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Commensal gut microbiota and probiotics have numerous effects on the host's metabolic and protective systems, which occur primarily through the intestinal epithelial cell interface. Prebiotics, like galacto-oligosaccharides (GOS) are widely used to modulate their function and abundance. However, important structure-function relations may exist, requiring a detailed structural characterization. Here, we detailed the structural characterization of bovine whey derived oligosaccharide preparations enriched with GOS or not, dubbed GOS-enriched milk oligosaccharides (GMOS) or MOS, respectively. We explore GMOS's and MOS's potential to improve intestinal epithelial barrier function, assessed in a model based on barrier disruptive effects of the Clostridioides difficile toxin A. GMOS and MOS contain mainly GOS species composed of β1-6- and β1-3-linked galactoses, and 3'- and 6'-sialyllactose. Both GMOS and MOS, combined with lactobacilli, like Lactobacillus rhamnosus (LPR, NCC4007), gave synergistic epithelial barrier protection, while no such effect was observed with Bifidobacterium longum (BL NCC3001), Escherichia coli (Nissle) or fructo-oligosaccharides. Mechanistically, for barrier protection with MOS, (i) viable LPR was required, (ii) acidification of growth medium was not enough, (iii) LPR did not directly neutralize toxin A, and (iv) physical proximity of LPR with the intestinal epithelial cells was necessary. This is the first study, highlighting the importance of structure-function specificity and the necessity of the simultaneous presence of prebiotic, probiotic and host cell interactions required for a biological effect.

Endothelial sialomucin CD34 functions as an L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x (6-sulfo SLe(x)). It is thought that multivalent 6-sulfo SLe(x) expression promotes high-affinity binding to L-selectin by enhancing avidity. However, the reported low amount of 6-sulfo SLe(x) in total human CD34 is inconsistent with this model and prompted us to re-evaluate CD34 glycosylation. We separated CD34 into 2 glycoforms, the L-selectin-binding and nonbinding glycoforms, L-B-CD34 and L-NB-CD34, respectively, and analyzed released O- and N-glycans from both forms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represented less than 10% of total tonsillar CD34. MECA-79, a mAb to sulfated core-1 O-glycans, bound exclusively to L-B-CD34 and this form contained all sulfated and fucosylated O-glycans. 6-Sulfo SLe(x) epitopes occur on core-2 and extended core-1 O-glycans with approximately 20% of total L-B-CD34 O-glycans expressing 6-sulfo SLe(x). N-glycans containing potential 6-sulfo SLe(x) epitopes were also present in L-B-CD34, but their removal did not abolish binding to L-selectin. Thus, a minor glycoform of CD34 carries relatively abundant 6-sulfo SLe(x) epitopes on O-glycans that are important for its recognition by L-selectin.

Carbohydrates present on cell surfaces participate in numerous biological recognition phenomena including cell-cell interactions, cancer metastasis and pathogen invasion. Therefore, synthetic carbohydrates have a potential to act as pharmaceutical substances for treatment of various pathological phenomena by inhibiting specifically the interaction between cell surface carbohydrates and their protein receptors (lectins). However, the inherently low affinity of carbohydrate-protein interactions has often been an obstacle for successful generation of carbohydrate based pharmaceuticals. Multivalent glycoconjugates, i.e. structures carrying several copies of the active carbohydrate sequence in a carrier molecule, have been constructed to overcome this problem. Here we present two novel types of multivalent carbohydrate conjugates based on chondroitin oligomer and cyclodextrin carriers. These carriers were modified to express primary amino groups, and oligosaccharides were then bound to carrier molecules by reductive amination. Multivalent conjugates were produced using the human milk type oligosaccharides LNDFH I (Lewis-b hexasaccharide), LNnT, and GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc.

Leukocyte traffic into lymph nodes and sites of inflammation is guided by L-selectin. Experiments performed in vitro and with gene-deleted mice suggest that CD34 recognizes L-selectin if decorated by 6-sulfo sialyl Lewis x (sLex) saccharides and the MECA-79 epitope. However, very little is known about glycosylation of human L-selectin ligands. We report here on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiles of N- and O-linked oligosaccharide fractions from human tonsillar endothelial CD34. All detected O-glycans were sialylated; some were also monosulfated or monosulfated and monofucosylated. If a given CD34-glycan may carry all requirements for L-selectin recognition, that is, both 6-sulfo-sLex and MECA-79 epitopes, only one O-glycan fraction, O-9, SA(2)Hex(3)HexNAc(3)- Fuc(1)(SO(3))(1), meets the criteria. A candidate structure is SAalpha2-3Galbeta1-4(Fucalpha1-3)(6-sulfo)GlcNAcbeta1-3Galbeta1-3(SAalpha2-3Galbeta1-4GlcNAcbeta1-6)GalNAc. However, if sulfo sLex glycans are supplemented with separate sulfated, nonfucosylated O-glycans, saccharides in O-6, O-8, or O-9, putatively carrying MECA-79 epitopes, could form multiglycan binding epitopes for L-selectin.

Human alpha3-fucosyltransferases (Fuc-Ts) are known to convert N-acetyllactosamine to Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis x antigen); some of them transfer fucose also to GalNAcbeta1-4GlcNAc, generating GalNAcbeta1-4(Fucalpha1-3)GlcNAc determinants. Here, we report that recombinant forms of Fuc-TV and Fuc-TVI as well as Fuc-Ts of human milk converted chitin oligosaccharides of 2-4 GlcNAc units efficiently to products containing a GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-4R determinant at the nonreducing terminus. The product structures were identified by mass spectrometry and nuclear magnetic resonance experiments; rotating frame nuclear Overhauser spectroscopy data suggested that the fucose and the distal N-acetylglucosamine are stacked in the same way as the fucose and the distal galactose of the Lewis x determinant. The products closely resembled a nodulation factor of Mesorhizobium loti but were distinct from nodulation signals generated by NodZ-enzyme.

HPLC, MALDI-TOF MS and NMR spectroscopy were used to investigate the hydrolysis of cello- and mannooligosaccharides by Cel7A and Man5A from Trichoderma reesei. The experimental progress curves were analysed by fitting the numerically integrated kinetic equations, which provided cleavage patterns for oligosaccharides. This data evaluation procedure accounts for product inhibition and avoids the initial slope approximation. In addition, a transglycosylation step had to be included in the model to reproduce the experimental progress curves. For the hydrolysis of manno-oligosaccharides, Man4-6, by Man5A no mannose was detected at the beginning of the reaction showing that only the internal linkages are hydrolysed. For cellotriose and cellotetraose hydrolysis by Cel7A, the main product is cellobiose and glucose is released from the non-reducing end of the substrate. Intermediary products longer than the substrates were detected by MALDI-TOF MS when oligosaccharides (Glc4-6 or Man4-6) were hydrolysed by either Cel7A or Man5A. Interestingly, two distinct transglycosylation pathways could be observed. Cel7A produced intermediates that are one unit longer than the substrate, whereas Man5A produced intermediates that are two units longer than the substrate.

We have previously isolated and partially characterised the components of a highly purified interferon-alpha (IFN-alpha) preparation produced by Sendai-virus-induced human peripheral blood leukocytes. Nine IFN-alpha species were identified, and two of these were found to be glycosylated [Nyman, T. A., Tölö, H., Parkkinen, J. & Kalkkinen, N. (1998) Identification of nine interferon-alpha subtypes produced by Sendai-virus-induced human peripheral blood leukocytes, Biochem. J. 329, 295-302]. Here, we isolated the N-linked oligosaccharides of IFN-alpha14c and the O-linked chains of IFN-alpha2b, and the glycans were characterised by electrospray tandem mass spectrometry and by specific glycosidase digestions monitored by matrix-assisted laser desorption ionisation time of flight mass spectrometry. The IFN-alpha14c N-glycans were shown to exhibit core-fucosylated biantennary glycans, with about 10% carrying an additional alpha1,3-linked fucose unit at the antennae. The IFN-alpha2b was shown to carry about 50% core type-1 disialyltetrasaccharides, 30% core type-1 monosialyltrisaccharides and 20% core type-2 monosialylpentasaccharides.

The polylactosamine sLex beta1-3'(sLex beta1-6')LacNAc beta1-3'(sLex beta1-6')LacNAc beta1-3'(sLex beta1-6')LacNAc (7) (where sLex is Neu5Ac alpha2-3Gal beta1-4(Fuc alpha1-3)GlcNAc and LacNAc is Gal beta1-4GlcNAc) is a nanomolar L-selectin antagonist and therefore a potential anti-inflammatory agent (Renkonen et al. (1997) Glycobiology, 7, 453). Here we describe an improved synthesis of 7. The octasaccharide LacNAc beta1-3'LacNAc beta1-3'LacNAc beta1-3'LacNAc (4) was converted into the triply branched undecasaccharide LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (5) by incubation with UDP-GlcNAc and the midchain beta1,6-GlcNAc transferase activity of rat serum. Glycan 5 was enzymatically beta1,4-galactosylated to LacNAc beta1-3'(LacNAc beta1-6')LacNAc beta1-3'(LacNAc beta1-6')LacNAc beta1-3'(LacNAc beta1-6')LacNAc (6). Combined with the enzymatic conversion of 6 to 7 (Renkonen et al., loc. cit.) and the available chemical synthesis of 4, our data improve the availability of 7 for full assessment of its anti-inflammatory properties.

By using two different reaction pathways, we generated enzymatically three sialylated and site-specifically alpha 1-3-fucosylated polylactosamines. Two of these are isomeric hexasaccharides Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc and Neu5Ac(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4) GlcNAc, containing epitopes that correspond to VIM-2 and sialyl Lewis (x), respectively. The third one, nonasaccharide Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc, is a sialylated and internally difucosylated derivative of a trimeric N-acetyllactosamine. All three oligosaccharides have one fucose-free N-acetyllactosaminyl unit and can be used as acceptors for recombinant alpha 1-3-fucosyltransferases in determining the biosynthesis pathways leading to polyfucosylated selectin ligands.

A novel saccharide was synthesized by incubating globo-N-tetraose, GalNAc beta1-3Gal alpha1-4Gal beta1-4Glc, and UDP[3H]GlcNAc with hog gastric mucosal microsomes, known to contain beta1,6-N-acetylglucosaminyltransferase activity of a broad acceptor specificity. Chromatography and MALDI-TOF mass spectrometry of the product, as well as the amount of incorporated radioactivity indicated that one [3H]GlcNAc residue was transferred to the acceptor saccharide. One- and two-dimensional 1H NMR-spectroscopic analysis of the product and ESI-CID mass spectrometry of the pentasaccharide in permethylated form established its structure as GalNAc beta1-3([3H]GlcNAc beta1-6)Gal alpha1-4Gal beta1-4Glc. The new enzyme activity possesses substrate specificity features common to a purified beta1,6-GlcNAc-transferase from bovine tracheal epithelium, which forms branches at the subterminal beta1,3-substituted galactose and accepts both GlcNAc- and Gal-configuration at the terminal residue of the acceptor (Ropp et al. (1991) J. Biol. Chem., 266, 23863-23871). The new beta1,6-GlcNAc-branch was readily galactosylated by bovine milk beta1,4-galactosyltransferase, revealing a pathway to novel hybrid type glycans with N-acetyllactosamine chains on globotype saccharides. This pathway may lead to the rare IP blood-group antigen and to globoside-like molecules mediating cell adhesion.

Lymphocyte infiltration is a hallmark of acute rejections in solid organ transplants, such as cardiac allograft. We have previously shown that lymphocyte extravasation to cardiac grafts undergoing rejection is largely due to interactions between lymphocyte L-selectin and its sialyl Lewis x (sLex) decorated ligands. Our previous work demonstrated further that an enzymatically synthetized tetravalent sLex glycan of a branched polylactosamine backbone is a highly efficient inhibitor of L-selectin-dependent lymphocyte adhesion to graft endothelium. To improve the availability of multivalent sLex glycans for anti-inflammatory indications, we now report enzymatic synthesis of another tetravalent sLex glycan that can be potentially produced on a large scale, and show that even the new saccharide is a nanomolar inhibitor of L-selectin-dependent lymphocyte adhesion. The novel antagonist is sLex beta 1-3' (sLex beta 1-6') LacNAc beta 1-3' (sLex beta 1-6') LacNAc beta 1-3' (sLex beta 1-6') LacNAc (8) (where LacNAc is the disaccharide Gal beta 1-4GlcNac and sLex is the tetrasaccharide Neu5Ac alpha 2-3Gal beta 1-4 (Fuc alpha 1-3) GlcNAc). Its five-step synthesis was started from the octameric polylactosamine LacNAc beta 1-3' (GlcNAc beta 1-6') LacNAc beta 1-3' (GlcNAc beta 1-6') LacNAc (3), which in turn is accessible in one step from the hexasaccharide LacNAc beta 1-3'LacNAc beta 1-3'LacNAc. Importantly, the hexasaccharide primer has been synthesized chemically (Alais and Veyrieres, Tetrahedron Lett., 24, 5223, 1983). Hence, our data outline a route to glycan 8, consisting of a combination of chemical and enzymatic methods of oligosaccharide synthesis. In addition, our data show that polylactosamine backbones are able to present multiple sialyl Lewis x determinants to L-selectin in high-affinity mode, without a requirement for uniqueness in the backbone structure.

Proposing to study the molecular mechanisms of mouse gamete adhesion with the aid of high affinity adhesion inhibitors of saccharide nature, we report here the enzymatic synthesis of a bivalent oligosaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (4), consisting of two long arms that link together two distal alpha 1,3-galactose residues. Binding data reported elsewhere (E. Litscher et al., Biochemistry, 1995, 34, 4662-4669) show that 4 is a high affinity inhibitor of mouse gamete adhesion in vitro (IC50 = 9 microM), while a related octasaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc, consisting of two short arms is of very low inhibitory activity. The data highlight the importance of the two alpha-galactose residues of 4, and the length of the sugar chains joining them.

Hog gastric mucosal microsomes contain beta-N-acetylglucosaminidase activity which cleaves GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc at the terminal GlcNAc beta 1-3Gal linkage faster than at the GlcNAc beta 1-6Gal bond, producing mainly GlcNAc beta 1-6Gal beta 1-4GlcNAc. In a marked contrast, GlcNAc beta 1-3(GlcNAc beta 1-6)Gal is cleaved primarily at the GlcNAc beta 1-6Gal bond, while partial hydrolysis of GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc reveals similar rates of cleavage for the (1-3) and (1-6) linkages. Our data support the notion that the terminal beta 1,6-linked GlcNAc unit of GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc may interact with the reducing end GlcNAc unit intramolecularly in water solution.

Partial reactions catalyzed by a (1----3)-N-acetyl-beta-D- glucosaminyltransferase (EC2.4.1.149), known to be present in human serum, were studied by use of biantennary "backbone" saccharides of oligo-N-acetyllactosamine-type as acceptors. Incubation of the radiolabeled blood-group I-active hexasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-Galp- (1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1----4)-D-GlcNAc (1) and UDP-GlcNAc with serum gave first a transient 1:1 mixture of two isomeric heptasaccharides, beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D- GlcpNAc-(1----3)-[beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D- Galp-(1----4)-D-GlcNAc (2) and beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-GlcpNAc-(1----3)- beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1----4)-D-Glc NAc (3), showing that both branches of 1 react equally well. The two heptasaccharides reacted further in the incubation mixture to form the radiolabeled octasaccharide, beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[be ta-D- GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Ga lp- (1----4)-D-GlcNAc (4); during this second reaction, the composition of the heptasaccharide mixture remained unchanged, indicating that 2 and 3 reacted at approximately equal rates. The heptasaccharides 2 and 3 could not be separated from each other, but they could be detected, identified, and quantitatively determined by stepwise enzymic degradations. Partial (1----3)-N-acetyl-beta-D-glucosaminylation reactions, carried out with another acceptor, the branched pentasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-Galp-(1----4)-beta- D- GlcpNAc-(1----6)]-beta-D-Gal (11), revealed that it reacted also equally well at both branches.(ABSTRACT TRUNCATED AT 400 WORDS)

Four radiolabeled pentasaccharides, GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc, Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc, GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4Glc, and Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc, were prepared in virtually pure form. They were obtained by partial enzymic beta 1,4-galactosylations of the appropriate tetrasaccharide acceptors or by partial enzymic degalactosylations of the appropriate hexasaccharides, followed by paper chromatographic separations. All four pentasaccharides contain two nonidentical distal branches, making them valuable primers for enzymatic in vitro synthesis of larger oligo(N-acetyllactosaminoglycans).

Affinity chromatography of unreduced oligosaccharides on a small column of immobilized wheat germ agglutinin (WGA) revealed high-binding affinities for several radiolabeled molecules containing at the reducing end either beta-D-GlcpNAc-(1----6)-D-Gal, beta-D-GlcpNAc-(1----6)-beta- D-Galp-(1----4)-D-GlcNAc, beta-D-GlcpNAc-(1----6)-beta-D-Galp-(1----4)DGlc, D-GlcpNAc-(1----3)-[beta-D-GlcpNAc-(1----6)]-D-Gal, beta-D-GlcpNAc-(1----6)- D-GalNAc, or beta-D-Galp-(1----3)-[beta-D-GlcpNAc-(1----6)]-D-GalNAc sequences. Reduction changed the binding affinities remarkably: The sequences carrying a D-galactose or 2-acetamido-2-deoxy-D-galactose residue at the reducing end lost most of their affinities, but the sequences containing a D-glucose or 2-acetamido-2-deoxy-D-glucose residue at the reducing end gained additional affinity upon reduction. These findings emphasize the role of the unreduced, 6-o-substituted D-galactose and 2-acetamido-2-deoxy-D-galactose residues for the binding of saccharides to WGA, which has been recognized previously as a lectin specific for oligosaccharides containing a 2-acetamido-2-deoxy-D-glucose or sialic acid unit. The results suggested also that WGA-agarose chromatography of alditols may become a valuable method for the fractionation of oligo-N-acetyllactosaminoglycans and related saccharides.

The branch specificity of Escherichia coli beta-galactosidase (EC 3.2.1.23) was studied by analyzing the cleavage of the branched hexasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc (1). This hexasaccharide was cleaved to pentasaccharides Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6) [14C(U)]Gal beta 1-4GlcNAc (3) and GlcNAc beta 1-3(Gal-beta 1-4GlcNAc beta 1-6) [14C(U)]Gal beta 1-4GlcNAc (4) without any appreciable branch specificity. Even the further conversions of the pentasaccharides 3 and 4 into the tetrasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc seemed to proceed at similar rates, without any appreciable branch specificity. In marked contrast to the hexasaccharide 1, the pentasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)[14C(U)]Gal (2), missing the reducing end GlcNAc, is known to be cleaved selectively at the 6-branch; this finding was confirmed in the present study. The different behaviour of hexasaccharide 1 and pentasaccharide 2 reflects differences in the reactivity of their 6-branches; the preferred conformations of these closely related molecules may be quite different.

GlcNAc beta 1-3(GlcNAc beta 1-6) [14C(U)]Gal and GlcNAc beta 1-3(GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc were prepared by in vitro synthesis. They were characterized by enzymatic sequencing, by partial acid hydrolysis, and by periodate oxidation experiments. The two saccharides were isolated also from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine embryonal carcinoma cells (line PC 13). The tetrasaccharide was retarded in a column of agarose-linked wheat germ agglutinin; the trisaccharide was strongly bound. Chromatography in this column separated the trisaccharide into two distinct peaks, which represented interconvertible molecules. Together with our previous data on linear teratocarcinoma saccharides, these findings show that affinity chromatography with immobilized wheat germ agglutinin can be advantageously used in fractionating radiolabeled oligo-N-acetyllactosaminoglycans and saccharides related to them.

A novel linear tetrasaccharide, Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4GlcNAc, was isolated from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine teratocarcinoma cells. It was characterized by exo-glycosidase sequencing and by mild acid hydrolysis followed by identification of all partial cleavage products. The tetrasaccharide, and likewise labelled GlcNAc beta 1-6Gal beta 1-4GlcNAc, resisted the action of endo-beta-galactosidase (EC 3.2.1.103) from E. freundii at a concentration of 125 mU/ml, while the isomeric, radioactive teratocarcinoma saccharides Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc and GlcNAc beta 1-3Gal beta 1-4GlcNAc were cleaved in the expected manner.

Five pure oligosaccharides derived from poly-N-acetyllactosaminoglycans of teratocarcinoma cells were chromatographed on immobilized wheat germ agglutinin (WGA). Three of them, Gal beta 1-4GlcNAc, GlcNAc beta 1-3Gal, and GlcNAc beta 1-3Gal beta 1-4GlcNAc, revealed only weak binding, but GlcNAc beta 1-6Gal beta 1-4GlcNAc was bound moderately and GlcNAc beta 1-6Gal was bound quite strongly. The differences in the binding affinities were large enough to allow chromatographic separation of the five oligosaccharides into three distinct fractions in a small column of WGA-agarose.