Structures of the asparagine-linked sugar chains of laminin

Abstract

This investigation describes the isolation and characterization of oligosaccharides of the basement membrane glycoprotein, laminin. Pronase-released glycopeptides of isolated laminin, from a mouse Engelbreth-Holm-Swarm tumor, were fractionated using a combination of gel permeation chromatography and Con A-Sepharose affinity chromatography. The glycopeptides were analyzed for sugar linkage patterns by methylation analysis. Glycopeptides and hydrazine-released oligosaccharides were further analyzed using endo-β-galactosidase, endo- β- N-acetylglucosaminidase H and specific exoglycosidases in conjunction with calibrated gel permeation chromatography. Based on these experiments, murine tumor laminin was shown to contain asparagine-linked oligosaccharides with the following structures: (1) bi-, tri- and tetraantennary complex-type oligosaccharides; (2) polylactosaminyl side chains containing Gal( β1→4)GlcNAc( β1→3) repeating units attached to the trimannose core portion of the bi-, tri- and tetraantennary complex-type oligosaccharides; (3) unusual complex-type oligosaccharides terminated at the nonreducing end with sialic acid, α-galactose and β- N-acetylglucosamine; α-galactosyl residues linked to N-acetyllactosamine sequences; (4) high-mannose-type oligosaccharides. These result, in conjunction with analytical data, indicate that most of the carbohydrate of this laminin is N-linked to asparagine and that there are about 43 such N-linked oligosaccharides per laminin molecule.