Reduced utilization of Man5GlcNAc2-P-P-lipid in a Lec9 mutant of Chinese hamster ovary cells: analysis of the steps in oligosaccharide-lipid assembly.
Recently we reported that CHB11-1-3, a Chinese hamster ovary cell mutant defective in glycosylation of asparagine-linked proteins, is defective in the synthesis of dolichol [Quellhorst et al., 343:19-26, 1997: Arch Biochem Biophys]. CHB11-1-3 was found to be in the Lec9 complementation group, which synthesizes polyprenol rather than dolichol. In this paper, levels of various polyprenyl derivatives in CHB11-1-3 are compared to levels of the corresponding dolichyl derivatives in parental cells. CHB11-1-3 was found to maintain near normal levels of Man5GlcNAc2-P-P-polyprenol and mannosylphosphorylpolyprenol, despite reduced rates of synthesis, by utilizing those intermediates at a reduced rate. The Man5GlcNAc2 oligosaccharide attached to prenol in CHB11-1-3 cells and to dolichol in parental cells is the same structure, as determined by acetolysis. Man5GlcNAc2-P-P-polyprenol and Man5GlcNAc5-P-P-dolichol both appeared to be translocated efficiently in an in vitro reaction. Glycosylation of G protein was compared in vesicular stomatitus virus (VSV)-infected parent and mutant; although a portion of G protein was compared in vesicular stomatitus virus (VSV)-infected parent and mutant; although a portion of G protein was normally glycosylated in CHB11-1-3 cells, a large portion of G was underglycosylated, resulting in the addition of either one or no oligosaccharide to G. Addition of a single oligosaccharide occurred randomly rather than preferentially at one of the two sites.
Hall
C W
CW
Laboratory of Biochemistry and Metabolism, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
McLachlan
K R
KR
Krag
S S
SS
Robbins
A R
AR
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells / Q000378:metabolism* / Q000821:virology; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004286:Dolichols / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism; D006031:Glycosylation; D045782:Hemiterpenes; D050356:Lipid Metabolism; D008358:Mannose; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D000439:Pentanols / Q000378:metabolism; D011105:Polyisoprenyl Phosphate Sugars / Q000378:metabolism*; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
D004286:Dolichols; D045782:Hemiterpenes; D000439:Pentanols; D011105:Polyisoprenyl Phosphate Sugars; D014764:Viral Proteins; C009034:prenol; D008715:Methionine; D019204:GTP-Binding Proteins; D008358:Mannose