions and were fully identified by nuclear magneticresonance (NMR). The novel trisaccharide determinantGlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-OR proved to be a versatileacceptor for a number of glycosyltransferase reactions, whichgenerated several types of polylactosamine analogs withinternal LdN units. The distal GlcNAcβ1-3′LdN determinantwas cleaved quite slowly by jackbean β-N-acetylhexosaminidase.This observation will be helpful in searching for putativeinternal LdN determinants among natural polylactosamines.

Results

Structural overview of the key oligosaccharides

The key oligosaccharides are presented and numbered in Table I.They represent three subgroups of different proximal (reducing)ends. All of them carry the underlined GalNAcβ1-4GlcNAcunit (LdN). The monosaccharide residues are identified byone-letter symbols to facilitate discussion of the NMR spectra.

1Present address: Institute of Biotechnology, Research Program in Cellular Biotechnology, Yeast Laboratory, FIN-00014 University of Helsinki, Finland2To whom correspondence should be addressed at Rational Drug Design Program, Biomedicum Helsinki

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Conversion of GalNAcβ1-4GlcNAcβ1-OR type saccharides into products of GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-OR type

The trisaccharide GalNAcβ1-4GlcNAcβ1-3Galβ1-OMe(Glycan 1) was incubated with UDP-GlcNAc and humanserum, which is known to contain β3-GlcNAc transferaseactivity capable of elongation of polylactosamine i-chains(Yates and Watkins, 1983; Hosomi et al., 1984; Piller et al.,1984; Seppo et al., 1990). Biogel P2 gel filtration (data notshown) suggested that 13 mol% of Glycan 1 were convertedinto a tetrasaccharide product (Glycan 2). Indeed, the purifiedGlycan 2 revealed in matrix-assisted laser desorption ionizationand time-of-flight (MALDI-TOF) mass spectrum two majorsignals that were assigned to the molecular ions [M+Na]+ and[M+K]+ of a tetrasaccharide of the compositionHex1HexNAc3OMe (Figure 1A).

Structural reporter group signals of Glycan 2 are shown inthe one-dimensional proton spectrum (Figure 2B, Table II).The H1 doublet of the e-GlcNAc shows a J1,2 coupling constantof 8.4 Hz, confirming that this unit is β-linked to the acceptor.Full assignment of the 1H and 13C signals from various spectra(Table III) and the clear interglycosidic correlations in theheteronuclear multiple bond correlation (HMBC) spectrum

(Figure 3) established the positions of all glycosidic linkagesof Glycan 2. The correlation between the distal e-GlcNAc H1(eH1 in the spectrum of Figure 3) and the d-GalNAc C3 (dC3in the spectrum of Figure 3) shows that the novel β-glycosidicbond of Glycan 2 was GlcNAc1-3GalNAc.

The disaccharide GalNAcβ1-4GlcNAcβ1-OMe (Glycan 9)was β1,3-N-acetylglucosaminylated in the same way asGlycan 1, yielding 12 mol% of a trisaccharide (Glycan 10)(data not shown). The MALDI-TOF mass spectrum revealedthe molecular ions [M+Na]+ and [M+K]+ of HexNAc3OMe(Figure 1B).

The NMR signals of the structural reporter groups ofGlycan 10 are recorded in Table IV. A series of 2D NMRexperiments analogous to those of Glycan 2 were performedwith Glycan 10, and the 1H and the 13C resonances wereassigned (Table III). The data show a great similarity betweenthe analogous resonances of the distal e-GlcNAc of Glycans 2and 10. For example, the e-GlcNAc H1 resonance of Glycan 10showed a J1,2 of 8.4 Hz, establishing the presence of a β-linkagealso in Glycan 10. The analogous resonances of the d-GalNAcunits in Glycans 2 and 10 were similar, too. A concluding HMBCexperiment with Glycan 10 revealed a long range correlation

Table I. Structures of the key oligosaccharides and denotation of the monosaccharide residues.

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between the distal e-GlcNAc H1 and the d-GalNAc C3 (datanot shown), identifying the novel linkage of Glycan 10 also asGlcNAcβ1-3GalNAc.

Incubation of the tetrasaccaharide GalNAcβ1-4GlcNAcβ1-3Galβ1-4Glc (Glycan 14) with UDP-GlcNAc and a concentrateof the β3-GlcNAc transferase activity from human serumyielded a mixture of a pentasaccharide (Glycan 15, 27 mol%)and the unreacted Glycan 14 (73 mol%), which were separatedby Bio-Gel P2 chromatography (not shown). The compositionof pure Glycan 15 was Hex2HexNAc3 as revealed by theMALDI-TOF mass spectrum (Figure 1C). One dimensional 1HNMR spectrum (Table IV) contained the reporter groupresonances of the acceptor (Glycan 14) and additionally oneequivalent resonances at 4.574 and 4.174 ppm as well as athree equivalents resonance at 2.022 ppm. In analogy withGlycans 2 and 10, the new signals of Glycan 15 were assignedto the distal e-GlcNAc H-1, to the peridistal d-GalNAc H-4,

and the NAc protons of e-GlcNAc, respectively. Beingidentical with the analogous signals of Glycans 2 and 10, thesereporter group resonances of Glycan 15 suggest that a distalGlcNAcβ1-3GalNAc determinant was present also here.

Reactions of GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-OR glycans with glycosyltransferases

An overview in Figure 4 shows enzymatic reactions thatconverted the novel GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-ORglycans into several types of polylactosamine analogs carryinginternal LdN determinants.

Enzymatic β 1,4-galactosylation of GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-OR type saccharides. Incubation of Glycan 2 withUDP-Gal and purified bovine milk β4-galactosyltransferasegave the pentasaccharide Galβ1-4GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-3Galβ1-OMe (Glycan 3) in a yield of 96 mol%.

Fig. 1. Positive-ion mode MALDI-TOF mass spectra. (A) Glycan 2; (B) Glycan 10; (C) Glycan 15.

Fig. 2. Selected areas of 1D 1H NMR spectra of Glycans 1–7 showing major reporter group resonances. The two sections of the spectra are drawn on different scales.

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After high-performance liquid chromatography (HPLC) gelfiltration, the purified Glycan 3 revealed in the MALDI-TOFmass spectrum a major signal (observed m/z 988.3) that wasassigned to [M+Na]+ of Hex2HexNAc3OMe (calculated m/z 988.4).

The 1H NMR spectrum of Glycan 3 (Figure 2C, Table II) revealedthe reporter group resonances of Glycan 2 and the additionalH1 doublet (at 4.474 ppm) of the novel, β4-linked f-Gal unit(J1,2 = 7.8 Hz). The reaction caused also a downfield shift of the

Table II. 1H chemical shifts (ppm) at 23°C in 2H2O of reporter groups in selected glycans.

aThe assignments may have to be exchanged.—, Not appropriate.

Glycan

Reporter proton Mono saccharide 1 2 3 6 7

H-1 b 4,297 4,296 4,296 4,274 4,295

c 4,691 4,685 4,685 4,683 4,682

c′ — — — 4,537 —

d 4,522 4,524 4,524 4,518 4,514

e — 4,574 4,595 4,590 4,568

e′ — — — 4,590 —

f — — 4,474 4,475 —

g — — — — 4,514

H-4 b 4,127 4,123 4,124 4,095 4,124

d — 4,173 4,177 4,175 4,161

NAc c 2,023 2,023 2,019 2,028 2.018a

c′ — — — 2,028 —

d 2,065 2,069 2,068 2,067 2,065

e′ — — — 2,060 —

g — — — – 2,065

Table III. 1H and 13C chemical shifts (ppm) of Glycans 2 and 10 at 23°C.

aFrom HMQC.bAssignments may have to be exchanged.

Glycan Residue H1 H2 H3 H4 H5 H6 H6′ CH3

2 OMe 3.555

10 OMe 3.495

2 b-Gal 4.296 3.532 3.695 4.123 3.676 3.727 3.779 —

10 b-Gal — — — — — — — —

2 c-GlcNAc 4.685 3.776 3.729 3.636 3.498 3.636 3.828 2.023

10 c-GlcNAc 4.429 3.724 3.70a 3.612 3.508 3.647 3.850 2.023

2 d-GalNAc 4.524 3.976 3.819 4.174 3.723 3.75a 3.75a 2.069

10 d-GalNAc 4.516 3.976 3.818 4.172 3.712 3.76a 3.76a 2.074

2 e-GlcNAc 4.574 3.692 3.543 3.463 3.413 3.758 3.889 2.023

10 e-GlcNAc 4.573 3.694 3.544 3.456 3.415 3.756 3.885 2.023

Glycan Residue C1 C2 C3 C4 C5 C6 CH3

2 OMe 58.53

10 OMe 58.52

2 b-Gal 105.22 71.03 83.63 69.66 76.00 62.22 —

10 b-Gal — — — — — — —

2 c-GlcNAc 103.92 56.29 73.64 80.11 75.64 61.30 23.53b

10 c-GlcNAc 103.33 56.05 74.07 80.47 75.91 61.49 23.54

2 d-GalNAc 102.82 52.50 80.42 69.07 76.13 62.22 23.59

10 d-GalNAc 102.97 52.56 80.58 69.19 76.20 62.26 23.60

2 e-GlcNAc 103.80 56.90 74.71 70.97 76.96 61.72 23.51b

10 e-GlcNAc 103.89 56.92 74.87 71.06 77.05 61.79 23.54

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H1 of the e-GlcNAc from 4.573 ppm in Glycan 2 to 4.595 ppm inGlycan 3; a similar change is associated also with distal β4-galac-tosylation of ordinary i-type polylactosamines (Leppänen et al.,1997).

Enzyme-assisted β1,4-galactosylation of Glycan 10 gave thetetrasaccharide Galβ1-4GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-OMe(Glycan 11). In the MALDI-TOF mass spectrum, the majormolecular ion signal at m/z 826.2 was assigned to [M+Na]+ ofHex1HexNAc3OMe (calculated m/z 826.3). The 1H-NMRspectrum of Glycan 11 (Table IV) revealed the H1 and H4doublets of the acceptor and an additional doublet at 4.475 ppm(J1,2 = 7.9 Hz) that was assigned to the H1 of the β4-linked f-Galunit. Even here, β1,4-galactosylation caused a downfield shiftof the e-GlcNAc H1 resonance from 4.573 ppm in Glycan 10to 4.595 ppm in Glycan 11.

β1,4-Galactosylation of Glycan 15 gave the hexasaccharideGalβ1-4GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-3Galβ1-4Glc(Glycan 17). The MALDI-TOF mass spectrum of the productrevealed a major signal of m/z 1136.3 that was assigned to hexa-saccharide Hex3HexNAc3 (calculated m/z 1136.4). The 1H NMRspectra of Glycans 15 and 17 (Table IV) confirmed successfultransfer of β4-bonded galactose to the distal GlcNAc of Glycan 15.The conversion of Glycan 15 to Glycan 17 was also accompaniedby a downfield shift of the resonance of e-GlcNAc H1.

Enzymaticβ 21,4-N-acetylgalactosaminylation of Glycan gener-ated Glycan 7 with two successive LdN units. Incubation ofGlycan 2 with UDP-GalNAc and bovine milk β1,4-galactosyl-transferase gave a pentasaccharide (Glycan 7) in a yield thatexceeded 95 mol%. (The reaction was performed by using apurified sample of β1,4-galactosyltransferase from bovinemilk [Palcic and Hindsgaul, 1991]. It appears that also β1,4-N-acetylgalactosaminytransferase was present in the enzyme[van den Nieuwenhof et al., 1999].) The MALDI-TOF massspectrum revealed a major signal of m/z 1029.4 that wasassigned as [M+Na]+ of the pentasaccharide Hex1HexNAc4OMe(calculated m/z 1029.4). The NMR spectrum (Figure 2F, Table II)confirmed that Glycan 7 represented the pentasaccharideGalNAcβ1-4GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-3Galβ1-OMe,a polylactosamine analog with two adjacent LdN units.

Enzymatic β1,3-galactosylation of Glycan 2. Glycan 2 wasβ1,3-galactosylated by incubating it with UDP-Gal and alysate of Colo 205 cells known to contain β4GalT- andβ3GalT-activities, the latter representing β3Gal T5 (Isshikiet al., 1999). The β1,4-galactosylated product (Glycan 3) wasremoved from the desired β1,3-galactosylated product, Glycan 8,by converting it back to Glycan 2 with the linkage-specificβ1,4-galactosidase from Diplococcus pneumoniae (Hughesand Jeanloz, 1964; Renkonen et al., 1989). Glycan 2, in turn,

Fig. 3. HMBC spectrum of the purified Glycan 2. The 1H and 13C resonances of each monosaccharide unit of Glycan 2 are listed in Table III. The interglycosidic H1-C1-O1-Cx-correlations are boxed, identifying all glycosidic linkages present. The unmarked correlations represent intraresidual correlations.

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was successfully removed from Glycan 8 by hydrolysis catalyzedby a recombinant form of β2,3,4,6-N-acetylglucosaminidasefrom Streptococcus pneumoniae, followed by chromatography(not shown). The MALDI-TOF mass spectrum of purifiedGlycan 8 revealed a major signal at m/z 988.3; it was assignedto [M+Na]+ of Galβ1-3GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-3Galβ1-OMe (calculated m/z 988.4). In high-pH anionexchange (HPAE) chromatography on a PA-1 column, known

to separate the slowly eluting type 1 glycans from the type 2isomers (Townsend et al., 1988), Glycan 8 revealed a majorcomponent emerging in 40 mM NaOH at 4.77 min. In a similarrun, purified Glycan 3 gave a major peak at 2.56 min. Theseexperiments confirmed the identity of Glycan 8 as a penta-saccharide of type 1.

Enzymatic conversion of linear Glycan 3 into the doubly branched Glycan 6. Mammalian blood serum contains β1,6-GlcNActransferase activity (cIGnT) that generates midchain branchesto ordinary linear polylactosamines (Leppänen et al., 1991,1997; Gu et al., 1992). To see whether also internal LdN deter-minants participate in the branching reactions, we incubatedthe linear Glycan 3 with UDP-GlcNAc and rat serum andsubjected the resulting saccharide mixture to gel filtration in acolumn of Bio-Gel P-4. The saccharide fraction emerging firstfrom the column (Glycan 6) revealed in MALDI-TOF MS amajor signal of m/z 1394.4 that was assigned to [M+Na]+ ofHex2HexNAc5OMe (calculated m/z 1394.5). Its 1H NMRspectrum (Figure 2E; Table II) revealed seven H1 resonances,two H4 doublets, and four singlet peaks arising from the fiveN-acetyl groups. The structural reporter group resonances ofthe branched trisaccharide determinant GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-OMe at the proximal end of Glycan 6were identical with their counterparts in the synthetic tri-saccharide GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-Ome (Maaheimo,1998). Importantly, the branch-bearing b-Gal of Glycan 6revealed H1 and H4 resonances that were distinct from theiranalogs in the spectrum of linear Glycan 3. These resonancespermitted a comparison of the kinetics of generation of the twobranches of Glycan 6 as will be described.

Table IV. 1H Chemical shifts (ppm) at 23°C in 2H2O of reporter groups in selected glycans.

aThe assignments may have to be exchanged.—, Not appropriate.

Glycan

Reporter proton Monosaccharide 9 10 11 12 14 15 17 18

H-1 a 5.218 5.218 5.217 5.217

— — — — 4.661 4.660 4.660 4.660

b — — — — 4.435 4.432 4.433 4.43

c 4.436 4.429 4.429 4.436 4.680 4.675 4.675 4.667

d 4.516 4.516 4.518 4.509 4.524 4.525 4.526 4.46

e — 4.573 4.595 4.578a — 4.574 4.596 4.591

e′ — — — 4.566a — — — —

f — — 4.475 — — — 4.475 4.477

i — — — — — — — 5.119

H-2 a — — — — 3.277 3.277 3.277 3.276

H-4 b — — — — 4.139 4.135 4.135 4.132

d — 4.172 4.176 4.168 — 4.174 4.178 4.132

H-5 i — — — — — — — 4.845

H-6 i — — — — — — — 1.252

NAc c 2.027 2.023 2.021a 2.021a 2.028 2.025 2.025 2.014

d 2.069 2.074 2.072 2.071 2.066 2.070 2.068 2.030

e — 2.023 2.024a 2.039a — 2.022 2.019 2.014

e′ — — — 2.061 — — — —

Fig. 4. Enzymatic conversions of the novel GlcNAcβ1-3′LdNβ1-OR saccharides into a number of distinct polylactosamine analogs with internal LdN units.

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Another branched product generated from the linear Glycan 3emerged from the gel filtration column soon after Glycan 6. Itrevealed in MALDI-TOF MS a major signal of m/z 1191.5 thatwas assigned to [M+Na]+ of Hex2HexNAc4OMe (calculatedm/z 1191.5). The 1D 1H NMR spectrum (Figure 2D) suggestedthat this material represented largely a mixture of two hexa-saccharide isomers (Glycans 4 and 5) with single GlcNAcbranches at different sites of the linear acceptor. Particularlyrelevant resonances were those of H1 and H4 of the b-Gal.Based on the spectra of Glycans 3 and 6 we interpret the dataas follows: in the spectrum of Glycan 4, where the b-Galcarries the c′-GlcNAc substituent at position 6, the b-Gal H4resonates at 4.096 ppm, whereas in the spectrum of Glycan 5,where the b-Gal does not carry any GlcNAc branch, the b-Gal H4resonance is observed at 4.124 ppm. Also in the H1-resonances ofthe branch-bearing and the branch-free b-Gal units, the differencesin the chemical shifts were quite clear. Completely analogousdistinction has been described in the H1 and H4 resonances ofbranch-bearing and branch-free galactose units of ordinarypolylactosamines (Leppänen et al., 1997). By contrast, we couldnot establish in the present experiments any major differencesbetween the H1 and the H4 signals of the branch-bearingGalNAc of Glycan 6 and the analogous signals of the linearGlycan 3.

The integrals of the H1 and H4 signals of the branch-bearingand the branch-free b-Gal units in the spectrum of the mixtureof Glycans 4 and 5 suggest that about one third of the hexa-saccharides of the mixture carry the c′-GlcNAc branch at theb-Gal (representing Glycan 4), whereas the majority of thehexasaccharides bear the e′-GlcNAc branch at the six d-GalNAc(representing Glycan 5). Hence, in the branching reaction ofthe linear Glycan 3, both the internal d-GalNAc and the proximalb-Gal served as acceptor sites, but the reaction proceededpreferentially at the internal d-GalNAc.

Purified recombinant human β 1,6-GlcNAc transferase of cIGnT-type appeared to transfer to the internal GalNAc of Glycan 11. The tetrasaccharide Galβ1-4GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-OMe (Glycan 11) was partially converted into apentasaccharide product Hex1HexNAc4OMe (putative Glycan 13)on incubation with UDP-GlcNAc and a purified sample of arecombinant form of cIGnT from human embryonal carcinomacells (Mattila et al., 1998). The MALDI-TOF mass spectrumof the desalted reaction mixture revealed that a product wasformed that contained a new GlcNAc (Figure 5). The datasuggest that in addition to rat serum cIGnT activity, thepurified recombinant cIGnT also worked with the internalGalNAc of an LdN analog of polylactosamines.

In another, shorter reaction, Glycan 11 gave 7% of the putativeGlycan 13, and a parallel incubation under identical conditionsconverted 97% of the polylactosamine Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc into the branched pentasaccharide Galβ1-4GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAc as established bythe MALDI-TOF mass spectrum of the reaction mixture (datanot shown). These data imply that the recombinant cIGnTtransferred the branch-forming GlcNAc about 30 times fasterto an internal Gal of the ordinary i-type polylactosamine thanto the internal GalNAc of the LdN-containing polylactosamineanalog. The β1,6-GlcNAc transferase activity of dIGnT type,present in hog gastric mucosal microsomes, converted thelinear Glycan 10 into the branched Glycan 12. Incubation of

the trisaccharide GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-OMe(Glycan 10) with UDP-GlcNAc and hog gastric mucosalmicrosomes, which contain dIGnT activity (Piller et al., 1984;Seppo et al., 1990; Helin et al., 1997), gave a branched tetra-saccharide GlcNAcβ1-3(GlcNAcβ1-6)GalNAcβ1-4GlcNAcβ1-OMe (Glycan 12) that was isolated. MALDI-TOF mass spectrumof the purified product revealed a major signal of m/z 867.3that was assigned to [M+Na]+ of HexNAc4OMe (calculatedm/z 867.4). The NMR signals of the structural reporter groups(Table IV) confirmed the identity of the product as a tetra-saccharide with four HexNAc residues. In the acceptor, Glycan10, both GlcNAc residues revealed NAc-proton singlets at2.023 ppm. But in the product, Glycan 12, the new β6-linkedGlcNAc, revealed a “low field” NAc proton resonance at2.061 ppm (Table IV). This signal was almost identical with theNAc proton resonance at 2.064 ppm of the β6-linked GlcNAc inthe closely related tetrasaccharide GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAcβ1-OMe, synthesized from the trisaccharideGlcNAcβ1-3Galβ1-4GlcNAcβ1-OMe in precisely the samemanner than Glycan 12 was generated from Glycan 10(Maaheimo, 1998). Hence, we conclude that in Glycan 12, thenovel e′-GlcNAc was probably β6-linked to the internalGalNAc residue.

α1,3-Fucosylation of internal LdN units. Treatment of Glycan 15(GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-3Galβ1-4Glc) with GDP-fucose and a partially purified recombinant form of humanFuc TIV resulted in site-specific α1,3-fucosylation at theinternal LdN unit, generating Glycan 16. The MALDI-TOFmass spectrum of the reaction mixture revealed that mono-fucosylated products were formed with an apparent yield of88%; difucosylated products were not evident (data notshown). Chromatography in a column of immobilized wheatgerm agglutinin (Niemelä et al., 1998), gave pure Glycan 16.Its MALDI-TOF mass spectrum revealed a major signal ofm/z 1120.4 that was assigned to [M+Na]+ of the hexasaccharideHex2HexNAc3Fuc1 (calculated m/z 1120.4).

Pure Glycan 16 was β4-galactosylated as described for theGlycans 2, 10, and 15, and the purified product, Glycan 18,was characterized. Its MALDI-TOF mass spectrum revealed amajor signal of m/z 1282.5 that was assigned to [M+Na]+ of theheptasaccharide Hex3HexNAc3Fuc1 (calculated m/z 1282.5). The

Fig. 5. Positive-ion MALDI-TOF mass spectrum of the oligosaccharides from a partial branching reaction of Glycan 11 with purified human recombinant cIGnT and UDP-GlcNAc.

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1D 1H NMR spectrum of Glycan 18 (Table IV) suggested that thesample obtained was a reasonably pure specimen of the hepta-saccharide Galβ1-4GlcNAcβ1-3GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc. Comparison of the spectra of Glycans 17 and 18in Table IV reveals that Glycan 18 contains the backbone ofGlycan 17 and bears the LdN determinant in the α3-fuco-sylated form (Bergwerff et al., 1993). The isomer, GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc, was absentor present only in very small amounts, as indicated, for example,by the absence of significant reporter group resonances ofGlc-bonded fucose [de Vries, 1995 577] in the 1D NMR spectrumof Glycan 18.

Enzymatic cleavage of LdN analogs of polylactosamines

We studied two enzymatic degradation reactions, hoping theywould prove promising for detection and isolation of putativenatural LdN analogs among ordinary polylactosamines.

Endo-β-galactosidase cleaved the internal GalNAcβ

1-4GlcNAc bond of Glycan 11. Incubation of the tetrasaccharideGalβ1-4GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-OMe (Glycan 11)with endo-β-galactosidase from B. fragilis (Boehringer,Germany) resulted in significant cleavage. The MALDI-TOFmass spectrum of the reaction mixture revealed a distinct signalthat was assigned to [M+Na]+ of a hydrolysis product, thetrisaccharide Hex1HexNAc2, that is, Galβ1-4GlcNAcβ1-3GalNAc(Figure 6A). Also the presence of another cleavage productwas obvious; the signal at m/z 683.3 was assigned to [M+Na]+ ofGalβ1-4GlcNAcβ1-3GalNAcβ1-O-glycerol (calculated m/z 683.2).This compound probably represents a product from a trans-glycosylation reaction with free glycerol that was carried to thereaction milieu with the enzyme. The signal at m/z 826.2represents the substrate (calculated m/z of [M+Na]+, 826.3);two-thirds of Glycan 11 apparently had survived the enzymetreatment in intact form. In a parallel control reaction, thepentasaccharide methyl glycoside Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-OMe of the ordinary polylactosaminetype was cleaved completely. The MALDI-TOF mass spectrumof this digest (Figure 6B). revealed no signal of the intactsubstrate (calculated m/z of [M+Na]+, 947.3) but revealedsignals of the hydrolysis products GlcNAcβ1-3Galβ1-OMe(observed m/z 419.9) and Galβ1-4GlcNAcβ1-3Gal (observedm/z 568.1), as well as the signal of a transglycosylation productGalβ1-4GlcNAcβ1-3Galβ1-O-Glycerol (observed m/z 642.1).The proximal Galβ1-OMe linkage of the substrate and of thecleavage product GlcNAcβ1-3Galβ1-OMe apparently resistedthe enzyme’s action, implying an important role for the adjacent,proximal GlcNAc in the normal cleavage of the internal galacto-sidic linkages of i-type polylactosamines.

The GlcNAcβ1-3GalNAc linkage of LdN-saccharides was unusually resistant toward jackbean β-N-acetylhexoxaminidase. The LdN-hexasaccharide Galβ1-4GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-3Galβ1-4Glc (Glycan 17) was treated with a mixture of β-galac-tosidase and β-N-acetylhexosaminidase of jackbean. MALDI-TOFmass spectrum of the resulting digest revealed large signalsthat were assigned to the molecular ions [M+Na]+ and [M+K]+ ofthe pentasaccharide Hex2HexNAc3 (Figure 7A), representingprobably GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-3Galβ1-4Glc(Glycan 15). Small signals (6%), assigned to the tetrasaccharide

Hex2HexNAc2, representing probably GalNAcβ1-4GlcNAcβ1-3Galβ1-4Glc (Glycan 14), were also present, but smallercleavage products of the original Glycan 17 could not bedetected. In a similar experiment, Glycan 18, Galβ1-4GlcNAcβ1-3GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc,was also completely degalactosylated, but the resultingproduct (Glycan 16) was de-N-acetylglucosaminylated only toa very limited degree (data not shown).

In a parallel control experiment, the heptasasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4GlcNAcof the ordinary polylactosamine type was treated in the sameway as Glycans 17 and 18. The resulting digest showed in theMALDI-TOF mass spectrum (Figure 7B) a complete loss ofthe distal galactose and also an almost complete loss of theperidistal GlcNAc of the heptasaccharide substrate. Consideredtogether, the cleavage data obtained with the jackbeanenzymes imply that the distal GlcNAcβ1-3GalNAc bond of theLdN-containing Glycans 15 and 16 are much more resistanttoward the β-N-acetylhexosaminidase than the distalGlcNAcβ1–3Gal linkage of the ordinary polylactosamineGlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4GlcNAc.The difference in the reaction rates is so large that desialylatedand defucosylated polylactosamine backbones of the ordinarytype can probably be eliminated by the combined action of

Fig. 6. Positive ion MALDI-TOF mass spectra of endo-β-galactosidase digests. (A)Digest of Glycan 11 (Galβ1-4GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-OMe, 1 nmol). (B) Digest of the pentasaccharide Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-OMe (1 nmol).

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β-N-acetylhexosaminidase and β-galactosidase of jackbean,leaving putative LdN analogs largely intact at the GlcNAcβ1-3′LdNβ1-OR stage of the “erosive” reaction.

Discussion

The present data show that distal GalNAcβ1-4GlcNAcβ1-ORdeterminants of unconjugated saccharides are acceptors inhuman blood serum-catalyzed β3-GlcNAc transferasereactions as shown in Equation 1.

(1) GalNAcβ1-4GlcNAcβ1-OR + UDP-GlcNAc → GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-OR + UDP

The purified oligosaccharide products were unambiguouslyidentified by a series of 1D and 2D NMR experiments culminatingin the measurement of some HMBC spectra, which revealedconsistently the long-range heteronuclear correlation over thenovel glycosidic bond, that is, between the distal GlcNAc-H1and the peridistal GalNAc-C3 (cf. Figure 3). Previous work hasestablished that human serum contains a β1,3-GlcNAc transferaseactivity that catalyzes the reactions of Equation 2 (Piller andCartron, 1983; Yates and Watkins, 1983; Hosomi et al., 1984;Seppo et al., 1990).

(2) Galβ1-4GlcNAcβ1-OR + UDP-GlcNAc → GlcNAcβ1-3Galβ1-4GlcNAcβ1-OR + UDP

We suggest that the serum activity responsible for the reactionsof Equation 2 may well be responsible also for the reactions ofEquation 1. This notion is based on previous data showing thatsome purified glycosyltransferases are genuinely multifunctional,transferring efficiently to GalNAc as well as to Gal; pertinentexamples include the sialyl α2,3-transferase known as ST3Gal II(Toivonen et al., 2001) and the β6-GlcNAc transferase generatingO-glycan core 4 as well as branched polylactosamines of I-type(Ropp et al., 1991; Yeh et al., 1999). It will be interesting tosee whether some of the purified recombinant β1,3-GlcNActransferases (GlcNAc to Gal) (Sasaki et al., 1997; Zhou et al.,1999; Shiraishi et al., 2001) will transfer also to distal LdNunits.

In the present experiments, human serum converted only 12.5%of the GalNAcβ1-4GlcNAcβ1-OR into GlcNAcβ1-3GalNAcβ1-4GlcNAcβ1-OR. Under comparable conditions, serum catalyzes40–70% conversion of i-type polylactosamines Galβ1-4GlcNAcβ1-OR into GlcNAcβ1-3Galβ1-4GlcNAcβ1-OR inour laboratory (Leppänen et al., 1997; Penttilä et al., unpublisheddata). These data suggest that internal LdN units may not beabundant in human polylactosamines. In vitro synthesis of theGlcNAcβ1-3GalNAc linkage has been described in the contextof O-glycan core 3 biosynthesis (Brockhausen et al., 1985).We do not know whether the serum β1,3-GlcNAc transferaseactivity catalyzing the reactions of Equation 1 can alsogenerate core 3.

Whether internal LdN units are actually present amongordinary mammalian polylactosamines is not yet known, butsome of the present observations may provide tools forenriching and recognizing putative glycoconjugates withinternal LdN units. In particular, the erosive treatment ofdesialylated and defucosylated backbones of polylactosamineswith β-galactosidase and β-N-acetylhexosaminidase of jackbeanwill destroy the ordinary polylactosamines (Niemelä et al.,1998) but will leave the GlcNAcβ1-3′LdNβ1-OR determinantsvirtually intact as shown by the present data. The “nude” poly-lactosamine backbones for this kind of erosive experimentscan be obtained in many cases by enzymatic desialylation andmild acid defucosylation (Hounsell et al., 1985). As shown inFigure 4, the synthetic GlcNAcβ1-3′LdNβ1-ORs proved to befunctional acceptors for enzyme-assisted β1,3-galactosyl-,β1,4-galactosyl-, β1,4-N-acetyl-galactosaminyl, α1,3-fucosyl-and various β1,6-N-acetyl-glucosaminyl transfer reactionscatalyzed by mammalian enzymes. Most of the products werepurified and characterized adequately in the present experiments.Hence, the data imply that many mammalian enzymes workingwith backbones of ordinary i-type polylactosamines catalyzeanalogous reactions also with internal LdN units of polylactosamineanalogs.

The apparent multifunctionality of polylactosamine-metabolizingenzymes may be partly responsible for the fact that internalLdN groups have not been observed in natural polylactosaminesso far. Their (small) total amount may be distributed amongtoo many distinct determinants. To conclude, the presentexperiments (1) represent the first successful synthesis ofGlcNAcβ1-3′LdN units, (2) show that all monomer units ofthis trisaccharide function as acceptor sites for various glyco-syltransferases known to synthesize ordinary mammalian

Fig. 7. Positive ion MALDI-TOF mass spectra of digests obtained by incubation of oligosaccharides with a mixture of β-galactosidase and β-N-acetylhexosaminidase of jackbean. (A) Digest of the LdN-containing Glycan 17. (B) Digest of a control saccharide Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4GlcNAc (calculated m/z of [M+Na]+ = 1282.5), representing an ordinary i-type polylactosamine.

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polylactosamines, and (3) provide methods for selectiveenzymatic removal of ordinary polylactosamines from putativemixtures containing polylactosamines and their analogs thatcarry internal LdN units.

Materials and methods

Oligosaccharides containg distal GalNAcb1-4GlcNAc determinants

The trisaccharide GalNAcβ1-4GlcNAcβ1-3Galβ1-OMe (Glycan 1),was synthesized by incubating GlcNAcβ1-3Galβ1-OMe,UDP-GalNAc, and purified β1,4 GalT (Sigma) from bovinemilk essentially as described in Palcic and Hindsgaul (1991).Its positive ion mode MALDI-TOF mass spectrum revealed amajor signal (m/z 623.1) that was assigned to the molecular ion[M+Na]+ of Hex1HexNAc2OMe (calculated m/z 623.2). The1D 1H NMR spectrum (Figure 2A, Table II) revealed only theexpected reporter group signals. The disaccharide GalNAcβ1-4GlcNAcβ1-OMe (Glycan 9) was synthesized from GlcNAcβ1-OMe (Sigma) in the same way as Glycan 1. Its MALDI-TOFmass spectrum revealed the molecular ion [M+Na]+ of theexpected m/z value (observed m/z 461.0, calculated m/z 461.2).The reporter group NMR signals (Table IV) were identicalwith those reported in (Hokke, 1993; van den Nieuwenhof et al.,1999). The tetrasaccharide GalNAcβ1-4GlcNAcβ1-Galβ1-4Glc(Glycan 14) was synthesized essentially as described in(Nyame et al., 1999). Its MALDI-TOF mass spectrum revealedthe molecular ion [M+Na]+ of the expected m/z value (observedm/z 771.3, calculated m/z 771.3). The 1D 1H NMR-spectrum(Table IV) revealed the expected reporter group signals.

β1,3-N-Acetylglucosaminyltransferase reactions with distal LdN determinants

Typically, 120 nmol of the acceptor and 2400 nmol of UDP-GlcNAc (Sigma) were incubated with 500 µl of fresh humanserum, which contains β1,3-N-acetylglucosaminyltransferaseactivity (Piller and Cartron, 1983; Yates and Watkins, 1983;Hosomi et al., 1984; Seppo et al., 1990). In some experiments,a concentrate of β3-GlcNAc transferase activity from humanserum was used; the concentrate was prepared by precipitationwith ammonium sulfate as described elsewhere (Yates andWatkins, 1983).

Glycosyltransferase reactions with GlcNAcβ1-3′ LdN acceptors

The β1,6N-acetylglucosaminyltransferase reactions of dIGnTtype, catalyzed by hog gastric mucosal microsomes, wereperformed essentially as in Seppo et al. (1990). The β1,6N-acetylglucosaminyltransferase reactions of cIGnT type, cata-lyzed by the recombinant enzyme, were performed asdescribed in Mattila et al. (1998); the reactions catalyzed by ratserum were performed as described in Leppänen et al. (1997).The β1,4-GalT reactions were performed with purifiedβ1,4GalT from bovine milk (Sigma) essentially as described inBrew et al. (1968). This enzyme was also used as the catalyst forthe β1,4-N-acetylgalactosaminyltransfer reactions (Palcic andHindsgaul, 1991; van den Nieuwenhof et al., 1999). The β1,3-GalT reactions were performed essentially as described inPykari et al. (2000). Colo 205 cell lysates were used as theenzyme, hence both β1,3- and β1,4-GalT reactions took place,

requiring subsequent separation of the two major products. Theα1,3-FucT reaction was performed by incubating the acceptorand GDP-fucose with a partially purified sample of recom-binant human Fuc TIV from BHK-21 cells (Grabenhorst et al.,1998).

Enzymatic degradation reactions

Incubations with the endo-β-galactosidase (B. fragilis, Boehringer,Germany) were performed as in Leppänen et al. (1997). Theexohydrolase reactions with mixed jackbean β-galactosidaseand jackbean β-N-acetylhexosaminidase were performedessentially as described in Niemelä et al. (1998). Recombinantβ2,3,4,6-N-acetylglucosaminidase from Streptococcus pneumoniae(Calbiochem) was used according to the manufacturer’srecommendations.

Origin of reference oligosaccharides

LNβ1-3′LNβ1-3Gal-OMe was obtained as described inNiemelä et al., 1998), LNβ1-3′(Fucα1-3)LN β1–3′LN wasobtained as described (Niemelä et al., 1999).

Chromatographic methods

Biogel P2 chromatography was performed essentially asdescribed (Leppänen et al., 1997). Gel filtration HPLC onSuperdex peptide HR 10/30 (Pharmacia) was performed asdescribed in Maaheimo et al. (1995) for the Superdex 75 HRcolumn. The HexNAc content of oligosaccharide peaks wasmeasured of UV absorption, standardized against externalGlcNAc and GalNAc. HPAE chromatography was performedas described in Maaheimo et al. (1995), but 40 mM NaOH wasused throughout as the eluant. Chromatography on immoblizedwheat germ agglutinin was performed as described elsewhere(Renkonen et al., 1988). Desalting of neutral oligosaccharideswas carried out on mixed beds of AG-1 (AcO-) and AG-50W(H+) (Bio-Rad, CA) as described in Renkonen et al. (1988).

MS and NMR spectroscopy

Positive-ion MALDI-TOF MS was performed as in Leppänenet al. (1997). The m/z values are monoisotopic. The nano-NMRexperiments were performed as described in Toivonen et al.(2001); the conventional NMR experiments were carried out asin Maaheimo et al. (1995).

Acknowledgments

This work was supported by Grants 38042, 40901, and 44318from the Academy of Finland; Grant 40896 from the TechnologyDevelopment Center, Helsinki; a Jubileum Grant of EmilAaltonen Foundation, Tampere, Finland; as well as the ViikkiGraduate School in Biosciences, University of Helsinki, andthe Graduate School of Bioorganic Chemistry, University ofTurku. Some of the data of this report were presented in posterform at the Internatonal Carbohydrate Symposium in SanDiego August 9–14, 1998. We thank Marja Makarow forcritically reading the manuscript.

Abbreviations

cIGnT, β1,6-GlcNAc transferase acting at midchain Gal unitsof polylactosamines; dIGnT, β1,6-GlcNAc transferase acting

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at peridistal Gal units of polylactosamines of the type ofGlcNAcβ1-3Galβ1-4OR; HMBC, heteronuclear multiple bondcorrelation; HPAE, high-pH anion exchange; HPLC, high-performance liquid chromatography; LdN, GalNAcβ1-4GlcNAc;LN Galβ1-4GlcNAc; MALDI-TOF matrix-assisted laserdesorption ionization and time-of-flight; NMR, nuclearmagnetic resonance.

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