Structural studies of the sugar chains of human parotid alpha-amylase.

107

Laminin, a high molecular weight (1,000,000) glycoprotein component of basement membranes, was isolated from the EHS murine tumor as a noncovalent complex with entactin by lectin affinity chromatography using the alpha-D-galactosyl binding lectin Griffonia simplicifolia I (GS I). Entactin was removed from this complex by passage over Sephacryl S-1000 in the presence of SDS. Compositional analysis showed that the affinity-purified laminin contained 25-30% carbohydrate by weight. Methylation analysis revealed that the oligosaccharides of laminin contained bi- and triantennary chains, the blood group I structure, and repeating sequences of 3Gal beta 1,4GlcNAc beta 1 units. Free oligosaccharides were derived from the asparagine-linked glycans of affinity-purified laminin by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. When fractionated by affinity chromatography on concanavalin A (Con A)-Sepharose, 80% of the oligosaccharides passed through the column unretarded and a single peak corresponding to 20% of the oligosaccharides was adsorbed and specifically eluted with a linear gradient of 0-30 mM methyl alpha-D-glucopyranoside. Further fractionation of the Con A reactive oligosaccharides on GS I-Sepharose demonstrated that 70% of these oligosaccharides possess at least one terminal nonreducing alpha-D-galactopyranosyl unit. The Con A reactive oligosaccharides were subjected to sequential digestion with endo- and exoglycosidases, and the reaction products were analyzed by gel filtration chromatography on a column of Bio-Gel P4. We thereby obtained evidence for a variety of structures not previously reported to exist on murine laminin including hybrid biantennary complex and biantennary complex structures containing poly(lactosaminyl) repeating units. The poly(lactosaminyl) units occur either on one or on both branches of the biantennary chains, as well as in more highly branched blood group I poly(lactosamine) structures. All sialic acid is present as N-acetylneuraminic acid linked alpha 2,3 to galactose.

Section: Analysis Of the Terminal Nonreducing Residues Of Lamininsupporting

confidence: 85%

Section: Analysis Of the Terminal Nonreducing Residues Of Lamininmentioning

confidence: 99%

Structure of the major concanavalin A reactive oligosaccharides of the extracellular matrix component laminin

Knibbs¹,

Perini²,

Goldstein³

1989

107

“…The methylation data of fraction A in Table I indicated that more than 30% of the total galactose residues in this fraction remained nonsialylated. Therefore, the fucosylated outer chain may not be sialylated as in the case of human parotid amylase (Yamashita et al, 1980) and mouse kidney -glutamyltranspeptidase (Yamashita et al, 1985).…”

Section: Resultsmentioning

confidence: 99%

“…X-Antigenic determinant is widely distributed in various tissues and cell types as free oligosaccharides (Kobata & Ginsburg, 1969;Hallgren & Lundblad, 1977), glycolipids (Yang & Hakomori, 1971), and glycoproteins (Yamashita et al, 1980(Yamashita et al, , 1985Endo et al, 1986). This trisaccharide is also designated as stage-specific embryonic antigen 1 (SSEA-1), and its expression on cell surface in early developmental stage is considered to function as adhesion molecules between the cell-cell interactions in normal developmental processes in embryo (Solter & Knowles, 1978;Fenderson et al, 1984).…”

Section: Discussionmentioning

confidence: 99%

Diversity of oligosaccharide structures linked to asparagines of the scrapie prion protein

Endo

Groth²,

Prusiner³

et al. 1989

288

201

Prion proteins from humans and rodents contain two consensus sites for asparagine-linked glycosylation near their C-termini. The asparagine-linked oligosaccharides of the scrapie isoform of the hamster prion protein (PrP 27-30) were released quantitatively from the purified molecule by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The radioactive oligosaccharides were fractionated into one neutral and three acidic oligosaccharide fractions by anion-exchange column chromatography. All oligosaccharides in the acidic fractions could be converted to neutral oligosaccharides by sialidase digestion. Structural studies on these oligosaccharides including sequential exoglycosidase digestion in combination with methylation analysis revealed that PrP 27-30 contains a mixture of bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6(GlcNAc beta 1----4)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4-(Fuc alpha 1----6)GlcNAc as their core. Variation is produced by the different combination of the oligosaccharides Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, GlcNAc beta 1----, Sia alpha 2----3Gal beta 1----4GlcNAc beta 1----, and Sia alpha 2----6Gal beta 1----4GlcNAc beta 1---- in their outer chain moieties. When both asparagine-linked consensus sites are glycosylated, the diversity of oligosaccharide structures yields over 400 different forms of the scrapie prion protein. Whether these diverse asparagine-linked oligosaccharides participate in scrapie prion infectivity or modify the function of the cellular prion protein remains to be established.

“…It releases only one GlcNAc/3l-2 residue linked to the Mano 1-3 branch from degalactosylated biand triantennary derivatives with bisecting GlcNAc and tetraantennary derivatives of oligosaccharides (Yamashita et al, 1981;Renwick et al, 1987). Jack bean o-mannosidase hydrolyzes one Man residue from the Gal/3l-4Glc-NAc/31 -2Mana 1 -6(Mana 1 -3)Man structure but not from the Gal/31-4GlcNAc/31-2Mana 1-3(Mana 1 -6)Man structure under the conditions used (Yamashita et al, 1980b).…”

mentioning

confidence: 99%

Structural Analysis of N-Linked Oligosaccharides of Equine Chorionic Gonadotropin and Lutropin .beta.-Subunits

Matsui¹,

Mizuochi²,

Titani³

et al. 1994

Equine chorionic gonadotropin (eCG) and lutropin (eLH) are composed of alpha- and beta-subunits with an identical amino acid sequence but show different biological activities. To elucidate the molecular difference between these gonadotropins, the structure of the N-linked oligosaccharides of each beta-subunit was determined. N-linked sugar chains, liberated as tritum-labeled oligosaccharides by hydrazinolysis followed by N-acetylation and reduction with NaB3H4, were neutralized by sialidase digestion and/or methanolytic desulfation. Neutralized oligosaccharides were fractionated by sequential chromatography on serial lectin affinity columns and on a Bio-Gel P-4 column. Each oligosaccharide structure was determined by sequential exoglycosidase digestion in conjunction with elution profiles on lectin columns and methylation analysis. Each beta-subunit contained a single N-glycosylation site, but a high degree of microheterogeneity was observed in the structure of its N-linked oligosaccharides. eCG beta contained mono-, bi-, tri-, and tetraantennary complex-type oligosaccharides in a ratio of 3:63:13:1. eCG beta oligosaccharides contained about 16% of the bisecting GlcNAc and about 20% of poly-N-acetyllactosamine structures. Elongation of N-acetyllactosamine units showed a preference to the Man alpha 1-->6 side rather than the Man alpha 1-->3 side. Triantennary chains had only a C-2, 4-branched structure. eLH beta contained only mono- and biantennary complex-type and hybrid-type oligosaccharides in a ratio of approximately 18:67:10. eLH beta also contained bisected structures in about 18%. Oligosaccharides derived from the sulfated fraction of eLH beta contained GalNAc residues at nonreducing termini. Oligosaccharides from the sialylated/sulfated fraction of eLH beta contained both Gal and GalNAc residues at nonreducing termini, and those GalNAc residues were preferentially distributed to the Man alpha 1-->3 side of the trimannosyl core. These results clearly indicate that eCG beta and eLH beta possess structurally distinct N-linked oligosaccharides in addition to different charge groups even though they have a protein moiety identical to each other. Our results suggest that the biological activity of these hormones might be modulated by its terminal charge groups and stem structures of carbohydrate moiety synthesized in different organs.