Mannosyl transfer by membranes of Aspergillus niger: mannosylation of endogenous acceptors and partial analysis of the products.
A smooth membrane fraction of Aspergillus niger catalyzed the transfer of mannose from GDP-mannose to endogenous lipid and protein acceptors. The mannolipid was acidic, as judged by diethylaminoethyl-cellulose chromatography, and had a mobility similar to ficaprenyl phosphate on thin-layer chromatograms. Mannose transfer occurred optimally at pH 6.5 to 7.5 and required Mn(2+) for use of the protein as acceptor, but either Mn(2+) or Mg(2+) with the lipid as acceptor. Glycopeptides of the mannosylated protein ([(14)C]gly) and of an alpha-glucosidase (alpha-glu) secreted by the organism were produced by Pronase digestion and separation of the products on Sephadex G-25. Because ovalbumin has a carbohydrate composition similar to that of alpha-glu and because the carbohydrate structure of ovalbumin is known, ovalbumin glycopeptides (Ov) were similarly obtained and used as standards in determining carbohydrate structures. Oligosaccharide chains of [(14)C]gly, alpha-glu, and Ov were obtained by treatment of the respective glycopeptides with endo-beta-N-acetylglucosaminidase, reduction with NaBT(4), and concanavalin A-Sepharose chromatography. The (3)H-labeled oligosaccharides obtained were subjected to the following treatments: (i) digestion with alpha- and beta-mannosidases, (ii) Smith degradation, and (iii) acetolysis. Subsequently, changes in paper chromatographic mobilities were detected. Also, alpha-glu was permethylated, and the partially methylated alditol acetates were analyzed by gas-liquid chromatography. The resultant proposed structure shows that the oligosaccharide chain of alpha-glu is almost identical to that of an Ov chain, while [(14)C]gly has a structure which is probably the same as that of alpha-glu. It is suggested that the transferase(s) involved in [(14)C]gly synthesis in vitro may be responsible for glycosylation of secreted enzymes.
D001234:Aspergillus niger / Q000378:metabolism*; D005656:Fungal Proteins / Q000032:analysis / Q000378:metabolism*; D006020:Glycopeptides / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D050356:Lipid Metabolism; D008345:Manganese / Q000494:pharmacology; D008358:Mannose / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism; D008745:Methylation; D009844:Oligosaccharides / Q000032:analysis; D000520:alpha-Glucosidases / Q000032:analysis
D005656:Fungal Proteins; D006020:Glycopeptides; D009844:Oligosaccharides; D008345:Manganese; D000520:alpha-Glucosidases; D008358:Mannose