Setsuzo Tejima

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.

Demonstration of a new glycopeptidase, from jack-bean meal, acting on aspartylglucosylamine linkages

An enzyme preparation from jack-bean meal hydrolyzed beta-aspartylglucosylamine linkages in glycopeptides. The enzyme could release sialic acid-containing complex-type oligosaccharides as well as high-mannose-type and hybrid-type oligosaccharides. The products were equimolar amounts of ammonia, oligosaccharide and peptide. The enzyme cleaved glycopeptides with three or more amino acid residues, whereas it did not hydrolyze GlcNAc-Asn. The mechanism of action of the enzyme and substrate specificity so far tested were similar to those of the glycopeptidase from almonds.

Either high-mannose-type or hybrid-type oligosaccharide is linked to the same asparagine residue in ovalbumin

After pepsin digestion, all of the carbohydrates in ovalbumin were recovered in two glycopeptides, Glu-Glu-Lys-Tyr-Asn(CHO)-Leu-Thr-Ser-Val and Glu-Gln-Lys-Tyr-Asn(CHO)-Leu-Thr-Ser-Val. Almond glycopeptidase released quantitatively oligosaccharides from the glycopeptides. The products from both glycopeptides contained both the high-mannose-type oligosaccharides and the hybrid-type oligosaccharides in the same ratio. Thus, either the high-mannose-type or the hybrid-type oligosaccharide is attached to the unique asparagine residue in the ovalbumin molecule.

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)

Synthesis of 1,6-anhydro-6-thio-β-lactose (6-thiolactosan)

Abstract A facile syntheiss of lactosan hexaacetate ( 4 ) is described. Zemplen degradation of 4 , followed by the Koenigs-Knorr reaction, was shown to yield 1,2,3,2′,3′,4′,6′-hepta- O -acetyl-β-lactose ( 5 ) in 62% yield. Compound 5 is a useful intermediate for modification of the hydroxymethyl group of the reducing moiety in lactose. Starting from 5 , via a crystalline 6-sulfonate (methyl, 6 , or p -tolyl, 7 ), the corresponding 2,3,2′,3′,4′,6′-hexa- O -acetyl-6- O -methyl- or p -tolylsulfonyl-β-lactosyl ethylxanthate ( 9 or 10 ) was synthesized. Alkaline treatment of 9 or 10 , followed by acetylation of the product gave 2,3,2′,3′,4′,6′-hexa- O -acetyl-1,6-anhydro-6-thio-β-lactose ( 11 ) in good yield. Deacetylation of 11 gave the title compound ( 12 ), a novel thio analog of lactosan. Crystalline phenyl α-lactoside ( 3 ), its peracetate ( 2 ), and 1,2,3,2′,3′,4′,6′-hepta- O -acetyl-6-deoxy-6-iodo-β-lactose ( 8 ) are also described.

Studies on the reactivities of the secondary hydroxyl groups in 1,6-anhydro-4′,6′-o-benzylidene-β-lactose by selective benzoylation☆

Abstract Selective benzoylation of 1,6-anhydro-4′,6′- O -benzylidene-β-lactose ( 1 ), using 2.1 molar equivalents of benzoyl chloride in pyridine at — 20°, yielded five benzoates which were designated 2 to 6 in order of decreasing R F value on t.l.c. After column chromatography on silica gel, compounds 2 – 6 were separated as the 2,2′,3,3′-tetra-benzoate ( 2 , 3%), 2,3,3′-tribenzoate ( 3 , 11%), 2,2′,3′-tribenzoate ( 4 , 5%), 2,3′-dibenzoate ( 5 , 30%), and 3′-benzoate ( 6 , 22%), respectively. Selective benzoylation of 5 , using 1.1 molar equivalents of benzoyl chloride, afforded 2 , 3 , and 4 in yields of 15, 56, and 8%, respectively, together with 5% of 5 . Thus, the order of reactivities of the secondary hydroxyl groups in 1 is 3′>3>2′. Compounds 3 – 6 have potential value in the chemical modification of lactose or the synthesis of lactose-containing oligosaccharides.

N-Acetylneuraminic acid and N-glycolylneuraminic acid in glycopeptides of colonic tumor and mucosa in rats treated with carrageenan and 1,2-dimethylhydrazine

N-linked glycopeptides were prepared from colonic tumor (adenocarcinoma) and mucosa in rats treated with carrageenan, an indigestible polysaccharide, and 1,2-dimethylhydrazine. Sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid, obtained by acid hydrolysis of the glycopeptides were determined by HPLC. The N-acetylneuraminic acid/N-glycolylneuraminic acid ratio in colonic tumor was 25.2, while each treated mucosa had the values between 0.29 and 0.55. Thus, necessity which observes the qualitative change of sialic acid in malignant transformation was suggested.