Substrate specificity of N-acetylglucosamine 1-phosphate transferase activity in Chinese hamster ovary cells.
The assembly pathway of the oligosaccharide chains of asparagine-linked glycoproteins in mammalian cells begins with the formation of GlcNAc-PP-dolichol in a reaction catalysed by the enzyme N-acetylglucosamine 1-phosphate transferase. We have investigated the efficiency of two lipid substrates for the transferase activity in an in vitro assay using Chinese hamster ovary (CHO) cell membranes as an enzyme source. Experiments were carried out with varying concentrations of dolichyl phosphate or its precursor, polyprenyl phosphate. We determined that enzyme activity was optimal at pH 9, where the enzyme exhibited a 3-fold higher Vmax and a 2-fold lower Km for the dolichol substrate. At pH 7.4, the Km and Vmax differences between the two lipids were 10-fold. Under all assay conditions tested, we found that GlcNAc-PP-lipid was the only product formed. We conclude from these results that dolichyl phosphate rather than polyprenyl phosphate is the preferred substrate for the transferase enzyme in CHO cells. This observation is significant in light of the fact that we have previously isolated CHO glycosylation mutants which fail to convert polyprenol into dolichol, and hence utilize polyprenyl derivatives for glycosylation reactions. Thus, these results contribute to our understanding of the glycosylation defects in the mutant cell lines.
McLachlan
K R
KR
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205.
Krag
S S
SS
D000818:Animals; D016466:CHO Cells / Q000201:enzymology*; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D006224:Cricetinae; D004288:Dolichol Phosphates / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015394:Molecular Structure; D010770:Phosphotransferases / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D017855:Transferases (Other Substituted Phosphate Groups)
D004288:Dolichol Phosphates; D011106:Polyisoprenyl Phosphates; C028306:dolichol monophosphate; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase