Biosynthesis, in calf pancreas microsomes, of three lipid-linked oligosaccharide diphosphates from a synthetic dolichyl diphosphate tetrasaccharide.
Incubation of calf pancreas microsomes with synthetic alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1 ----4)-alpha-D- GlcpNAc-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-beta-D-glucosaminidases D and H, by jack bean alpha-D-mannosidase and Aspergillus oryzae (1----2)-alpha-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: alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1 ----4)-alpha-D- GlcpNAc-PP-Dol + GDP-D-Man gave GDP + alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)- beta-D-GlcpNAc-(1----4)-alpha-D-GlcpNAc-PP-Dol. The next products to be formed were alpha-D-Manp-(1----2)-alpha-D-Manp-(1----3)-[alpha-D-Manp -(1----6)]-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-alpha-D-GlcpNAc-PP-Dol, followed by alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp+ ++-(1----3)- [alpha-D-Manp-(1----6)]-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-alpha- D-GlcpNAc-PP-Dol. The mannose incorporation was enhanced by Triton X-100 and inhibited by Mn2+, and it occurred in the presence of either Mg2+ 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.
Goussault
Y
Y
Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts.
Nakabayashi
S
S
Warren
C D
CD
Bugge
B
B
Jeanloz
R W
RW
D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050356:Lipid Metabolism; D008274:Magnesium / Q000494:pharmacology; D008361:Mannosidases / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008364:Mannosyltransferases / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis*; D010179:Pancreas / Q000378:metabolism*; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000378:metabolism*; D011105:Polyisoprenyl Phosphate Sugars / Q000378:metabolism*; D043323:alpha-Mannosidase
D009844:Oligosaccharides; D011104:Polyisoprenyl Phosphate Oligosaccharides; D011105:Polyisoprenyl Phosphate Sugars; C024962:dolichyl diphosphate oligosaccharides; D006155:Guanosine Diphosphate Mannose; D008364:Mannosyltransferases; D008361:Mannosidases; D043323:alpha-Mannosidase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008274:Magnesium