Structural Determination of the O‐Linked Sialyl Oligosaccharides Liberated from Fetuin with Endo‐α‐N‐Acetylgalactosaminidase‐S by HPLC Analysis and 600‐MHz ¹H‐NMR Spectroscopy

Abstract

The endo-a-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-I 1242 (endo-GalNAc-ase-S) hydrolyzed the 0-glycosidic linkage between GalNAc and Ser (Thr) in fetuin, liberating oligosaccharides. The 0-linked oligosaccharides liberated from the fetuin with endo-GalNAcase-S were pyridylaminated following fractionation on a Bio-Gel P-4 column. The structure of the pyridylaminated 0-linked oligosaccharides from fetuin has been determined by reverse-phase HPLC and 600-MHz 'H-NMR spectroscopy. The chemical shifts and the coupling constants of pyridylaminated (PA) NeuAca2-3GalpI -3GalNAc were refined by computer simulation of the spectrum. The structures of NeuAca2-3Ga1/31-3(NeuAca2-6)GalNAc-PA and NeuActr2-3GaljZ1-3(NeuAca2-3Gal~l-4GlcNAc~1-6)GalNAc-PA were determined by their structural reporter groups.Keywords: 0-linked sugar chain; 0-glycosidase; NMR; HPLC ; fetuin.The endo-a-N-acetylgalactosaminidase from a culture medium of Streptomyces sp. OH-I 1242 (endo-GalNAc-ase-S) was found to be capable of liberating not only Galpl-3GalNAc but other larger neutral oligosaccharides through the hydrolysis of the 0-glycosidic linkage between the GalNAc and Ser(Thr) residues [I, 21. The substrate specificity of endo-GalNAc-ase-S was different from that of endo-a-N-acetylgalactosaininidase (endoGalNAc-ase), i.e. 0-glycanase, from Diplococcus pneumoniae (endo-GalNAc-ase-D [3 -51) and endo-a-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNAc-ase-A) [6], which are both commercially available, and can hydrolytically cleave the a-N-acetylgalactosaminyl linkages of only Galpl-3Gal-NAcal-Ser/Thr in glycopeptides and glycoproteins to liberate the disaccharide moiety. The endo-GalNAc-ase-S has been partially purified, and its specific activity, molecular mass and some characteristics have been reported [2], but its action on the sialoglycoprotein has not been examined at all. Fetuin, leukosialin, glycophorin A, submaxillary mucin, intestinal mucin etc. [7-131 are sialoglycoproteins, which have 0-linked sialyl oligosaccharide5. The structure of the oligosaccharide moiety of these sialoglycoproteins has been partially clarified, and changes in the sialyl oligosaccharide with differentiation, malignant transformation and immunodeficiency have been extensively studied [7,[14][15][16][17][18][19][20][21]. To investigate the oligosaccharide structure of the mucin-type glycoproteins, the O-glycoside linkage was chemically cleaved through weak alkalinity obtained in the p-elimination reactions, and the liberated oligoCorrespondence to K. Hotta, Department of Biochemistry, School of Medicine, Kitasato University, 1-15-1, Kitasato, Sagamiharashi, Kanagawa, Japan 228 F u r +81 427 78 9372. Abbreviutions. PGM, pig gastric mucus glycoprotein; Np, p-nitrophenyl ; PA, pyridylaminated; endo-GalNAc-ase, endo-a-N-acetylgalactosaminidase.Enzymes. Endo-u-N-acetylgalactosaminidase (EC 3.2.1.97) ; sialidase (EC 3.2.1.18).saccharide-alditols were investigated by 'H-NMR spectroscopy [22]. The enzymic cleavage of the 0-glycosidi...

Psathyrella velutina Mushroom Lectin Exhibits High Affinity toward Sialoglycoproteins Possessing TerminalN-Acetylneuraminic Acid α2,3-Linked to Penultimate Galactose Residues of Trisialyl N-Glycans

“…As shown in Scheme 1A, human transferrin contains two N-linked complex-type glycans, and the major oligosaccharide has 2 mol of ␣2,6-linked NeuAc (Scheme 1A) (34), whereas bovine fetuin has three N-linked glycans with the major oligosaccharides of a sialylated triantennary complex-type (35), and three O-linked glycans of a sialylated core-type 1 on a molecule, respectively (Scheme 1B) (36,37). Total fetuin glycans contain approximately equal amounts of ␣2,3-and ␣2,6-linked NeuAc residues.…”

Identification and Molecular Cloning of a Novel Glycoside Hydrolase Family of Core 1 Type O-Glycan-specific Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum

“…All of the enzymes are secretory and high-molecular mass proteins (Ͼ110 kDa), and are able to release Gal␤1,3GalNAc disaccharide from glycoproteins. A similar enzyme was found in the culture medium of Streptomyces sp., which is capable of releasing longer sugar chains than the disaccharide from porcine mucin (18,19), although further studies are necessary to validate its action.Bifidobacteria are strictly anaerobic lactic acid-producing bacteria that constitute a major part of the intestinal microflora of human and animals, and have attracted a great deal of attention because of their many beneficial probiotic effects (20). Recently, we found that bifidobacteria widely have an endo-␣-GalNAcase specific for Core 1 mucin-type O-glycans.…”

Convenient Analytical Methods for Endo-type Glycosidase that Acts on Glycoconjugates and Their Application in Glycotechnology

“…The growth of Streptomyces sp. OH-11242 and preparation of PGM were described by Ishii-Karakasa et al [3][4][5] Crude enzyme from the culture fluid. The culture fluid of Streptomyces sp.…”

“…The crude enzyme was prepared by 80% (v/v) ammonium sulfate precipitation, gel chromatofocusing, DEAE-Toyopearl and N-(p-aminophenyl)-oxamic-acid-agarose chromatography as previously described. 4 Some of the pooled solution was concentrated by dialysis against poly(ethylene glycol) 20000.…”

“…OH-11242. 3,4 This strain was obtained by adding porcine gastric mucin to culture solution of soil fungus and isolating the culture that grew well. For Streptomyces sp.…”