The Structure of the Disaccharide Unit of the Renal Glomerular Basement Membrane

Abstract The disaccharide unit of the glomerular basement membrane has been studied in regard to its detailed structure and its linkage to the peptide portion. Graded acid hydrolysis of glycopeptides containing this unit resulted in a more rapid release of glucose than galactose and indicated that glucose was in the external position. Isolation of a disaccharide consisting of glucose and galactose with galactose in the reducing position confirmed the sequence of the unit. Studies by methylation, periodate oxidation, and galactose oxidase treatment of the isolated disaccharide, as well as of glycopeptides containing it, indicated that the glucose is linked glycosidically to C-2 of the galactose. The anomeric configuration of this linkage was shown to be α on the basis of studies with α- and β-glucosidases. These findings indicated that the isolated disaccharide was 2-O-α-d-glucopyranosyl-d-galactose. Alkaline hydrolysis of glycopeptides or of the entire basement membrane permitted isolation of glucosylgalactosylhydroxylysine in 80 to 90% yield. Upon mild acid hydrolysis of this compound, glucose was released preferentially, and galactosylhydroxylysine was obtained in about 80% yield. Studies of N-acetylated glycopeptides indicated that the stability to acid of the galactosylhydroxylysine bond is markedly influenced by the charge on the e-amino group of the hydroxylysine. Both α- and β-galactosidases were used to study the anomeric configuration of the galactosylhydroxylysine linkage. The β-galactosidase was effective in cleaving this bond, but only when the galactosylhydroxylysine was in its N-acetylated form. These studies are consistent with the following structure for the disaccharide unit and its peptide attachment: 2-O-α-d-glucopyranosyl-O-β-d-galactopyranosylhydroxylysine. Measurement of the glucosylgalactosylhydroxylysine content of the basement membrane could be performed directly on the amino acid analyzer after alkaline hydrolysis of the basement membrane. Such analyses indicated that there are 14.5 disaccharide units per 100 mg of the basement membrane (17.3/1000 amino acid residues). All of the glucose of the basement membrane occurs in the form of this hydroxylysine-linked disaccharide unit. The disaccharide units could be removed from the peptide chain by a single Smith degradation.