THE HYDROXY AMINO-ACID IN AN ASN-X-SER/THR SEQUON CAN INFLUENCE N-LINKED CORE GLYCOSYLATION EFFICIENCY AND THE LEVEL OF EXPRESSION OF A CELL-SURFACE GLYCOPROTEIN
THE HYDROXY AMINO-ACID IN AN ASN-X-SER/THR SEQUON CAN INFLUENCE N-LINKED CORE GLYCOSYLATION EFFICIENCY AND THE LEVEL OF EXPRESSION OF A CELL-SURFACE GLYCOPROTEIN
复制标题
DOI：
10.1074/jbc.270.24.14756
复制
发表时间：
1995-06-16
影响因子：
4.8
通讯作者：
SHAKINESHLEMAN, SH
中科院分区：
文献类型：
--
作者：
KASTURI, L;ESHLEMAN, JR;SHAKINESHLEMAN, SH
N-连接糖基化通常发生在序列子Asn-X-Ser/Thr。在这个序列中，羟基氨基酸（Ser或Thr）的侧链可能在核心寡糖酶促转移到Asn残基中发挥直接催化作用。使用狂犬病病毒糖蛋白（RGP）的重组变体，我们研究了羟基氨基酸对核心糖基化效率的影响。通过定点诱变将在Asn（37）处含有单个Asn-X-Ser序列子的RGP变体修饰为Asn-X-Cys或Asn-X-Thr。通过在无细胞转录/翻译/糖基化系统和转染的组织培养细胞中表达变体来评估这些变化对核心糖基化效率的影响。在位置39处的Cys的取代阻断糖基化，而Thr的取代显著增加了Asn（37）在膜锚定和分泌形式的RGP中的核心糖基化效率。当Asn（37）处的序列子是蛋白质中唯一的序列子时，Thr取代Ser也显著增强了RGP的表达水平和细胞表面递送。膜锚定和分泌的RGP，这是完全糖基化的所有三个序列的新形式也产生了Thr在位置39的取代。
N-Linked glycosylation usually occurs at the sequon, Asn-X-Ser/Thr. In this sequon, the side chain of the hydroxy amino acid (Ser or Thr) may play a direct catalytic role in the enzymatic transfer of core oligosaccharides to the Asn residue. Using recombinant variants of rabies virus glycoprotein (RGP), we examined the influence of the hydroxy amino acid on core glycosylation efficiency. A variant of RGP containing a single Asn-X-Ser sequon at Asn(37) was modified by site directed mutagenesis to change the sequon to either Asn-X-Cys or Asn-X-Thr. The impact of these changes on core glycosylation efficiency was assessed by expressing the variants in a cell-free transcription/translation/glycosylation system and in transfected tissue culture cells. Substitution of Cys at position 39 blocks glycosylation, whereas substitution of Thr dramatically increases core glycosylation efficiency of Asn(37) in both membrane-anchored and secreted forms of RGP. The substitution of Thr for Ser also dramatically enhances the level of expression and cell surface delivery of RGP when the sequon at Asn(37) is the only sequon in the protein. Novel forms of membrane-anchored and secreted RGP which are fully glycosylated at all three sequons were also generated by substitution of Thr at position 39.