Biosynthesis of lipid-linked oligosaccharides. I. Preparation of lipid-linked oligosaccharide substrates.
In order to purify the glycosyltransferases involved in the assembly of lipid-linked oligosaccharides and to be able to study the acceptor substrate specificity of these enzymes, methods were developed to prepare and purify a variety of lipid-linked oligosaccharides, differing in the structure of the oligosaccharide moiety. Thus, Man9 (GlcNAc)2-pyrophosphoryl-dolichol was prepared by isolation and enzymatic synthesis using porcine pancreatic microsomes, while Glc3Man9(GlcNAc)2-PP-dolichol was isolated from Madin-Darby canine kidney cells. Treatment of these oligosaccharide lipids with a series of selected glycosidases led to the preparation of Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6(Man alpha 1,3)Man alpha 1,6]Man beta 1,4GlcNAc beta 1,4GlcNAc-PP-dolichol; Man alpha 1,2Man alpha 1,2Man alpha 1,3[Man alpha 1,6]Man beta 1,4GlcNAc beta 1, 4GlcNac-PP-dolichol; and Man alpha 1,6(Man alpha 1,3)Man alpha 1, 6[Man alpha 1,3]Man beta 1,4GlcNAc-beta 1,4GlcNAc-PP-dolichol. The preparation, isolation, and characterization of each of these lipid-linked oligosaccharide substrates are described.
D'Souza
C
C
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205.
Sharma
C B
CB
Elbein
A D
AD
D000818:Animals; D002240:Carbohydrate Sequence; D002460:Cell Line; D004285:Dogs; D006001:Glycoconjugates / Q000096:biosynthesis* / Q000737:chemistry; D016695:Glycosyltransferases / Q000378:metabolism; D006868:Hydrolysis; D008055:Lipids / Q000737:chemistry*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D010179:Pancreas / Q000378:metabolism; D011105:Polyisoprenyl Phosphate Sugars / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D006001:Glycoconjugates; D008055:Lipids; D009844:Oligosaccharides; D011105:Polyisoprenyl Phosphate Sugars; C076885:Man(9)(GlcNAc)(2)-diphosphate-dolichol; D016695:Glycosyltransferases