Biosynthesis of dolichyl pentasaccharide diphosphate in calf pancreas microsomes.
Incubation of synthetic dolichyl pyrophosphate tetrasaccharide and GDP-[14C]mannose with calf pancreas microsomes gave three lipid-linked oligosaccharides, which could be extracted with chloroform/methanol (2:1) and separated on silica gel plates. The fastest migrating product was characterized as dolichyl pyrophosphate pentasaccharide based on gel filtration and high pressure liquid chromatography. The formation of the pentasaccharide-lipid was greatly stimulated by addition of synthetic tetrasaccharide-lipid and required the presence of Triton X-100. Dolichyl phosphate mannose could not replace GDP-mannose as a sugar donor. The structure of the pentasaccharide was determined by degradation with endo-beta-N-acetylglucosaminidase D, acetolysis, alpha-D-mannosidase, and concanavalin A-Sepharose chromatography, showing that the following reaction was taking place: alpha-D-Manp-(1 leads to 3)-beta-D-Manp-(1 leads to 4)-beta-D-GlcpNAc-(1 leads to 4)-alpha-D-GlcpNAcPPDol + GDPMan leads to GDP + alpha-D-Manp-(1 leads to 3)-[alpha-D-Manp-(1 leads to 6)]-beta-D-Manp-(1 leads to 4)-beta-D-GlcpNAc-(1 leads to 4)-alpha-D-GlcpNAcPPDol. The mannosyltransferase was partially characterized.
D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D008361:Mannosidases / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008364:Mannosyltransferases / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism*; D011105:Polyisoprenyl Phosphate Sugars / Q000096:biosynthesis*; D043323:alpha-Mannosidase
D011105:Polyisoprenyl Phosphate Sugars; D006155:Guanosine Diphosphate Mannose; D008364:Mannosyltransferases; D008361:Mannosidases; D043323:alpha-Mannosidase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase