Geetha-Habib M, Park H R, Lennarz W J
Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
J Biol Chem. 1990 Aug 15;265(23):13655-60.
The minimum primary structural requirement for a tripeptide to serve as a substrate for oligosaccharyl transferase is the sequence -Asn-X-Ser/Thr-. In the present study the activities of three structurally different tripeptides containing acceptor sequences for oligosaccharyl transferase were compared in three systems: Xenopus oocytes, in which they were introduced into the cytoplasm by microinjection, cultured mammalian cells, and isolated rat liver microsomes. In the last two systems, the peptides were added exogenously to the culture or to the incubation medium, respectively. On the basis of lectin column and paper chromatographic analysis it was established that the microinjected acceptor tripeptides were glycosylated in Xenopus oocytes. However, lectin column analysis and retention of sensitivity to endoglycosidase H revealed that none of the three glycopeptides was processed to complex oligosaccharide chains and none was subsequently secreted. Rather, over a 24-h period the glycopeptides were degraded. Chloroquine was found to block this degradation process, but even under these conditions, the glycopeptides were not secreted into the medium. In the isolated microsomes the glycosylation of the acceptor tripeptides was time-dependent and the tripeptide with an iodotyrosine residue in the X position was found to be a poor substrate. When added to cultured mammalian cells, all three of the tripeptides were taken up, glycosylated, and subsequently secreted. These results are discussed in the context of the wide differences in glycosylation of the three peptides and their lack of secretion after glycosylation in Xenopus oocytes.
三肽作为寡糖基转移酶底物的最低一级结构要求是序列 -Asn-X-Ser/Thr-。在本研究中，比较了三种含有寡糖基转移酶受体序列、结构不同的三肽在三种系统中的活性：非洲爪蟾卵母细胞（通过显微注射将它们引入细胞质）、培养的哺乳动物细胞和分离的大鼠肝微粒体。在最后两种系统中，分别将肽外源添加到培养物或孵育培养基中。基于凝集素柱和纸色谱分析确定，显微注射的受体三肽在非洲爪蟾卵母细胞中发生了糖基化。然而，凝集素柱分析以及对内切糖苷酶H的敏感性保留表明，三种糖肽均未加工成复杂的寡糖链，也没有随后被分泌。相反，在24小时内糖肽被降解。发现氯喹可阻断这一降解过程，但即使在这些条件下，糖肽也未分泌到培养基中。在分离的微粒体中，受体三肽的糖基化是时间依赖性的，并且发现在X位置带有碘酪氨酸残基的三肽是一种较差的底物。当添加到培养的哺乳动物细胞中时，所有三种三肽都被摄取、糖基化并随后被分泌。在三种肽糖基化的巨大差异及其在非洲爪蟾卵母细胞中糖基化后缺乏分泌的背景下讨论了这些结果。
