Efficiency of N-linked core glycosylation at asparagine-319 of rabies virus glycoprotein is altered by deletions C-terminal to the glycosylation sequon.
Efficiency of N-linked core glycosylation at asparagine-319 of rabies virus glycoprotein is altered by deletions C-terminal to the glycosylation sequon.
复制标题
狂犬病病毒糖蛋白天冬酰胺-319 处 N 连接核心糖基化的效率通过删除糖基化序列序列的 C 端而改变。
DOI：
10.1021/bi00087a026
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Spitalnik,SL
中科院分区：
文献类型：
--
作者：
Shakin-Eshleman,SH;Wunner,WH;Spitalnik,SL
1993年6月3日接收的修订版Mandalpt ®摘要：在N-连接的核心糖基化中，寡糖Glc 3 MansGlcNAc 2转移到三肽序列子Asn-X-Ser/Thr。然而，这一过程必须通过额外的蛋白质信号来调节，因为许多序列子要么糖基化程度很低，要么根本没有糖基化。由于N-连接的糖基化可以影响蛋白质的结构和功能，因此理解这些信号对于重组糖蛋白的设计和表达至关重要。核心糖基化通常发生在粗面内质网（RER）的新生蛋白质转运过程中。由于当核心糖基化发生时，只有紧邻序列子或其N-末端的蛋白质区域被认为是在RER中，因此大多数模型预测序列子C-末端的区域不会影响这一过程。我们测试了序列子的C末端区域是否会影响其核心糖基化。全长（505个氨基酸）狂犬病病毒糖蛋白（RGP）突变体，每个只含有一个的三个序列通常存在于RGP，用于这些研究。使用无细胞系统，确定每个序列子的核心糖基化效率。然后将终止密码子引入这些突变体的限定位点以产生C-末端截短，并评估这些截短中的每一个对每个序列子处的核心糖基化效率的影响。虽然C-末端跨膜和胞质结构域的缺失不影响核心糖基化，但更广泛的C-末端缺失确实导致以位点特异性方式改变的核心糖基化。具体而言，C-末端截短导致蛋白质含有386或344个氨基酸，降低了Asn 319处的核心糖基化效率。许多蛋白质通过在特定的天冬酰胺（Asn）残基上添加寡糖来修饰[综述见Kornfeld和Kornfeld（1985）]。该过程称为N-连接糖基化，可影响蛋白质的许多性质，包括细胞内转运（Machamer & Rose，1988; Dube et al. 1988; Semenkovich等人，1990; Ng等人，1990）、生物活性（Dube等人，1988; Semenkovich等人，1990; Tao和莫里森，1989; Matzuk埃塔尔，1989）、稳定性（Matzuk & Boime，1988; Pizer等人，1980）和抗原性（Wright埃塔尔，1989; Klenk，1990; Hobmanetal.，1991年）。N-连接的糖基化的生物学效应通常取决于多肽链内的糖基化位点（Machamer & Rose，1988; Dube et al. 1988; Ng等人，1990; Matzuk等人，1989年）。此外，在特定位点的低效糖基化可导致蛋白质的替代糖型的合成，所述糖型仅通过在特定位点存在或不存在寡糖而彼此不同（Plummer & Hirs，1964; Wunner等人，1985）;这些糖型在生物学功能方面可能彼此不同。因此，要完全理解糖蛋白合成和功能的调控，需要对在特定位点调节N-连接糖基化效率的蛋白信号进行表征。
Revised Manuscript Received June 3, 1993® abstract: In N-linked core glycosylation, the oligosaccharide Glc3MansGlcNAc2 is transferred to the tripeptide sequon Asn-X-Ser/Thr. However, this process must be regulated by additional protein signals, since many sequons are either poorly glycosylated or not glycosylated at all. Since N-linked glycosylation can influence protein structure and function, understanding these signals is essential for the design and expression of recombinant glycoproteins. Core glycosylation usually occurs cotranslationally in the rough endoplasmic reticulum (RER) during translocation of nascent proteins. Since only regions of a protein immediately near to a sequon or N-terminal to it are thought to be in the RER when core glycosylation occurs, most models predict that regions C-terminal to the sequon do not influence this process. We tested whether regions C-terminal to a sequon can influence its core glycosylation. Full-length (505 amino acid) rabies virus glycoprotein (RGP) mutants, each containing only one of the three sequons normally present in RGP, were used for these studies. Using a cell-free system, the core glycosylation efficiency ateach sequon was determined. Termination codons were then introduced into these mutants at defined sites to produce C-terminal truncations, and the effect of each of these truncations on the core glycosylation efficiency at each sequon was assessed. While deletion of the C-terminal transmembrane and cytoplasmic domains did not affect core glycosylation, more extensive C-terminal deletions did resultin altered core glycosylation in a site-specific fashion. Specifically, C-terminal truncations resulting in proteins containing 386 or 344 amino acidsdecreased the efficiency of core glycosylation at Asn319. This demonstrates that core glycosylation efficiency can be influenced by thepresence or absence of regions in a protein more than 68 amino acids C-terminal to a specific glycosylation site.Many proteins are modified by the addition of oligosac-charides to specific asparagine (Asn) residues [for review, see Kornfeld and Kornfeld (1985)]. This process, known as N-linked glycosylation, can influence many properties of proteins, including intracellular transport (Machamer & Rose, 1988; Dube et al., 1988; Semenkovich et al., 1990; Ng et al., 1990), biological activity (Dube et al., 1988; Semenkovich et al., 1990; Tao & Morrison, 1989; Matzuk etal., 1989), stability (Matzuk & Boime, 1988; Pizer et al., 1980), and antigenicity (Wright etal., 1989; Klenk, 1990; Hobmanetal., 1991). The biological effects of N-linked glycosylation often depend on the site of glycosylation within a polypeptide chain (Machamer & Rose, 1988; Dube et al., 1988; Ng et al., 1990; Matzuk et al., 1989). In addition, inefficient glycosylationat a specific site can lead to the synthesis of alternate glycoforms of a protein which differ from one another only by the presence or absence of an oligosaccharide at a specific site (Plummer & Hirs, 1964; Wunner et al., 1985); these glycoforms may differ from one another with regard to biological function. Therefore, complete understanding of the regulation of glycoprotein synthesis and function requires the characterization of the protein signals which regulate the efficiency of N-linked glycosylationat specific sites.