Biosynthesis, in calf pancreas microsomes, of three lipid-linked oligosaccharide diphosphates from a synthetic dolichyl diphosphate tetrasaccharide

Abstract

Incubation of calf pancreas microsomes with synthetic α- d-Man p-(1→6)-β- d-Man p-(1→4)-β- d-Glc pNAc-(1→4)-α- d- Glc pNAc- PP-Dol and GDP- d-[ 14C]-mannose gave three major lipid-linked oligosaccharide diphosphates. After release of the phospholipid residue by mild acid hydrolysis, the corresponding [ 14C]oligosaccharides were analyzed by gel-filtration, liquid chromatography, degradation by endo- N-acetyl-β- d-glucosaminidases D and H, by jack bean α- d-mannosidase and Aspergillus oryzae (1→2)-α- d-mannosidase, acetolysis, and binding to concanavalin A-Sepharose. From the results it could be inferred that the following reaction took place in calf pancreas microsomes: α- d-Man p-(1→6)-β- d-Man p-(1→4)-β- d-Glc pNAc-(1→4)-α- d-Glc pNAc- PP-Dol + GDP- d-Man gave GDP + α- d-Man p-(1→3)-[α- d-Man p-(1→6)]-β- d-Man p-(1→4)-β- d-Glc pNAc-(1→4)-α- d-Glc pNAc- PP- Dol. The next products to be formed were α- d-Man p-(1→2)-α- d-Man p-(1→3)-[α- d-Man p-(1→6)]-β- d-Man p- (1→4)-β- d-Glc pNAc-(1→4)-α- d-Glc pNAc- PP-Dol, followed by α- d-Man p-(1→2)-α- d-Man p-(1→2)-α- d-Man p-(1→3)-[α- d-Man p-(1→6)]-β- d-Man p-(1→4)- β- d-Glc pNAc-(1→4)-α- d-Glc pNAc- PP-Dol. The mannose incorporation was enhanced by Triton X-100 and inhibited by Mn 2+, and it occurred in the presence of either Mg 2+ or EDTA. It is likely that the mannose donor was GDP-mannose since, under the conditions used, the formation of dolichyl mannosyl phosphate was negligible and the dolichyl heptasaccharide diphosphate accumulated.