Ronin C, Granier C, Caseti C, Bouchilloux S, Van Rietschoten J
Eur J Biochem. 1981 Aug;118(1):159-64. doi: 10.1111/j.1432-1033.1981.tb05499.x.
The N-glycosylation of proteins is initiated by transfer of preassembled oligosaccharides from lipid carriers to an asparagine residue in the sequence Asn-Xaa-Ser(Thr). Various synthetic peptides were previously shown to act as effective acceptors when supplied to microsomal membranes. The present work was undertaken to delineate further within such peptides the structural determinants required for the N-glycosylation process. Several new peptides were synthesized to evaluate the effects of the chain length and of the modification on the asparagine, Xaa or threonine residues: asparagine was replaced by glutamine, Xaa by proline or N-methylated alanine, threonine by an homoserine residue. Furthermore, both side chains of asparagine and threonine were simultaneously replaced by those of glutamine and homoserine respectively to restore a putative hydrogen bonding between the carbonyl and hydroxyl groups. The assays were performed with a solubilized form of the oligosaccharide transferase and the acceptor capacities of the peptides expressed by the ratio V/Km of their apparent kinetic parameters V and Km. Results showed that N-acetyl-tripeptide amides are excellent substrates for the enzyme. All modifications in the -Asn-Xaa-Thr- region resulted in a loss of acceptor capacity. The importance of the conformational aspect is discussed.
蛋白质的N-糖基化是通过将预组装的寡糖从脂质载体转移到序列Asn-Xaa-Ser（Thr）中的天冬酰胺残基上开始的。先前已证明，当将各种合成肽提供给微粒体膜时，它们可作为有效的受体。开展本研究是为了在这类肽中进一步确定N-糖基化过程所需的结构决定因素。合成了几种新肽，以评估链长以及天冬酰胺、Xaa或苏氨酸残基修饰的影响：将天冬酰胺替换为谷氨酰胺，将Xaa替换为脯氨酸或N-甲基化丙氨酸，将苏氨酸替换为高丝氨酸残基。此外，分别将天冬酰胺和苏氨酸的两个侧链同时替换为谷氨酰胺和高丝氨酸的侧链，以恢复羰基和羟基之间假定的氢键。使用寡糖转移酶的可溶形式进行测定，并通过肽的表观动力学参数V和Km的比值V/Km来表示肽的受体能力。结果表明，N-乙酰三肽酰胺是该酶的优良底物。-Asn-Xaa-Thr-区域的所有修饰均导致受体能力丧失。讨论了构象方面的重要性。
