Mellquist J L, Kasturi L, Spitalnik S L, Shakin-Eshleman S H
Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Biochemistry. 1998 May 12;37(19):6833-7. doi: 10.1021/bi972217k.
Many eukaryotic proteins are modified by Asn-linked (N-linked) glycosylation. The number and position of oligosaccharides added to a protein by the enzyme oligosaccharyltransferase can influence its expression and function. N-Linked glycosylation usually occurs at Asn residues in Asn-X-Ser/Thr sequons where X not equal Pro. However, many Asn-X-Ser/Thr sequons are not glycosylated or are glycosylated inefficiently. Inefficient glycosylation at one or more Asn-X-Ser/Thr sequons in a protein results in the production of heterogeneous glycoprotein products. These glycoforms may differ from one another in their level of expression, stability, antigenicity, or function. The signals which control the efficiency of N-linked glycosylation at individual Asn residues have not been fully defined. In this report, we use a site-directed mutagenesis approach to investigate the influence of the amino acid at the position following a sequon (the Y position, Asn-X-Ser/Thr-Y). Variants of rabies virus glycoprotein containing a single Asn-X-Ser/Thr sequon at Asn37 were generated. Variants were designed with each of the twenty common amino acids at the Y position, with either Ser or Thr at the hydroxy (Ser/Thr) position. The core glycosylation efficiency of each variant was quantified using a cell-free translation/glycosylation system. These studies reveal that the amino acid at the Y position is an important determinant of core glycosylation efficiency.
许多真核生物蛋白质通过天冬酰胺连接（N 连接）的糖基化进行修饰。寡糖基转移酶添加到蛋白质上的寡糖数量和位置会影响其表达和功能。N 连接糖基化通常发生在 Asn-X-Ser/Thr 序列中的天冬酰胺残基处，其中 X 不等于脯氨酸。然而，许多 Asn-X-Ser/Thr 序列并未发生糖基化或糖基化效率低下。蛋白质中一个或多个 Asn-X-Ser/Thr 序列处糖基化效率低下会导致产生异质性糖蛋白产物。这些糖型在表达水平、稳定性、抗原性或功能上可能彼此不同。控制单个天冬酰胺残基处 N 连接糖基化效率的信号尚未完全明确。在本报告中，我们使用定点诱变方法来研究序列后位置（Y 位置，Asn-X-Ser/Thr-Y）的氨基酸的影响。产生了在 Asn37 处含有单个 Asn-X-Ser/Thr 序列的狂犬病病毒糖蛋白变体。设计的变体在 Y 位置具有二十种常见氨基酸中的每一种，在羟基（Ser/Thr）位置具有丝氨酸或苏氨酸。使用无细胞翻译/糖基化系统对每个变体的核心糖基化效率进行定量。这些研究表明，Y 位置的氨基酸是核心糖基化效率的重要决定因素。
