title: Substrate specificity studies of Flavobacterium chondroitinase C and heparitinases towards the glycosaminoglycan--protein linkage region. Use of a sensitive analytical method developed by chromophore-labeling of linkage glycoserines using dimethylaminoazobenzenesulfonyl chloride.
abstract: Bacterial chondroitinases and heparitinases are potentially useful tools for structural studies of chondroitin sulfate and heparin/heparan sulfate. Substrate specificities of Flavobacterium chondroitinase C, as well as heparitinases I and II, towards the glycosaminoglycan-protein linkage region -HexA-HexNAc-GlcA-Gal-Gal-Xyl-Ser (where HexA represents glucuronic acid or iduronic acid and HexNAc represents N-acetylgalactosamine or N-acetylglucosamine) were investigated using various structurally defined oligosaccharides or oligosaccharide-serines derived from the linkage region. In the case of oligosaccharide-serines, they were labeled with a chromophore dimethylaminoazobenzenesulfonyl chloride (DABS-Cl), which stably reacted with the amino group of the serine residue and rendered high absorbance for microanalysis. Chondroitinase C cleaved the GalNAc bond of the pentasaccharides or hexasaccharides derived from the linkage region of chondroitin sulfate chains and tolerated sulfation of the C-4 or C-6 of the GalNAc residue and C-6 of the Gal residues, as well as 2-O-phosphorylation of the Xyl residue. In contrast, it did not act on the GalNAc-GlcA linkage when attached to a 4-O-sulfated Gal residue. Heparitinase I cleaved the innermost glucosaminidic bond of the linkage region oligosaccharide-serines of heparin/heparan sulfate irrespective of substitution by uronic acid, whereas heparitinase II acted only on the glucosaminidic linkages of the repeating disaccharide region, but not on the innermost glucosaminidic linkage. These defined specificities of chondroitinase C, as well as heparitinases I and II, will be useful for preparation and structural analysis of the linkage oligosaccharides.
authors.affiliation: Department of Biochemistry, Kobe Pharmaceutical University, Kobe, Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Tsuda
authors.forename: S
authors.initials: S
authors.lastname: Yamada
authors.forename: H
authors.initials: H
authors.lastname: Miyazono
authors.forename: K
authors.initials: K
authors.lastname: Morikawa
authors.forename: K
authors.initials: K
authors.lastname: Yoshida
authors.forename: F
authors.initials: F
authors.lastname: Goto
authors.forename: J I
authors.initials: JI
authors.lastname: Tamura
authors.forename: K W
authors.initials: KW
authors.lastname: Neumann
authors.forename: T
authors.initials: T
authors.lastname: Ogawa
authors.forename: K
authors.initials: K
authors.lastname: Sugahara
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D002240:Carbohydrate Sequence; D002808:Chondroitin Lyases / Q000378:metabolism*; D002856:Chromatophores / Q000737:chemistry; D005417:Flavobacterium / Q000201:enzymology*; D006025:Glycosaminoglycans / Q000378:metabolism*; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000378:metabolism*; D012680:Sensitivity and Specificity; D012694:Serine / Q000737:chemistry*; D013379:Substrate Specificity; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
chemical_list: D001426:Bacterial Proteins; D006025:Glycosaminoglycans; D012694:Serine; C010572:4-N,N-dimethylaminoazobenzene-4'-sulfonyl chloride; D004124:p-Dimethylaminoazobenzene; D002808:Chondroitin Lyases; D011133:Polysaccharide-Lyases; C021349:heparitinsulfate lyase