Localization and structure of the asparagine-linked oligosaccharides of type IV collagen from glomerular basement membrane and lens capsule.
Analysis of the Sephacryl S-200 fractionated type IV collagen domains from bovine and human glomerular basement membranes (GBM) and calf anterior lens capsule (ALC) indicated that Asn-linked oligosaccharides are primarily or exclusively localized in the 7 S region, whereas the hydroxylysine-linked Glc alpha 1----2Gal disaccharides (Glc-Gal-Hyl) are present in all the major segments of the molecule (7 S, NC1, and helical domain); no Ser/Thr-linked saccharide were detected. The Asn-linked carbohydrate units observed in the 7 S domain (Mr approximately 300,000) occurred in a number equal to the 12 polypeptide chains constituting this cross-linked region, and this was consistent with lectin blots of the reduced electrophoretically resolved 7 S components. Fractionation of the N-glycanase and endo-beta-N-acetylglucosaminidase-released oligosaccharides by concanavalin A affinity and high performance liquid chromatography indicated that the Asn-linked carbohydrate occurred predominantly in the form of complex tri- and biantennary units, although submolar amounts of polymannose variants (Man5-7GlcNAc2) were also present in calf ALC and bovine GBM. Structural studies of the complex N-linked oligosaccharides employing hydrazine/nitrous acid fragmentation and glycosidase digestions indicated a pattern in which there was complete fucosylation of the innermost GlcNAc residue of the Man3GlcNAc2 core but only sparse substitution with capping groups of the nonrepeating N-acetyllactosamine branches. Whether tri- or biantennary, the oligosaccharides from bovine GBM contained only one capping residue, in the form of either NeuAc or alpha-D-Gal, whereas those from ALC had only a single alpha-D-Gal and no NeuAc; human GBM oligosaccharides were devoid of both NeuAc and alpha-D-Gal. The absence of terminal alpha-D-Gal in the human 7 S domain was reflected in its lack of reactivity with Bandeiraea simplicifolia I and from its failure to yield Gal alpha 1----3Gal beta 1----4 [3H]anhydromannitol after hydrazine/nitrous acid/NaB3H4 treatment. Application of the latter procedure to the collagen domains yielded, in addition to fragments from the N-linked oligosaccharides, a disaccharide (Glc alpha 1----2[3H]galactitol) derived from the Glc-Gal-Hyl units. The localization of Asn-linked carbohydrate units in the evolutionarily conserved 7S domain of type IV collagens suggests that these oligosaccharides may play a role in the assembly of the collagen network of basement membranes.
Nayak
B R
BR
Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts.
Spiro
R G
RG
D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001485:Basement Membrane / Q000737:chemistry; D002240:Carbohydrate Sequence; D002417:Cattle; D003094:Collagen / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D007678:Kidney Glomerulus / Q000737:chemistry*; D037102:Lectins / Q000737:chemistry; D007908:Lens, Crystalline / Q000737:chemistry*; D008969:Molecular Sequence Data; D009608:Nitrous Acid / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry
D006023:Glycoproteins; D006834:Hydrazines; D037102:Lectins; D009844:Oligosaccharides; D010446:Peptide Fragments; C029424:hydrazine; D001216:Asparagine; D003094:Collagen; D006026:Glycoside Hydrolases; D009608:Nitrous Acid