Mannosyl Transfer by Membranes of Aspergillus niger : Mannosylation of Endogenous Acceptors and Partial Analysis of the Products

Abstract

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 α-glucosidase (α-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 α-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, α-glu, and Ov were obtained by treatment of the respective glycopeptides with endo-β- 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 α- and β-mannosidases, (ii) Smith degradation, and (iii) acetolysis. Subsequently, changes in paper chromatographic mobilities were detected. Also, α-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 α-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 α-glu. It is suggested that the transferase(s) involved in [ 14 C]gly synthesis in vitro may be responsible for glycosylation of secreted enzymes.