Freeze H H, Koza-Taylor P, Jones J A, Loomis W F
Department of Medicine, University of California, San Diego, La Jolla 92093.
J Cell Biochem. 1990 May;43(1):27-42. doi: 10.1002/jcb.240430104.
N-glycosylation was measured in wild-type cell lysates of Dictyostelium discoideum and in two mutant strains that synthesize a truncated lipid-linked oligosaccharide, Man6GlcNAc2 lacking terminal mannose and glucose residues. Endogenous lipid-linked oligosaccharide (LLO) was transferred to octanoyl-Asn-[125I]Tyr-ThrNH2 by membrane fractions. About 50% of the glycopeptide product remained associated with membranes. Taurocholate and saponin promoted and preserved glycosylation, but NP-40 and Triton X-100 did not. Using this artificial assay, the rate and extent of transfer of the truncated lipid-linked oligosaccharide in extracts of the two mutant strains, HL241 and HL243, was reduced 5-10-fold relative to that of wild-type. The low activity found in the mutant strains appears to result from either reduced affinity of the truncated LLO for the transferase or from its improper topological localization in the membrane. When protein N-glycosylation is measured in living cells it is nearly normal in HL241, but it is 3-4-fold decreased in HL243. Although the results of the in vitro and in vivo assays differ, they are not in conflict. Rather, they suggest that the static in vitro assay may be capable of revealing subtleties in the productive positioning of LLO and the oligosaccharyl transferase. The decrease in glycosylation seen in intact HL243 cells may be a consequence of the pleiotropic effects of the primary mutation rather than a direct result of the altered LLO structure. Genetic analysis showed that the mutation in HL241 is recessive, while the mutation in HL243 is dominant and prevents normal development. Thus, the two mutants share a lesion in lipid-linked oligosaccharide biosynthesis and in cell-free glycosylation, but differ in their in vivo glycosylation. Their primary defects are probably different.
在盘基网柄菌的野生型细胞裂解物以及两种合成截短型脂连接寡糖（缺少末端甘露糖和葡萄糖残基的Man6GlcNAc2）的突变菌株中测量了N-糖基化。内源性脂连接寡糖（LLO）通过膜组分转移至辛酰基-Asn-[125I]Tyr-ThrNH2。约50%的糖肽产物仍与膜结合。牛磺胆酸盐和皂苷促进并维持糖基化，但NP-40和Triton X-100则不然。使用这种人工测定法，相对于野生型，两种突变菌株HL241和HL243提取物中截短型脂连接寡糖的转移速率和程度降低了5至10倍。在突变菌株中发现的低活性似乎是由于截短型LLO对转移酶的亲和力降低或其在膜中的拓扑定位不当所致。当在活细胞中测量蛋白质N-糖基化时，HL241中的糖基化几乎正常，但在HL243中则降低了3至4倍。尽管体外和体内测定的结果不同，但它们并不矛盾。相反，它们表明静态体外测定法可能能够揭示LLO和寡糖基转移酶生产性定位中的细微差别。在完整的HL243细胞中观察到的糖基化降低可能是主要突变的多效性效应的结果，而不是LLO结构改变的直接结果。遗传分析表明，HL241中的突变是隐性的，而HL243中的突变是显性的，并阻止正常发育。因此，这两种突变体在脂连接寡糖生物合成和无细胞糖基化方面有共同损伤，但在体内糖基化方面有所不同。它们的主要缺陷可能不同。
