Toshiaki Osawa

Purification and characterization of an anti-H(O) phytohemagglutinin of Ulex europeus☆

Two anti-H(O) phytohemagglutinins have been separated by (NH4)2SO4 fractionation of extracts of Ulex europeus seeds. One was found to be inhibited by L-fucose and the other was inhibited most by di-N-acetylchitobiose. The former was further purified by chromatography successively on CM-cellulose. Sephadex G-200 and Biogel P-200. The homogeneity of the purified hemagglutinin was ascertained by ultracentrifugal analysis and electrophoresis on polyacrylamide gel. The purified hemagglutinin had an s020,w value of 6.5 S. This hemagglutinin was found to contain 5.2% carbohydrate in which mannose (1.6%) and glucosamine (1.4%) were the predominant sugars, with smaller amounts of fucose, glucose, xylose, arabinose and galactose.

Purification of galactose-binding phytoagglutinins and phytotoxin by affinity column chromatography using sepharose

Phytoagglutinine, welche Galaktose spezifisch binden, wurden mit Chromatographie an Sepharose gereinigt. Zwei Fraktionen ausRicinus communis-Agglitomom wiurden durch Gelfiltration mittels Bio-gel getrennt: die früh eluierte Fraktion zeigte starke Agglutinationsaktivität und schwache Toxizität, während die später eluierte Fraktion eine schwache Agglutinatinsaktivität bei starker Toxizität zeigt.

Carbohydrate binding specificity of the so-called galactose-specific phytohemagglutinins

The carbohydrate-binding specificities of various so-called galactose-specific phytohemagglutinins were investigated by means of hemagglutination-inhibition assays. As hapten inhibitors, glycopeptides prepared by pronase-digestion of various glycoproteins (porcine submaxillary mucin, bovine submaxillary mucin, and porcine thyroglobulin), and several glycosides of D-galactose and 2-acetamido-2-deoxy-D-galactose were employed. The results indicate that these galactose-specific phytohemagglutinins may recognize the sugar residue penultimate to D-galactose or 2-acetamido-2-deoxy-D-galactose residues of the sugar chain with which they interact, and that they can be classified into three groups based on the type of sugar sequence which they primarily recognize.

Carbohydrate structures of bovine submaxillary mucin

The structures of carbohydrate chains derived from bovine submaxillary mucin (BSM) were investigated. Oligosaccharide-alditols released from BSM by alkaline borohydride treatment were separated into three acidic (A-1-A-3) and five neutral (N-1-N-5) oligosaccharide-alditol fractions by liquid chromatography on columns of an ion-exchange resin and Bio-Gel P-4. On the basis of the data obtained on compositional and methylation analyses, and digestion by exoglycosidase, the following structures were assigned to these oligosaccharide-alditols: A-1, alpha-NeuAc (or NeuGc)-(2----6)-GalNAc-ol; A-2, beta-Gal-(1----3)-[alpha-NeuAc-(2----6)]-GalNAc-ol; A-3, beta-GlcNAc-(1----3)-[alpha-NeuAc-(2----6)]-GalNAc-ol; N-1, GalNAc-ol; N-2, beta-Gal-(1----3)-GalNAc-ol; N-3, beta-GlcNAc-(1----3)-GalNAc-ol; N-4, beta-Gal-(1----3)-[beta-GlcNAc-(1----6)]-GalNAc-ol; and N-5, alpha-Fuc-(1----2)-beta-Gal-(1----3)-[beta-GlcNAc-(1----6)]-GalNAc-ol. These results showed the heterogeneous nature of the carbohydrate chains of BSM.

Strong affinity of Maackia amurensis hemagglutinin (MAH) for sialic acid‐containing Ser/Thr‐linked carbohydrate chains of N‐terminal octapeptides from human glycophorin A

The interaction of the Maackia amurensis hemagglutinin (MAH) with various glycopeptides and oligosaccharides was investigated by means of immobilized lectin affinity chromatography. An amino terminal octapeptide obtained from human glycophorin A having three Neu5Acα→3Galβ1→3(Neu5Acα2→6)GalNAc tetrasaccharide chains, designated as CB‐II, was found to have an extremely strong affinity for MAH. Therefore, it is strongly suggested that hemagglutination by MAH was caused by its interaction with Ser/Thr‐linked carbohydrate chains of human glycophorin A on erythrocyte membranes.

Some properties of purified phytohemagglutinin from Lens culinaris seeds

Abstract Lens culinaris phytohemagglutinin was purified by specific adsorption on Sephadex G-100 and subsequent displacement with d -glucose. The homogeneity of the purified hemagglutinin was ascertained by ultracentrifugal analysis and polyacrylamide gel electrophoresis. The purified hemagglutinin had a s020,w value of 3.4 S. This hemagglutinin was disclosed to be a kind of glycoprotein containing 1.5% glucose and 0.5% glucosamine. The specificity of the purified hemagglutinin was tested by hemagglutination-inhibition assays and found to be essentially the same as that of concanavalin A. In each step of the purification, the mitogenic activity against human peripheral lymphocytes was found to be always confined to the fraction which had the hemagglutinating activity. Morphological studies on hemagglutinin-stimulated cultures indicated that at 72 h about 15% of the cell population had been transformed by the purified hemagglutinin.

Purification and characterization of a Cytisus-type anti-H(O) phytohemagglutinin from Ulex europeus seeds

Abstract A Cytisus -type anti-H(O) phytohemagglutinin, which is inhibited most by di- N -acetylchitobiose, has been purified from the seeds of Ulex europeus . The preparation was homogeneous by ultracentrifugal analysis and disc electrophoresis, and had a S 20, w value of 6.5 S. This purified hemagglutinin was found to contain 21.7% carbo-hydrate in which mannose (8.7%) and galactose (6.3%) were the predominant sugars, with smaller amounts of arabinose, glucosamine, glucose, fucose, and xylose. Treatment of human O erythrocytes with a purified H-decomposing enzyme (α- l -fucosidase) from Bacillus fulminans destroyed the agglutinability of the cells by the Cytisus-type anti-H(O) hemagglutinin. This fact indicates that the l -fucosyl residue is important even for the H-specificity detected by the Cytisus-type anti-H(O) reagents.

Carbohydrate-binding specificities of five lectins that bind to O-glycosyl-linked carbohydrate chains. Quantitative analysis by frontal-affinity chromatography

The carbohydrate-binding specificities of lectins purified from Agaricus bisporus (ABA-I), Arachis hypogaea (PNA), Bauhinia purpurea (BPA), Glycine max (SBA), and Vicia villosa (VVA-B4) have been studied by affinity chromatography on columns of the immobilized lectins, and quantitatively analyzed by frontal affinity chromatography. These five lectins could be classified into two groups with respect to their reactivities with typical mucin-type glycopeptides, beta-D-Galp-(1----3)-alpha-D-GalpNAc-(1----3)-Ser/Thr (2) and alpha-D-GalpNAc-(1----3)-Ser/Thr (3). One group, which consists of ABA-I, PNA, and BPA, preferentially binds to 2, and the other, which consists of SBA and VVA-B4, shows higher affinity for 3 than for 2. Among the lectins tested, only ABA-I was found to bind to a sialylated glycopeptide, whic which was prepared from human erythrocyte glycophorin A and contains three three tetrasaccharide chains having the structure of alpha-NeuAc-(2----3)-beta-D-GAlp-(1----3)-NeuAC-(2----6)]-alpha-D-Galp NAc-(1----, with an association constant of 15 microM, whereas the association constants of the other four lectins for this sialylated glycopeptide were less than 3.5 mM. On the other hand, removal of the beta-D-galactopyranosyl group from a glycopeptide containing sequence 2 resulted in decreased association constants for the three lectins of the first group, especially ABA-I and PNA. The two lectins of the second group showed a high affinity for 3, but SBA preferentially interacted with oligosaccharides containing the alpha-D-GalpNAc-(1----3)-beta-D-Galp-(1----3)-D-GlapNAc sequence, prepared from a blood group A-active oligosaccharide.

Immunochemical and chemical investigations of the structure of glycoprotein fragments obtained from epiglycanin, a glycoprotein at the surface of the TA3-Ha cancer cell☆

The structures of the carbohydrate chains present in fragments of a large-molecular-weight glycoprotein, epiglycanin, cleaved from the surface of viable TA3-Ha murine mammary carcinoma ascites cells and purified by gel filtration, were studied by immunochemical and chemical methods. Inhibitory activities for neuraminidase-treated and untreated glycoprotein material in the hemagglutination of NN-specific human erythrocytes by eight purified lectins were determined. Excellent inhibition was obtained in the Bauhinia purpurea, Arachis hypogaea, Iberis amara, and Wistaria floribunda systems, and weak inhibition against the Ricinus communis and Glycine max lectins. No activity against hemagglutination by the Phaseolus vulgaris and Phaseolus limensis lectins was observed. These results, when compared with those obtained by periodate oxidation, alkaline borohydride reduction, and partial methylation, suggest the possible presence of six different carbohydrate chains of 1 to 5 components in length, having as terminal groups N-acetylneuraminic acid, galactose, and 2-acetamido-2-deoxygalactose. All chains are attached to a single polypeptide chain by O-glycosyl bonds involving a 2-acetamido-2-deoxygalactose residue and a serine or threonine residue. It is suggested that the native molecule of epiglycanin of molecular weight 500,000 contains more than 500 carbohydrate chains attached to a single polypeptide chain of similar to 1,300 amino acid units.

The specific purification of various carbohydrate-binding hemagglutinins

Abstract The specific affinity adsorbent prepared by cross-linking the inhibitory sugar of the hemagglutinin to be purified to insoluble starch with epichlorhydrin was applied to the purification of various carbohydrate-binding hemagglutinins. The hemagglutinin can be isolated in essentially one step, and the adsorbent can be used several times with little changes in its capacity. If the effective inhibitory substance for the hemagglutinin is glycoprotein, the adsorbent prepared by coupling the glycoprotein to activated Sepharose is recommended for the purification of the hemagglutinin.