Lau J T, Welply J K, Shenbagamurthi P, Naider F, Lennarz W J
J Biol Chem. 1983 Dec 25;258(24):15255-60.
In a microsome system rendered competent in protein translation by the addition of rabbit reticulocyte lysate, co-translational insertion and glycosylation of N-linked glycoproteins is observed when the appropriate mRNA is supplied. We have utilized this system to examine the ability of acceptor tripeptides of the type Asn-X-Thr/Ser to inhibit co-translational glycosylation. Using endogenous oligosaccharide-lipid as the carbohydrate donor, dog pancreas microsomes efficiently glycosylated N alpha-[3H]Ac-Asn-Leu-Thr-NHCH3 (apparent Km = 100 microM). Glycopeptide formation was essentially complete within 20 min. In the presence of mRNA from vesicular stomatitis virus or chicken ovalbumin, a similar tripeptide, N alpha-Ac-Asn-Leu-Thr-NH2, inhibited co-translational glycosylation. Translocation of the nascent chains was not affected. Thus, in the absence of peptide, all translated G protein was glycosylated and found within the microsomes, whereas in the presence of the peptide a mixture of glycosylated and nonglycosylated G protein was sequestered. Inhibition of nascent chain glycosylation was competitive and not merely the result of oligosaccharide lipid depletion, because preincubation of the microsomes with the peptide followed by its removal did not affect subsequent glycosylation of ovalbumin or G protein. Six derivatives of Asn-Leu-Thr-NH2, three of which were acceptors and three of which were not, were tested for their ability to inhibit co-translational glycosylation. The three acceptor peptides, N alpha-Ac-Asn-Leu-Thr-NH2, N alpha-Oc-Asn-Leu-Thr-NH2, and N alpha-Bz-Asn-Leu-Thr-NH2, effectively inhibited nascent chain glycosylation. In contrast, the three nonacceptors, N alpha-Ac-Gln-Leu-Thr-NH2, N alpha-Ac-Asn(N beta-Me)-Leu-Thr-NH2, and Asn-Leu-Thr-NH2, had no effect. Taken together, these data indicate that the inhibition of co-translational glycosylation by a peptide is dependent on its ability to compete for the active site of the oligosaccharyl transferase.
在通过添加兔网织红细胞裂解物而具备蛋白质翻译能力的微粒体系统中，当提供合适的信使核糖核酸（mRNA）时，可观察到N - 连接糖蛋白的共翻译插入和糖基化过程。我们利用该系统研究了Asn - X - Thr/Ser类型的受体三肽抑制共翻译糖基化的能力。以内源性寡糖脂作为碳水化合物供体时，犬胰腺微粒体能够高效地将Nα - [³H]乙酰 - 天冬酰胺 - 亮氨酸 - 苏氨酸 - NHCH₃（表观米氏常数 = 100微摩尔）糖基化。糖肽的形成在20分钟内基本完成。在存在水疱性口炎病毒或鸡卵清蛋白的信使核糖核酸时，一种类似的三肽Nα - 乙酰 - 天冬酰胺 - 亮氨酸 - 苏氨酸 - NH₂可抑制共翻译糖基化。新生肽链的转位不受影响。因此，在没有肽的情况下，所有翻译的G蛋白都被糖基化并存在于微粒体中，而在有肽存在时，糖基化和未糖基化的G蛋白的混合物被隔离。新生肽链糖基化的抑制是竞争性的，并非仅仅是由于寡糖脂耗竭所致，因为微粒体与肽预孵育后再去除肽并不影响随后卵清蛋白或G蛋白的糖基化。对天冬酰胺 - 亮氨酸 - 苏氨酸 - NH₂的六种衍生物进行了测试，其中三种是受体，三种不是，以检测它们抑制共翻译糖基化的能力。三种受体肽，Nα - 乙酰 - 天冬酰胺 - 亮氨酸 - 苏氨酸 - NH₂、Nα - 辛酰 - 天冬酰胺 - 亮氨酸 - 苏氨酸 - NH₂和Nα - 苯甲酰 - 天冬酰胺 - 亮氨酸 - 苏氨酸 - NH₂，有效地抑制了新生肽链的糖基化。相比之下，三种非受体肽，Nα - 乙酰 - 谷氨酰胺 - 亮氨酸 - 苏氨酸 - NH₂、Nα - 乙酰 - 天冬酰胺（Nβ - 甲基） - 亮氨酸 - 苏氨酸 - NH₂和天冬酰胺 - 亮氨酸 - 苏氨酸 - NH₂则没有作用。综上所述，这些数据表明肽对共翻译糖基化的抑制取决于其竞争寡糖基转移酶活性位点的能力。
