McLachlan K R, Krag S S
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205.
Glycobiology. 1992 Aug;2(4):313-9. doi: 10.1093/glycob/2.4.313.
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.
哺乳动物细胞中天冬酰胺连接糖蛋白的寡糖链组装途径始于在N-乙酰葡糖胺1-磷酸转移酶催化的反应中形成GlcNAc-PP-多萜醇。我们使用中国仓鼠卵巢（CHO）细胞膜作为酶源，在体外测定中研究了两种脂质底物对转移酶活性的效率。实验使用了不同浓度的磷酸多萜醇或其前体，聚异戊二烯磷酸。我们确定酶活性在pH 9时最佳，此时该酶对多萜醇底物的Vmax高3倍，Km低2倍。在pH 7.4时，两种脂质之间的Km和Vmax差异为10倍。在所有测试的测定条件下，我们发现GlcNAc-PP-脂质是唯一形成的产物。从这些结果我们得出结论，磷酸多萜醇而非聚异戊二烯磷酸是CHO细胞中转移酶的首选底物。鉴于我们之前分离出未能将聚异戊二烯醇转化为多萜醇，因此利用聚异戊二烯衍生物进行糖基化反应的CHO糖基化突变体，这一观察结果具有重要意义。因此，这些结果有助于我们理解突变细胞系中的糖基化缺陷。
