INTRACELLULAR FOLDING OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR - EFFECTS OF DISULFIDE BOND FORMATION ON N-LINKED GLYCOSYLATION AND SECRETION
INTRACELLULAR FOLDING OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR - EFFECTS OF DISULFIDE BOND FORMATION ON N-LINKED GLYCOSYLATION AND SECRETION
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DOI：
10.1074/jbc.270.9.4797
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发表时间：
1995-03-03
影响因子：
4.8
通讯作者：
BULLEID, NJ
中科院分区：
文献类型：
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作者：
ALLEN, S;NAIM, HY;BULLEID, NJ
N-连接的核心寡糖添加到膜和分泌糖蛋白中，在新生多肽易位到内质网腔后不久，在三肽序列Asn-Xaa-Ser/Thr中的天冬酰胺残基处协同发生。然而，序列子的存在并不能自动确保核心糖基化，因为许多蛋白质含有保持未糖基化或糖基化至可变程度的序列子。为了研究细胞内蛋白质折叠是否会影响序列子的利用，我们在细胞培养中表达了组织型纤溶酶原激活剂（t-PA），在温和浓度的还原剂二硫苏糖醇的存在下，以防止在内质网中形成α-翻译二硫键。我们表明，防止二硫键形成的条件下，导致完全糖基化的序列子，否则在未经处理的细胞中经历可变的糖基化。这表明t-PA的折叠和二硫键的形成决定了其核心N-连接糖基化的程度。当从细胞中除去二硫苏糖醇时，还原和过糖基化的t-PA形成二硫键，折叠并分泌。我们还表明，细胞内存在的t-PA比分泌的t-PA更容易被低浓度的二硫苏糖醇还原。
The addition of N-linked core oligosaccharides to membrane and secretory glycoproteins occurs co-translationally at asparagine residues in the tripeptide sequon Asn-Xaa-Ser/Thr soon after translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum. However, the presence of the sequon does not automatically ensure core glycosylation, as many proteins contain sequons that remain either unglycosylated or glycosylated to a variable extent. To investigate whether intracellular protein folding can influence sequon utilization, we have expressed tissue-type plasminogen activator (t-PA) in cell culture in the presence of mild concentrations of the reducing agent dithiothreitol to prevent cc-translational disulfide bond formation in the endoplasmic reticulum. We show that conditions that prevent disulfide bond formation lead to complete glycosylation of a sequon that otherwise undergoes variable glycosylation in untreated cells. This demonstrated that folding and disulfide bond formation of t-PA determines its extent of core N-linked glycosylation. When dithiothreitol was removed from the cells, the reduced and overglycosylated t-PA formed disulfide bonds, folded and was secreted We also show t-PA present within cells is more susceptible to reduction with low concentrations of dithiothreitol than secreted t-PA.