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Structure and distribution of N-linked oligosaccharide chains on various domains of mouse tumour laminin.

ABSTRACT: Asparagine-linked oligosaccharides were liberated from laminin and some of its fragments by hydrazinolysis, and after purification characterized by exoglycosidase digestions. This demonstrated the presence of nine forms of complex oligosaccharide chains, which differed in antennary and oligolactosamine structure, and of small amounts of high-mannose-type oligosaccharides. Additional variations were found with regard to substitutions by terminal alpha-galactose and sialic acid residues. Each of the various laminin fragments showed a unique but less complex repertoire of carbohydrate structures. These fragments also differed in mass, carbohydrate content, localization within the laminin molecule and functional activities such as cell-binding (fragments 1 and 6) and heparin- and collagen-binding (fragments 3 and 4). Fragment 7 with a particularly high carbohydrate content (72%) also showed the highest complexity of tri- and tetra-antennary structures. Further differences between the fragments were detected with human antibodies against the Gal alpha 1-3Gal epitope, which was expressed in either a high-affinity or a low-affinity form. Such differences in carbohydrate structure of topologically distinct laminin domains may have implications for their functions and in the regulation of post-translational modification events.

SUBMITTER: Fujiwara S

PROVIDER: S-EPMC1149166 | biostudies-other | 1988 Jun

REPOSITORIES: biostudies-other

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Project description:Hepatic apolipoprotein (apo) B-100 isolated from human plasma is known to contain N-linked oligosaccharides of high-mannose-type and complex-type structures. Sequencing data have revealed that apo B-48 of small-intestinal origin, which represents about 48% of apo B-100 polypeptide from the N-terminus, possesses six potential sites for N-linked oligosaccharides, of which five are likely to be glycosylated. The characterization of the carbohydrate moiety of apo B-48 is the focus of this study. Apo B-48 was labelled with L-[35S]methionine and D-[3H]glucosamine in organ culture of human small-intestinal explants. N-Glycanase treatment resulted in loss of radioactivity from D-[3H]glucosamine-labelled but not L-[35S]methionine-labelled apo B-48 secreted into the medium, and caused no distinct change in mobility of apo B-48 upon electrophoresis on 5% polyacrylamide gel. Analysis of monosaccharide content revealed the presence of 16.8, 17.8, 13.4, 3.4, 2.4 and 2.3 residues of N-acetylglucosamine, mannose, galactose, fucose, xylose and N-acetylgalactosamine respectively. Small-intestinal apo B-48 from human lymph chylomicrons bound to [14C]concanavalin A, and the binding could be inhibited with methyl alpha-D-mannoside. In addition, wheat-germ, peanut, Limulus, soya-bean and Ulex lectins bound apo B-48 specifically. To characterize the carbohydrate moiety further, N-linked oligosaccharides were released by N-Glycanase treatment and reduced with NaB3H4. Labelled oligosaccharides were separated on a concanavalin A-Sepharose column. The majority (78%) were biantennary complex-type structures, 16% were high-mannose type and 6% (not retained by the column) most probably represented higher-branched oligosaccharides. These results suggest the presence of one high-mannose-type and four biantennary complex-type oligosaccharides, as well as probable O-linked sugars in apo B-48. By the use of h.p.l.c., exoglycosidase treatments and ion-exchange chromatography, a mixture of high-mannose-type species with predominant Man8GlcNAc2 as well as monosialylated, desialylated and fucosylated forms of complex-type oligosaccharides were detected.

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Structure and distribution of N-linked oligosaccharide chains on various domains of mouse tumour laminin.

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