Detection of carbohydrate recognition molecules on the plasma membrane of boar sperm by dextran-based multivalent oligosaccharide probes

Two kinds of molecules, one recognizing the sialo-/asialo-N-acetyllactosamine structures and the other recognizing the Lewis X structure in a divalent cation-independent manner, were detected on the head of boar sperm prepared from cauda epididymis by fluorescence-labeled or biotinylated dextran-based multivalent oligosaccharide probes. The N-acetyllactosamine recognition molecule(s) is weakly detected on uncapacitated sperm and becomes strongly detectable on capacitated sperm. On the other hand, the Lewis X recognition molecule is detected at a moderate level before capacitation and at a high level after capacitation. Both molecules disappear from the sperm head after induction of acrosome reaction and also by mild detergent treatment. Thus, the two kinds of carbohydrate molecules are expressed on the plasma membrane of boar sperm depending on their physiological state. Inhibition study of the oligosaccharide-dextran probe binding to isolated sperm plasma membrane by various glycoproteins, oligosaccharides, and sulfated polysaccharides also supported the occurrence of the two distinct kinds of molecules.     

A detailed analysis of neutral and acidic carbohydrates in human milk.

Reverse- and normal-phase chromatography have been used to separate a number of standard human milk oligosaccharides derivatized via a reductive amination reaction with 2-aminoacridone (2-AMAC). Analytes were detected by spectrofluorimetry and injected simultaneously with a hydrolyzed dextran ladder derivatized with methyl-4-aminobenzoate. The latter probe does not fluoresce at the wavelength of emission by the 2-AMAC derivatives, and the derivatized, hydrolyzed dextran components were visualized by their ultraviolet absorbance. This procedure gave precise measurements of the "size" of 2-AMAC oligosaccharides in terms of their glucose equivalent values. Analytical amounts of 2-AMAC oligosaccharide standards were also isolated for further characterization by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry. MS-MS was also used to provide information on oligosaccharide sequences. This methodology was used successfully to characterize mixtures of neutral and acidic oligosaccharides from samples of human milk. This approach could be usefully applied to the study of glycoforms from a variety of samples such as those released from glycoproteins/glycolipids; these have been reported to be altered in a number of diseases, for example, cancer, cystic fibrosis, and autoimmune disease such as rheumatoid arthritis.     

寡糖8-氨基萘基-1,3,6-三磺酸衍生物在毛细管区带电泳中迁移时间相互关系

确定了寡糖８－氨基萘基－１,３,６－三磺酸（ＡＮＴＳ）衍生物在毛细管区带电泳中迁移时间的相互关系．分别将葡聚糖、甘露聚糖、木聚糖和甲壳质部分酸水解产生的不同聚合度寡糖的混合物,用ＡＮＴＳ胺化还原衍生,然后用毛细管电泳,在５０ｍｍｏｌ／Ｌ,ｐＨ２．５磷酸缓冲液中分离衍生物,分别得到１至２１个聚合度的寡聚葡糖衍生物电泳梯度图,以及聚合度均为１至７的寡聚甘露糖、寡聚木糖和寡聚Ｎ－乙酰氨基葡糖衍生物电泳梯度图．同类寡糖衍生物相对迁移时间（ｔｍ）ｒ与聚合度ｎ均成线性关系．寡聚葡糖衍生物相对迁移时间与其他３类寡糖衍生物的相对迁移时间存在线性关系     

Fluorescent derivatives of carbohydrates in the analysis of the structure of glycoconjugates. N-(4-methylcoumarin-7-yl) glycamines in the study of asparagine-bound carbohydrate chains of glycoproteins

4-(N-Methylcoumarin-7-yl) glycamines were employed in studying asparagine-linked carbohydrate chains of acid desialylated fetuin. The procedure was optimised for the reductive amination of oligosaccharides with 7-amino-4-methylcoumarin in the presence of Na(CN)BH3 to lead to oligosaccharide glycamines (AMC-OS). AMC-OS were obtained from dextran oligosaccharides and from oligosaccharides released by hydrazinolysis of asparagine-linked sugar chains of asialofetuin. Reverse-phase HPLC and exclusion HPLC with fluorimetric quantitation of AMC-OS is described. TSK Gel 2000 SW column was calibrated using dextran AMC-OS to give linear relationship ln Ni = k(ti/tr)+b, where ti/tr is retention time of the AMC-OS relatively to the reference AMC-trisaccharide, and Ni is calibration unit value, characterizing molecular size of AMC-OS. Three AMC-OS, Gal3GlcNAc3Man3GlcNAc2-AMC (I) and (II), and Gal2GlcNAc3Man3GlcNAc2AMC (III), were obtained from asialofetuin in a molar ration of 1:1.8:0.1. Acid treatment of AMK-OS (II) in desialylation conditions also gave AMC-OS (III), thus suggesting a partial degalactosylation of the glycoprotein sugar chains during the desialylation. Consequent digestion of AMC-OS (II) and (III) with Jack bean beta-galactosidase and beta-N-acetylhexosaminidase led to the same AMC-OS, Man3GlcNAc2AMC. The final digestion product of AMC-OS (I) was GalGlcNAcMan3GlcNAc2AMC, suggesting a structural difference in one of the antennas of the minor sugar chain of asialofetuin. The monosaccharide quantitation and exoglycosidase sequencing were carried out at a 100 pmole level.     

Chemoenzymatic synthesis of neoglycoproteins using transglycosylation with endo-beta-N-acetylglucosaminidase A

A novel chemoenzymatic approach to synthesize neoglycoproteins containing high-mannose-type oligosaccharides is described. p-Isothiocyanatophenyl-beta-d-glucopyranoside (Glc-ITC) was transferred to the reducing end of the high-mannose-type oligosaccharides using a transglycosylation activity of endo-beta-N-acetylglucosaminidase A (Endo-A). A novel oligosaccharide, Man(6)GlcNAc-Glc-ITC, was synthesized as a coupling reagent for lysyl and N-terminal residues of the protein moiety. The neoglycoconjugate was coupled with several nonglycosylated proteins such as ribonuclease A, lysozyme, and alpha-lactalbumin. Between one and four high-mannose-type oligosaccharides were incorporated per molecule of these proteins. This method should be very useful for the synthesis of neoglycoproteins with homogeneous high-mannose-type oligosaccharides.     

Characterization of carbohydrates using highly fluorescent 2-aminobenzoic acid tag following gel electrophoresis of glycoproteins.

Application of the most sensitive fluorescent label 2-aminobenzoic acid (anthranilic acid, AA) for characterization of carbohydrates from the glycoproteins ( approximately 15 pmol) separated by polyacrylamide gel electrophoresis is described. AA label is used for the determination of both monosaccharide composition and oligosaccharide map. For the monosaccharide determination, bands containing the glycoprotein of interest are excised from the polyvinylidene fluoride (PVDF) membrane blots, hydrolyzed in 20% trifluoroacetic acid, derivatized, and analyzed by C-18 reversed-phase high-performance liquid chromatography. For the oligosaccharide mapping, bands were digested with peptide N-glycosidase F (PNGase F) in order to release the N-linked oligosaccharides, derivatized, and analyzed by normal-phase anion-exchange chromatography. For convenience, the PNGase F digestion was performed in 1:100 diluted ammonium hydroxide overnight. The oligosaccharide yield from ammonium hydroxide-PNGase F digestion was better or equal to all the other reported procedures, and the presumed "oligosaccharide-amine" product formed in the reaction mixture did not interfere with labeling of the oligosaccharides under the conditions used for derivatization. Sequencing of oligosaccharides can be performed using the same mapping method following treatment with an array of glycosidases. In addition, the mapping method is useful for determining the relative and simultaneous distribution of sialic acid and fucose.     

Enzymatic glycosylation of reducing oligosaccharides linked to a solid phase or a lipid via a cleavable squarate linker.

Reducing oligosaccharides were converted into their corresponding glycosylamines, and these were reacted with 3,4-diethoxy-3-cyclobuten-1,2-dione (squaric acid diethyl ester). The resulting derivatives could be linked to amino-functionalized lipids, solids, or proteins. Treatment of the obtained lipid or solid conjugates with aqueous bromine or, alternatively, with ammonia-ammonium borate cleaved the linkage and regenerated the oligosaccharide glycosylamines, which were in turn rapidly hydrolyzed to the reducing oligosaccharides. To demonstrate the usefulness of this linkage in enzymatic oligosaccharide synthesis, lactose was linked to a lipid or a solid phase, the obtained conjugates were then subjected to two enzymatic glycosylations (either consecutively or 'one-pot'). The resulting materials were then cleaved to give, in both cases, the expected reducing tetrasaccharide (lacto-N-neotetraose) in good yield.     

A uniform approach to the synthesis of carbohydrate conjugates of polyhedral boron compounds as potential agents for boron neutron capture therapy

A uniform approach to the synthesis of carbohydrate conjugates with polyhedral boron compounds (PBCs) was developed. Oligosaccharide derivatives with an aglycone moiety amino group can be coupled with PBC carboxyl derivatives using N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride as a coupling agent. Both N- and O-glycosides differing in the conformational mobility around the glycoside bond were shown to be useful as oligosaccharides with a functional group in the aglycone moiety. This allows the application of this approach to the synthesis of PBC conjugates with a wide range of oligosaccharides. For example, not only oligosaccharides obtained by chemical synthesis but also reducing oligosaccharides isolated from natural sources can be transformed into N-glycosides. The approach was tested by the example of conjugation of the carboxyl derivatives of ortho-carborane and dodecaborate anion with lactose as a model oligosaccharide. Lactose, an easily available disaccharide, is a ligand for lectins expressed on the surface of melanoma cells. The approach suggested is the first example of the synthesis of such conjugates that does not require protective groups for the carbohydrate residue. It is especially important for obtaining dodecaborate-carbohydrate conjugates for which the removal of protective groups is often a non-trivial task.     

Fixation of various aldehydic dextrans onto human hemoglobin: Study of conjugate stability

Formation and stability of different aldehydic dextran-hemoglobin conjugates were studied. Two types of polymers were used: sulfated or unsulfated oxidized dextrans and 4-carboxamidobenzaldehyde dextran. Periodate-oxidized dextran forms imine and ketoamine linkages by reaction with hemoglobin and the obtained conjugates are not completely stable, as their molecular size increases with time or decreases after incubation with lysine. The sulfated conjugates are more sensitive to lysine action than the unsulfated ones, which is consistent with the decreased possibilities of Amadori rearrangement. Therefore, this proves the importance of ketoamines for ensuring the cohesion of oxidized dextran-based conjugates. Carboxamidobenzaldehyde dextran forms only imine linkages with hemoglobin and the corresponding conjugates possess a marked instability in the absence of reductive treatment. The different types of conjugates could be stabilized by a sodium borohydride treatment in a satisfying manner.     

Structural analyses of asparagine-linked oligosaccharides of porcine pancreatic kallikrein

The structures of asparagine-linked oligosaccharides of porcine pancreatic beta-kallikrein are reported. Asparagine-linked neutral oligosaccharides were released by N-oligosaccharide glycopeptidase digestion, and the reducing ends of the oligosaccharides were derivatized with a fluorescent reagent, 2-aminopyridine. The mixture of pyridylamino oligosaccharides was separated by reverse-phase and amide-adsorption high-performance liquid chromatography. The pyridylamino oligosaccharides were separated into more than 50 kinds of oligosaccharides. The structures of 5 kinds of triantennary and 12 kinds of tetraantennary oligosaccharides were determined by the use of high-resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. Furthermore, the structures of five kinds of oligomannose-type oligosaccharides were elucidated by a combination of exoglycosidase digestion and high-performance liquid chromatography. 1H NMR data for 14 out of the 17 kinds of N-acetyllactosamine-type oligosaccharides reported here have not previously been described in the literature. (1) It has been shown that fucose containing tri- and tetraantennary oligosaccharides is predominant in porcine pancreatic beta-kallikrein B. (2) It has also been shown that the heterogeneity of the structure in these types of oligosaccharides is derived from the variety of the positions of galactose residues linked to outer N-acetylglucosamine residues. (3) The distribution of oligosaccharides into two glycosylation sites, asparagine-95 and asparagine-239, of beta-kallikrein B was determined. It has been found that oligomannose-type oligosaccharides are exclusively present at asparagine-239, although N-acetyllactosamine-type oligosaccharides occur at both glycosylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Leuconostoc mesenteroides NRRL B-512F Dextransucrase Carboxy-Terminal Deletions on Dextran and Oligosaccharide Synthesis

Dextransucrase (DSR-S) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes synthesis of soluble dextran from sucrose. In the presence of efficient acceptor molecules, such as maltose, the reaction pathway is shifted toward glucooligosaccharide synthesis. Like glucosyltransferases from oral streptococci, DSR-S possesses a C-terminal glucan-binding domain composed of a series of tandem repeats. In order to determine the role of the C-terminal region of DSR-S in dextran or oligosaccharide synthesis, four DSR-S genes with deletions at the 3′ end were constructed. The results showed that the C-terminal region modulated the initial velocity of dextran synthesis but that the K_m for sucrose, the optimum pH, and the activation energy were all unaffected by the deletions. The C-terminal domain modulated the rate of oligosaccharide synthesis whatever acceptor molecule was used (a good acceptor molecule such as maltose or a poor acceptor molecule such as fructose). The C-terminal domain seemed to play no role in the catalytic process in dextran and oligosaccharide synthesis. In fact, it seems that the role of the C-terminal domain of DSR-S may be to facilitate the translation of dextran and oligosaccharides from the catalytic site.     

Rapid and sensitive analyses of glycoprotein-derived oligosaccharides by liquid chromatography and laser-induced fluorometric detection capillary electrophoresis

Asparagine-type oligosaccharides are released from core proteins as N-glycosylamines in the initial step of the action of the peptide N(4)-(N-acetyl-β-D-glucosaminyl)asparagine amidase F (PNGase F). The released N-glycosylamine-type oligosaccharides (which are exclusively present at least during the course of the enzyme reaction) could therefore be derivatized with amine-labeling reagents. Here we report a method using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a labeling reagent for glycosylamine-type oligosaccharides. We applied the method for the sensitive analysis of some oligosaccharide mixtures derived from well-characterized glycoproteins including human transferrin, α(1)-acid glycoprotein, bovine fetuin, and ribonuclease B. NBD-labeled oligosaccharides were successfully separated on an amide-bonded column or a diol-silica column. This labeling method included the release of oligosaccharides from glycoproteins and derivatization of oligosaccharides in a one-pot reaction and was completed within 3h. The method showed approximately fivefold higher sensitivity than that involving labeling with ethyl p-aminobenzoate (ABEE) in HPLC using fluorometric detection and a one order of magnitude higher response in ESI-LC/MS. We also applied this method for the sensitive analysis of glycoprotein-derived oligosaccharides by capillary electrophoresis with laser-induced fluorometric detection (LIF-CE). The limit of detection in HPLC and LIF-CE were 100fmol and 4fmol, respectively.     

Oligosaccharide-derivatized dendrimers: defined multivalent inhibitors of the adherence of the cholera toxin B subunit and the heat labile enterotoxin of E. coli to GM1

Poly(propylene imine) dendrimers having four or eight primary amino groups and a Starburst^TM (PAMAM) dendrimer having eight primary amino groups were used as core molecules, to which phenylisothiocyanate derivatized (PITC) galβ1-3galNAcβ1-4[sialic acidβ2-3]-galβ1-4glc (oligo-GM1) residues were covalently attached to yield multivalent oligosaccharides. The synthesis of the oligo-GM1-PITC derivatized dendrimers was monitored using high performance thin layer chromatography, infrared spectroscopy, sialic acid content, and mass spectroscopy. The ability of multivalent oligo-GM1-PITC dendrimers to inhibit the binding of ¹²⁵I-labeled cholera toxin B subunit and the heat labile enterotoxin of E. coli to GM1-coated microtiter wells was determined. IC₅₀s obtained for the oligo-GM1-PITC dendrimers, GM1, and the oligosaccharide moiety of GM1 indicated that the derivatized dendrimers inhibited binding of the choleragenoid and the heat labile enterotoxin to GM1-coated wells at a molar concentration five- to 15-fold lower than native GM1 and more than 1,000-fold lower than that of the free oligosaccharide. This revised version was published online in November 2006 with corrections to the Cover Date.     

A combined strategy for glycan profiling: a model study with pyridylaminated oligosaccharides

Structural glycomics plays a fundamental role in glycoscience and glycotechnology. In this paper, a novel strategy for the structural characterization of glycans is described, in which MS2 analysis involving a LIFT-TOF/TOF procedure is combined with frontal affinity chromatography (FAC). As model compounds, 20 neutral pyridylaminated (PA) oligosaccharides were chosen, which included four groups of structural isomers differing in sequence, linkage, position, or branching features. By depicting significant diagnostic ions on MS2, most of the analyzed oligosaccharides were successfully differentiated, while two pairs of linkage isomers, i.e., LNT/LNnT, and LNH/LNnH were not. For subsequent analysis by FAC, 14 lectins showing significant affinity to either LNT (type 1) or LNnT (type 2) were screened, and a galectin from the marine sponge Geodia cydonium (GC1) and a plant seed lectin from Ricinus communis (RCA-I) were used for determination of type 1 and 2 chains, respectively. With these specific probes, both of the isomeric pairs were unambiguously differentiated. Furthermore, a pair of triantennary, asparagine-linked oligosaccharide isomers could also be successfully differentiated. Thus, the combination of MS2 and FAC is a practical alternative for the structural characterization of complex glycans.     

A universal approach to the synthesis of carbohydrate conjugates of polyhedral boron compounds as potential agents for boron neutron capture therapy

A universal approach to the synthesis of carbohydrate conjugates with polyhedral boron compounds (PBCs) was developed. Oligosaccharide derivatives with amino group in aglycone moiety can be conjugated with PBC carboxy derivatives using N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride as a condensing agent. Both N-and O-glycosides differing in the conformation mobility around the glycoside bond were shown to be useful as oligosaccharides with a functional group in the aglycone moiety. This allows the application of this approach to the synthesis of PBC conjugates with a wide range of oligosaccharides isolated from natural sources can be transformed into N-glycosides with a functional group in aglycone. The approach was tested by conjugation of the carboxy derivatives of ortho-carborane and dodecaborate anion with lactose as a model oligosaccharide. Lactose, an easily available disaccharide, is a ligand of lectins expressed on the surface of melanoma cells. The approach suggested is the first example of the synthesis of such conjugates that does not require protective groups for the carbohydrate residue. It is especially important for obtaining dodecaborate-carbohydrate conjugates for which the removal of protective groups is often a non-trivial task.     

Structural analysis of N-linked oligosaccharides by a combination of glycopeptidase, exoglycosidases, and high-performance liquid chromatography

A simple, sensitive, and rapid method for the analysis of structures of N-linked carbohydrates is reported. The method involves four steps: preparation of carbohydrate chains from glycopeptides by N-oligosaccharide glycopeptidase digestion; derivatization of the reducing ends of carbohydrate chains with a fluorescent reagent, 2-aminopyridine, by using sodium cyanoborohydride; separation of oligosaccharide derivatives by reverse-phase high-performance liquid chromatography; and structural analysis of oligosaccharides by sequential exoglycosidase digestion. The elution positions of 50 standard oligosaccharide derivatives were determined by HPLC. The structure of an unknown oligosaccharide can be characterized by comparison of its elution position with those of the standard compounds. The method was applied to elucidate the structures of oligosaccharides in the myeloma IgG protein, Yot.     

Electrophoretic resolution and fluorescence detection of N-linked glycoprotein oligosaccharides after reductive amination with 8-aminonaphthalene-1,3,6-trisulphonic acid.

The relative mobilities of various N-linked oligosaccharides reductively aminated to the charged fluorophore 8-amino-naphthalene-1,3,6-trisulphonic acid (ANTS) were determined by electrophoresis on high-density polyacrylamide slab gels. Each ANTS-derivatized oligosaccharide was assigned a relative migration index (RMI) expressed in terms of glucose equivalents, which was conveniently estimated by reference to a homologous series of ANTS--maltooligosaccharides run on each gel as oligosaccharide size standards. High-mannose-, complex- and hybrid-type structures were generally well resolved and easily visualized at picomole levels by simple UV light excitation. Application of these methods for the qualitative analysis of the oligosaccharides released from bovine fetuin and bovine asialofetuin by peptide-N-glycosidase F illustrates the usefulness of these techniques as fast, simple, and inexpensive tools for the characterization of N-linked oligosaccharides attached to glycoproteins.     

Interaction assay of oligosaccharide with lectins using glycosylasparagine

Glycosyl amino acids having natural glycan were useful for the interaction assay of oligosaccharides. A glycochip containing the whole structure of an oligosaccharide was easily prepared by the immobilization of the glycosyl amino acid. Furthermore, fluorescence probes were introduced into the glycosyl amino acid while maintaining the whole structure of oligosaccharide. By using these labeled oligosaccharides, fluorescence polarization (FP) and fluorescence correlation spectroscopy (FCS) analyzed the carbohydrate-lectin interaction in a solution assay system.     

Synthesis of glycocluster-tumor antigenic peptide conjugates for dendritic cell targeting

The use of dendritic cells (DC) for the development of therapeutic cancer vaccines is attractive because of their unique ability to present tumor epitopes via the MHC class I pathway to induce cytotoxic CD8+ T lymphocyte responses. C-Type membrane lectins, DC-SIGN and the mannose receptor (MR), present on the DC surface, recognize oligosaccharides containing mannose and/or fucose and mediate sugar-specific endocytosis of synthetic oligolysine-based glycoclusters. We therefore asked whether a glycotargeting approach could be used to induce uptake and presentation of tumor antigens by DC. To this end, we designed and synthesized glycocluster conjugates containing a CD8+ epitope of the Melan-A/Mart-1 melanoma antigen. These glycocluster-Melan-A conjugates were obtained by coupling glycosynthons: oligosaccharyl-pyroglutamyl-beta-alanine derivatives containing either disaccharides, a dimannoside (Manalpha-6Man) or lactoside, or a Lewis oligosaccharide, to Melan-A 16-40 peptide comprising the 26-35 HLA-A2 restricted T cell epitope, extended with an oligolysine stretch at the C-terminal end. We showed by confocal microscopy and flow cytometry that fluorescent-labeled Melan-A glycoclusters containing either dimannoside or Lewis oligosaccharide were taken up by DC and concentrated in acidic vesicles; conversely lactoside glycopeptides were not at all taken up. Furthermore, using surface plasmon resonance, we showed that dimannoside and Lewis-Melan-A conjugates bind MR and DC-SIGN with high affinity. DC loaded with these conjugates, but not with the lactose-Melan-A conjugate, led to an efficient presentation of the Melan-A epitope eliciting a CD8+ T-lymphocyte response. These data suggest that synthetically designed glycocluster-tumor antigen conjugates may induce antigen cross-presentation by DC and represent a promising tool for the development of tumor vaccines.     

Structure of asparagine-linked oligosaccharides on human and rabbit testosterone-binding globulin

We have previously demonstrated, using two-dimensional polyacrylamide gel electrophoresis, that much of the microheterogeneity of human (h) and rabbit (rb) testosterone-binding globulin (TeBG) is due to differential glycosylation of a single protomer. Since glycosylation has been shown to be a physiologically important modification of proteins, we have examined the structure of the oligosaccharide chains attached to hTeBG and rbTeBG to facilitate future studies on the mechanisms of action of the proteins. The structures of the oligosaccharides attached to TeBG were determined by using serial lectin chromatography. About 10% of the TeBG from castrated male rabbits and about 20% of the TeBG from pregnant rabbits and from a human sample were not retained on a column of immobilized concanavalin-A (Con-A). This fraction would consist of TeBG with attached asparagine (Asn)-linked tri- and tetraantennary complex and serine/threonine (O)-linked oligosaccharides as well as non-glycosylated forms. None of the lectins used to subfractionate these species was effective. Forty to 50% of the TeBG applied to Con-A possessed biantennary complex oligosaccharides as indicated by the fact that it could be eluted with 10 mM 1-O-methyl-alpha-D-glucopyranoside and by its retention on wheat germ agglutinin (WGA). About 8% of the biantennary complex oligosaccharides on hTeBG and none of those on rbTeBG were fucosylated on the chitobiose core, as determined by chromatography on Lens culinaris lectin (LcH). Galactosylated oligosaccharides were also present on the TeBG in this fraction as indicated by its interaction with Ricinus communis-I (RCA-I). Thirty to 40% of the TeBG applied to Con-A was retained and could be eluted with 0.5 M methyl-alpha-D-mannopyranoside. This fraction contains TeBG possessing high mannose-type, hybrid-type, and complex galactosylated glycans as determined by chromatography on Con-A, WGA, and RCA-I. Evidence based on the binding of mannoside-eluted TeBG to Con-A, WGA, and RCA-I indicated that at least the TeBG in this fraction contained two glycosylation sites and that the sites were differentially glycosylated.