Agglutinin from Limulus polyphemus. Purification with formalinized horse erythrocytes as the affinity adsorbent.

We have purified an agglutinin from the hemolymph of Limulus polyphemus about 1500-3000-fold by adsorption to formalinized horse erythrocytes, elution with N-acetylneuraminic acid and subsequent fractionation on Sephadex G-200. Recovery was in the range of 50 percent. On ultracentrifugation the agglutinin behaves as an homogenous protein with a molecular weight of about 460 000. On polyacrylamide gel electrophoresis of the dissociated protein in sodium dodecylsulfate we found a single prominent diffuse band with an apparent molecular weight of 22 000 plus or minus 2000. This band contained carbohydrate as determined by periodic acid-Schiff staining. The intensity of staining compared with standards suggested a carbohydrate content of less than 4 percent. The protein contains a preponderance of acidic amino acids and has an isoelectric point of 4.83.5 residues per 1000 of glucosamine were detected on amino acid analysis. Agglutination of formalinized horse erythrocytes by the purified protein is inhibited not only by N-acetylneuraminic acid but also by D-glucuronic acid; but not by a number of other monosaccharides. D-Glucuronic acid may be used in place of N-acetylneuraminic acid as the eluting sugar in the purification procedure.     

Structural studies on the carbohydrate units of armadillo submandibular glycoprotein.

The structure of carbohydrate units of the major glycoprotein fraction of armadillo submandibular gland was investigated. Alkaline borohydride reductive cleavage of the glycoprotein resulted in the release of O-glycosidically linked mono- and disaccharide units. The monosaccharide was identified as N-acetylgalactosaminitol, whereas disaccharide contained of N-acetylneuraminic acid and N-acetylgalactosaminitol. Treatment of the native and desialyzed glycoprotein with alpha-N-acetylgalactosaminidase resulted in the removal of 60% and 96% of N-acetylgalactosamine, respectively. No cleavage of this sugar was affected by the action of beta-N-acetylhexosaminidase. Both N-acetylgalactosamine and N-acetylneuraminic acid were susceptible to oxidation with periodate. Analyses of the partially methylated N-acetylgalactosamine derivatives, obtained from the permethylated native glycoprotein, showed the presence of 3,4,6-tri-O-methyl-N-methylacetamidogalactose and 3,4-di-O-methyl-N-methylacetamidogalactose in a ratio of 1 : 0.4. Only 3,4,6-tri-O-methyl-N-methylacetamidogalactose was found in the hydrolysates of permethylated desialyzed glycoprotein. These results together with our previous data on chemical composition of the glycoprotein suggest that about 30% of the oligosaccharide chains consist of NeuAc alpha 2 leads to 6GalNAc alpha 1 leads to O-Thr(Ser) and 70% of GalNAc alpha leads to O-Thr(Ser).     

A monoclonal antibody to free N-acetylneuraminic acid

A monoclonal antibody (70-A) to free N-acetylneuraminic acid was obtained by immunizing mice with its synthetic beta-glycoside, sodium O-[(5-acetamido-3,5-dideoxy-D-glycero-beta-D-galacto-2- nonulopyranosyl)onate]-(2----3)-1,2-di-O-tetradecyl-sn-glyce rol, followed by fusing the isolated spleen cells with mouse myeloma cells and cloning positive fusions. 70-A reacted with various synthetic beta-glycosides of N-acetylneuraminic acid and also with cytidine-5'-monophosphate-N- acetylneuraminic acid, known as its sole naturally occurring beta-glycoside. The inhibition assay showed that N-glycolylneuraminic acid had slightly lower reactivity than N-acetylneuraminic acid, but other monosaccharides tested, such as N-acetylglucosamine, N-acetylgalactosamine or N-acetylmannosamine, had no reactivity toward 70-A. Reactivity of 70-A with free N-acetylneuraminic acid was confirmed by measuring the specific binding of N-[14C]acetylneuraminic acid to the antibody. The association constant of 70-A with N-acetylneuraminic acid was determined to be 5.96.10(4) M-1 by equilibrium dialysis.     

Amino sugars in the glycoprotein toxin from Bacillus thuringiensis subsp. israelensis.

The carbohydrate content of purified Bacillus thuriniensis subsp. israelensis crystal toxin was determined by six biochemical tests, column chromatography on an amino acid analyzer, and the binding of 11 fluorescent lectins. The crystals contained approximately 1.0% neutral sugars and 1.7% amino sugars. The amino sugars consisted of 70% glucosamine and 30% galactosamine. No N-acetylneuraminic acid (sialic acid) was detected. The presence of amino sugars was confirmed by the strong binding of fluorescent wheat germ agglutinin and the weak binding of fluorescent soybean agglutinin. These lectins recognize N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, respectively. The lectin-binding sites appeared evenly distributed among the protein subunits of the crystal. The sugars were covalently attached to the crystal toxin because wheat germ agglutinin still bound alkali-solubilized toxin which had been boiled in sodium dodecyl sulfate, separate by polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. This study demonstrates the covalent attachment of amino sugars and indicates that the B. thuringiensis subsp. israelensis protein toxins should be viewed as glycoprotein toxins. The crystals used in the present study were purified on sodium bromide density gradients. Studies employing crystals purified on Renografin density gradients can give artificially high values for the anthrone test for neutral sugars.     

Studies on the composition of egg-white ovomucin

1. Purified ovomucin was isolated as an insoluble glycoprotein complex from thick egg white. 2. A homogeneous glycoprotein, designated alpha-ovomucin, of molecular weight 210000 and containing N-acetylglucosamine (6.7%, w/w), N-acetylgalactosamine (0.6%, w/w), galactose (1.8%, w/w), mannose (4.6%, w/w), N-acetylneuraminic acid (1.0%, w/w) and sulphate (0.7%, w/w), was isolated from preparations of reduced ovomucin by sedimentation equilibrium in a density gradient of caesium chloride formed in the presence of 4m-guanidine hydrochloride. 3. A carbohydrate-rich fraction, designated beta-ovomucin (which is homogeneous by sedimentation-velocity analysis in 5m-guanidine hydrochloride but which is heterogeneous by analytical sedimentation equilibrium in a density gradient of caesium chloride in the presence of 4m-guanidine hydrochloride), containing N-acetylglucosamine (11.0%, w/w), N-acetylgalactosamine (8.7%, w/w), galactose (19.2%, w/w), mannose (4.1%, w/w), N-acetylneuraminic acid (13.8%, w/w) and sulphate (2.7%, w/w), was also obtained from preparations of reduced ovomucin by the density-gradient method. 4. Mild acid hydrolysis of the unfractionated ovomucin complex showed that N-acetylneuraminic acid occupied a terminal position of the oligosaccharide chains. 5. Alkaline beta-elimination reactions with the unfractionated ovomucin complex indicated that N-acetylgalactosamine was linked by alkali-labile bonds to hydroxy amino acids.     

Deglycosylation studies on tracheal mucin glycoproteins

Following several model experiments, conditions were developed for optimal deglycosylation of tracheal mucin glycoproteins. Exposure of rigorously dried material to trifluoromethanesulfonic acid at 0 degree C for up to 8 h results in cleavage of essentially all fucose, galactose, and N-acetylglucosamine, about 80% of the N-acetylneuraminic acid (NeuNAc), and a variable amount of N-acetylgalactosamine (GalNAc), the sugar involved in linkage to protein. Residual N-acetylneuraminic acid is sialidase susceptible and apparently in disaccharide units, presumably NeuNAc2----GalNAc. The remaining N-acetylgalactosamine is mostly present as monosaccharides, and a few Gal beta 1----3GalNAc alpha units are also present; both are cleaved by appropriate enzymatic treatment. The saccharide-free proteins obtained from either human or canine mucin glycoproteins have molecular weights of about 100,000 and require chaotropic agents or detergents for effective solubilization.     

Isolation and identification of a fucose-containing ganglioside from bovine thyroid gland.

Bovine thyroid glands are known to contain a complex array of gandliosides. One of the predominant gangliosides was isolated analyzed by gas-liquid chromatography and mass spectrometry. The carbohydrate composition was fucose, N-acetylneuraminic acid, galactose, N-acetylgalactosamine, and glucose in molar ratios of 1:1:2:1:1. The structure of the ganglioside was identified as: (Formula: see text).     

Purification and properties of N-acyl-D-mannosamine dehydrogenase from Flavobacterium sp. 141-8

A new enzyme, N-acyl-D-mannosamine dehydrogenase, was purified to apparent homogeneity from a cell-free extract of Flavobacterium sp. 141-8 and some of its properties were investigated. The enzyme showed optimum activity at pH 8.0-9.5. N-Acetyl- and N-glycolyl-D-mannosamine were oxidized but other commonly existing sugars, such as N-acetylglucosamine, N-acetylgalactosamine, amino sugars, neutral hexoses, and pentoses, were not oxidized. NAD+ was specifically utilized as an effective hydrogen acceptor. The apparent Km values for N-acetyl- and N-glycolyl-D-mannosamine, and NAD+ were 1.0, 13.3, and 0.41 mM, respectively. The stoichiometry data showed that 1 mol each of N-acetyl-D-mannosamine and NAD+ were converted to 1 mol each of N-acetyl-D-mannosaminic acid and NADH, respectively. Although the formation of lactone was detected in the enzyme reaction mixture, the reverse reaction of the enzyme, the reduction of N-acetyl-D-mannosamino-lactone, was not observed. The enzyme activity was strongly inhibited by Hg2+ and SDS, but metal-chelating reagents and sulfhydryl-group-blocking reagents had almost no effect. The molecular weight of the enzyme was estimated to be 120,000 on gel filtration and 29,000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was at pH 4.8. On trial application of the enzyme, it was indicated that N-acetylneuraminic acid can be determined quantitatively with the combined enzyme system involving the new enzyme and N-acetylneuraminic acid aldolase.     

Purification and properties of N-acetylneuraminate lyase from Escherichia coli

N-Acetylneuraminate lyase [N-acetylneuraminic acid aldolase EC 4.1.3.3] from Escherichia coli was purified by protamine sulfate treatment, fractionation with ammonium sulfate, column chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA 44, and preparative polyacrylamide gel electrophoresis. The purified enzyme preparation was homogeneous on analytical polyacrylamide gel electrophoresis, and was free from contaminating enzymes including NADH oxidase and NADH dehydrogenase. The enzyme catalyzed the cleavage of N-acetylneuraminic acid to N-acetylmannosamine and pyruvate in a reversible reaction. Both cleavage and synthesis of N-acetylneuraminic acid had the same pH optimum around 7.7. The enzyme was stable between pH 6.0 to 9.0, and was thermostable up to 60 degrees C. The thermal stability increased up to 75 degrees C in the presence of pyruvate. No metal ion was required for the enzyme activity, but heavy metal ions such as Ag+ and Hg2+ were potent inhibitors. Oxidizing agents such as N-bromosuccinimide, iodine, and hydrogen peroxide, and SH-inhibitors such as p-chloromercuribenzoic acid and mercuric chloride were also potent inhibitors. The Km values for N-acetylneuraminic acid and N-glycolylneuraminic acid were 3.6 mM and 4.3 mM, respectively. Pyruvate inhibited the cleavage reaction competitively; Ki was calculated to be 1.0 mM. In the condensation reaction, N-acetylglucosamine, N-acetylgalactosamine, glucosamine, and galactosamine could not replace N-acetylmannosamine as substrate, and phosphoenolpyruvate, lactate, beta-hydroxypyruvate, and other pyruvate derivatives could not replace pyruvate as substrate. The molecular weight of the native enzyme was estimated to be 98,000 by gel filtration methods. After denaturation in sodium dodecyl sulfate or in 6 M guanidine-HCl, the molecular weight was reduced to 33,000, indicating the existence of 3 identical subunits. The enzyme could be used for the enzymatic determination of sialic acid; reaction conditions were devised for determining the bound form of sialic acid by coupling neuraminidase from Arthrobacter ureafaciens, lactate dehydrogenase, and NADH.     

Botulinum neurotoxin type B. Its purification, radioiodination and interaction with rat-brain synaptosomal membranes

Neurotoxin from Clostridium botulinum type B was purified to homogeneity by by affinity and ion-exchange chromatography; specific neurotoxicity of this protein (Mr of approximately equal to 155 000) following trypsinisation attained a level of 2 X 10(8) mouse LD50 units/mg protein. 125I-iodination of the toxin to high specific radioactivities (19-63 TBq/mmol) yielded typically greater than 65% of its original toxicity; dodecyl sulphate gel electrophoresis under reducing conditions, after trypsinisation, showed that the larger polypeptide (Mr of approximately equal to 101 000) was labelled preferentially. Saturable binding of the 125I-labelled neurotoxin to rat cerebrocortical synaptosomes was observed and Scatchard analysis showed a low content of acceptors with high affinity (Kd = 0.3-0.5 nM;Bmax approximately equal to 30-60 fmol/mg protein, together with a much larger population of weak-affinity sites. No significant differences in binding affinity were seen in competition experiments using native or fully activated (trypsinized) neurotoxin, indicating that chain cleavage is not essential for acceptor-toxin interaction. Type A botulinum neurotoxin showed a limited capacity to inhibit the synaptosomal binding of labelled type B toxin, even at high concentrations (1 muM), and other neurotoxins were without effect, emphasising the acceptor selectivity. Near-complete loss of specific toxin binding was produced by preincubation of synaptosomes with neuraminidase whereas inhibition of the low-affinity sites with wheat-germ agglutinin was less pronounced; such inactivation was prevented by inclusion of selective inhibitors (2,3-dehydro-2-deoxy-N-acetylneuraminic acid and N-acetylglucosamine, respectively). These observations implicate N-acetylneuraminic acid and, possibly, other sugar moieties as constituents of the toxin acceptors. Trypsinisation of synaptosomes gave incomplete inhibition of binding when assayed with 1 nM or 10 nM 125I-iodinated toxin. Detailed analysis of the actions of neuraminidase, trypsin and heat treatment on the concentration dependence of toxin binding suggest the existence of at least two distinguishable populations of sites that contain N-acetylneuraminic acid, with a protein component being associated with the acceptors of lower affinity. These findings are discussed in relation to those previously reported for type A neurotoxin and to the possible physiological significance of such membrane acceptors.     

Interaction of sulfated glycosaminoglycans with lectins

The sulfated glycosaminoglycans, such as keratan sulfate and chitin sulfate having 3-hydroxy free N-acetyl-beta-D-glucosaminyl residues as constituents, reacted with wheat germ agglutinin and Solanum tuberosum agglutinin by sugar-specific interaction. The glycosaminoglycans showed different inhibitory activities to the hemagglutination reaction of these lectins and keratan sulfate and its modified products formed insoluble complexes with both of the lectins at pH 7.0 in physiological saline solutions (0.15 M NaCl). S. tuberosum agglutinin was precipitated within a particularly narrow concentration range of keratan sulfate, and the formation of a soluble complex was observed by gel chromatography. These interactions were specifically inhibited by N,N'-diacetylchitobiose but not by 2 M NaCl. The specific interactions of the glycosaminoglycans with S. tuberosum agglutinin were confirmed by their ultraviolet difference spectra with two peaks at 285 and 298 nm attributable to the tryptophan residues in the binding site of the agglutinin. It was also found that S. tuberosum agglutinin and wheat germ agglutinin have different binding specificities. The presence of sulfate groups in either keratan sulfate or chitin sulfate did not interfere with their specific interactions with S. tuberosum agglutinin as strongly as with wheat germ agglutinin. The N-acetylneuraminic acid residues in keratan sulfate were found to be receptor sites for wheat germ agglutinin but not for S. tuberosum agglutinin.     

Identification of N-acetylhexosamines produced by enzymes of the N-acetylneuraminic acid metabolic pathway by borate complex anion-exchange chromatography of the corresponding N-acetylhexosaminitols

A mixture of hexosaminitols obtained by reducing N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine with sodium borohydride was resolved by borate complex anion-exchange chromatography. This procedure yielded a complete separation of N-acetylglucosaminitol, N-acetylgalactosaminitol, and N-acetylmannosaminitol and provided a rapid and accurate means for identifying and measuring N-acetylhexosamines in biological samples. This method was applied to studies on N-acetylneuraminic acid metabolism in human skin fibroblasts. It was used to identify reaction products in two enzymatic reactions: the conversion of UDP-N-acetylglucosamine to N-acetylmannosamine and UDP by UDP-N-acetylglucosamine 2-epimerase and the conversion of N-acetylneuraminic acid to N-acetylmannosamine and pyruvate by N-acetylneuraminate pyruvate-lyase. It was also used to identify the free 3H-labeled N-acetylhexosamines found in fibroblasts cultured in the presence of N-[3H]acetylmannosamine.     

Gracilaria tikvahiae agglutinin. Partial purification and preliminary characterization of its carbohydrate specificity

A potent agglutinin of rabbit and sheep red blood cells, obtained from the red alga Gracilaria tikvahiae, was purified by ammonium sulfate fractionation, ion exchange, gel filtration, and hydroxylapatite chromatography. Human A and B blood group erythrocytes were also agglutinated, whereas human O blood group erythrocytes were not agglutinated. The hemagglutination titer was not significantly affected by the addition of EDTA or the divalent cations Ca2+, Mg2+, or Mn2+. The carbohydrate specificity was characterized by hemagglutination inhibition using various monosaccharides, glycoproteins, and glycopeptides. The results suggested that the agglutinin has affinity for N-acetylneuraminic acid as well as glycoconjugates containing N-acetylneuraminic acid.     

Factors influencing lethality of Bacillus thuringiensis kurstaki toxin for eggs and larvae of Trichostrongylus colubriformis (Nematoda)

A toxin from Bacillus thuringiensis kurstaki was lethal to eggs and first- and second-stage larvae of the ruminant nematode Trichostrongylus colubriformis. Sheathed and exsheathed third-stage larvae were also killed by the toxin. However, susceptibility of the ova to the toxin decreased after several hours of development. Heating at 65 C for 1 hr or freezing at 0 C for 3 mo did not affect stability of the toxin. Ovicidal activity of the toxin was not altered by treatment with 13 microbial or mammalian enzymes, but toxicity was reduced by the antibiotics streptomycin or penicillin G and the enzyme inhibitor L-1-tosylamide 2-phenylethylchloromethyl ketone. Cuprous, ferrous, and zinc chlorides also inhibited the ovicidal activity of the toxin. Increased osmolarity of the assay media or solubilization of the toxin from pH 3 to 11 had no effect on toxicity for eggs. The membrane agents sodium vanadate and 4,4'-diisothiocyano-2,2' disulfonic acid stilbene increased (9-fold) and decreased (333-fold) toxicity, respectively. N-acetylneuraminic acid was the only tested sugar that reduced the toxicity of B. t. kurstaki.     

Lectin-Binding Sites Involved in Paramecium primaurelia Mating Pair Formation

ABSTRACT. Mating pair formation in Paramecium primaurelia was shown to be inhibited by incubating mating-competent mating type (mt) I and mt U cells with Limulus polyphemus agglutinin (LPA) or wheat germ agglutinin (WGA). Preincubation of LPA and WGA with their specific binding-monosaccharides, N-acetylneuraminic acid (NeuAc) and N-acetylglucosamine (GlcNAc), respectively, prevented the lectin effect on pair formation. Addition either of NeuAc or GlcNAc resulted in a reversal of cell pairing inhibition by LPA or WGA, respectively. Both NeuAc and GlcNAc monosaccharides inhibited pair formation when their concentration exceeded a threshold value. The pattern of the relative distribution of NeuAc and GlcNAc molecules on the cell surface was analyzed using fluorescence resonance energy transfer techniques combined with imaging systems. Mt I1 cells showed a high lectin-binding site density localized just on the surface region engaged in conjugation. The pattern of mt I cells, consisting of a quite homogeneous lectin-binding site density spread on the cell surface, was also common to nonmating-competent cells and to immature cells. These findings suggest that in P. primaurelia pair formation involves both NeuAc and GlcNAc residues and that the development of mating-competence is related to a modification in NeuAc and GlcNAc relative distribution on the cell surface of mt 11 cells.     

Human embryonal prealbumin-1. Chemical and immunochemical characteristics

General chemical and immunochemical characterization of human embryonic prealbumin-1 (EPA-1) isolated from abortive blood is presented. EPA-1 was found to exist as glycosylated and non-glycosylated forms, which are immunochemically identical. Sugar moiety of the glycosylated form contains residues of fucose (3.0%), mannose (3.2%), galactose (7.8%), N-acetylglucosamine (5.4%), N-acetylgalactosamine (1.2%) and N-acetylneuraminic acid. Using methylation studies, types of bonds between the sugar residues were elucidated.     

Interaction of Bacillus thuringiensis toxins with larval midgut binding sites of Helicoverpa armigera (Lepidoptera: Noctuidae)

In 1996, Bt-cotton (cotton expressing a Bacillus thuringiensis toxin gene) expressing the Cry1Ac protein was commercially introduced to control cotton pests. A threat to this first generation of transgenic cotton is the evolution of resistance by the insects. Second-generation Bt-cotton has been developed with either new B. thuringiensis genes or with a combination of cry genes. However, one requirement for the "stacked" gene strategy to work is that the stacked toxins bind to different binding sites. In the present study, the binding of (125)I-labeled Cry1Ab protein ((125)I-Cry1Ab) and (125)I-Cry1Ac to brush border membrane vesicles (BBMV) of Helicoverpa armigera was analyzed in competition experiments with 11 nonlabeled Cry proteins. The results indicate that Cry1Aa, Cry1Ab, and Cry1Ac competed for common binding sites. No other Cry proteins tested competed for either (125)I-Cry1Ab or (125)I-Cry1Ac binding, except Cry1Ja, which competed only at the highest concentrations used. Furthermore, BBMV from four H. armigera populations were also tested with (125)I-Cry1Ac and Cry1Ab to check the influence of the insect population on the binding results. Finally, the inhibitory effect of selected sugars and lectins was also determined. (125)I-Cry1Ac binding was strongly inhibited by N-acetylgalactosamine, sialic acid, and concanavalin A and moderately inhibited by soybean agglutinin. In contrast, (125)I-Cry1Ab binding was only significantly inhibited by concanavalin A. These results show that Cry1Ac and Cry1Ab use different epitopes for binding to BBMV.     

Purification and characterization of a sialic acid specific lectin from the hemolymph of the freshwater crab Paratelphusa jacquemontii.

A naturally occurring hemagglutinin was detected in the serum of the freshwater crab, Paratelphusa jacquemontii (Rathbun). Hemagglutination activity with different mammalian erythrocytes suggested a strong affinity of the serum agglutinin for horse and rabbit erythrocytes. The most potent inhibitor of hemagglutination proved to be bovine submaxillary mucin. The lectin was purified by affinity chromatography using bovine submaxillary mucin-coupled agarose. The molecular mass of the purified lectin was 34 kDa as determined by SDS/PAGE. The hemagglutination of purified lectin was inhibited by N-acetylneuraminic acid but not by N-glycolylneuraminic acid, even at a concentration of 100 mm. Bovine submaxillary mucin, which contains mainly 9-O-acetyl- and 8,9 di-O-acety-N-acetyl neuraminic acid was the most potent inhibitor of the lectin. Sialidase treatment and de-O-acetylation of bovine submaxillary mucin abolished its inhibitory capacity completely. Also, asialo-rabbit erythrocytes lost there binding specificity towards the lectin. The findings indicated an O-acetyl neuraminic acid specificity of the lectin.     

The structure and composition of cartilage keratan sulphate

Keratan sulphate was isolated from bovine intervertebral disc and bovine nasal septum after hydrolysis with proteinases and treatment with dilute alkali. Each preparation was found to contain, per keratan sulphate chain: (a) 1 residue of mannose; (b) 3 residues of N-acetylneuraminic acid (2 residues after alkali treatment); (c) 1 residue of N-acetylgalactosamine (lost after alkali treatment); (d) 1 residue or less of fucose. N-Acetyl-neuraminic acid residues were at non-reducing termini and were bonded to keratan sulphate through galactose residues. Evidence is presented for two different types of linkage between skeletal keratan sulphate and protein. Consideration of molecular parameters and compositions leads to a proposed structure for keratan sulphate-protein as found in skeletal proteoglycans.     

Isolation and partial characterization of the major sialoglycoprotein of human T-lymphoblastoid cells of a MOLT-4B cell line.

A sialoglycoprotein with an approx. mol.wt. of 95000 was isolated from human lymphoblastoid cells of a MOLT-4B cell line, which was of human T-lymphocyte origin, by ion-exchange chromatography, affinity chromatography on a column of wheat-germ agglutinin-Sepharose and preparative slab-gel electrophoresis. The localization of this glycoprotein on the cell surface was indicated by surface labelling by the periodate/NaB3H4 and lactoperoxidase-catalysed iodination methods. Carbohydrate analyses of this glycoprotein revealed that its total carbohydrate content is 28% (w/w), and it contains fucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid in molar proportions 1.0:4.0:3.7:3.5:1.2:2.5, suggesting that it has two types of sugar chain, i.e. sugar chains like those of serum glycoproteins and sugar chains of the type found in mucins. Actually, alkaline borohydride treatment of this glycoprotein yielded tri- and tetra-saccharide, the latter containing 1 molecule of fucose in addition to each molecule of galactose, N-acetylgalactosamine and sialic acid. This glycoprotein bound to Ricinus communis agglutinin and concanavalin A as well as to wheat-germ agglutinin.