ENZYMATIC CONVERSION OF PROTEINS TO GLYCOPROTEINS

蛋白质酶促转化为糖蛋白

• 批准号: 3484769
• 负责人: WILLIAM J LENNARZ
• 金额: $ 22.52万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-08-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3484769
• 关键词: Drosophilidae; affinity labeling; antibody formation; binding proteins; chemical binding; chickens; endoplasmic reticulum; enzyme inhibitors; enzyme mechanism; enzyme model; enzyme reconstitution; enzyme structure; enzyme substrate; fallopian tubes; genetic translation; glycolipids; glycoprotein biosynthesis; glycosylation; glycosyltransferase; hormone regulation /control mechanism; laboratory rat; membrane proteins; molecular cloning; oligosaccharides; peptide chemical synthesis; protein metabolism; protein sequence; tissue /cell culture; yeasts

Cell surface glycoproteins are believed to play a role in cell recognition events during both normal and abnormal growth and differentiation. A key step in the assembly of N-linked glycoproteins is the co-translational transfer of a preassembled oligosaccharide chain from dolichylpyrophosphate to an appropriate -Asn-X-Ser/Thr- site in the growing polypeptide chain. If the mature N-linked glycoprotein is destined to be of the polymannose type, subsequent processing in the Golgi complex is very limited. In contrast, if it is destined to be of the complex type extensive processing, involving removal of all but three of the Man residues and the addition of "capping sugars", occurs. The proposed studies will focus on three events in this overall process. First, the enzyme that catalyzes the transfer of the oligosaccharide chain, oligosaccharyltransferase, will be isolated from hen oviduct and characterized. Antibody to the enzyme and active site photoreactive peptide substrates will be employed in the isolation. The antibody will also be used to study the biosynthesis of this enzyme in oviduct cells. Peptides that inhibit the enzyme in vitro will be developed and studied as in vivo inhibitors of glycosylation in a number of cell types. Second, efforts will be made to better understand why certain -Asn-X-Ser/Thr- sites in proteins are not acted upon by oligosaccharyltransferase in vivo. Using ovalbumin as a model it will be determined if spatial factors prevent glycosylation of a second acceptor site when ovalbumin is synthesized in vivo. In addition, the role of temporal factors in the glycosylation of the second site of ovalbumin synthesized in vitro will be investigated. Third, the factors that control the processing of oligosaccharide chains after their transfer to protein will be studied. It is known that the polymannose chain of native bovine pancreatic RNase B is not processed by Golgi membranes from bovine pancreas, but is processed by rat liver membranes. The possibility will be investigated that this is due to differences in the properties of the GlcNAc transferase I from these 2 sources. In addition, efforts will be made to identify structural features in the polypeptide chain of bovine pancreatic RNase B that prevent its oligosaccharide chain from being processed by the pancreatic enzyme. Finally, these processing studies will be extended to 3 other high mannose glycoproteins and Golgi membranes from sources that are either homologous or heterologous with respect to the source of glycoproteins.

据信细胞表面糖蛋白在细胞识别中起作用 正常生长和分化异常的事件。 钥匙 逐步组装N连接的糖蛋白是共同翻译 从多乙基磷酸盐的寡糖链转移 在生长的多肽链中适当的-ASN-X-SER/THR-位置。 如果成熟的N连接糖蛋白注定为多曼诺糖 类型，随后在高尔基体复合物中进行的处理非常有限。 在 对比，如果它注定为复杂类型的广泛处理，则 涉及清除除三个男子残留物以外的所有人以及添加 发生“封糖”。 拟议的研究将集中于三个事件 在整个过程中。 首先，催化转移的酶 寡糖链，寡糖胆碱转移酶，将与 母鸡的卵形和特征。 酶和活性位点的抗体 分离中将采用光反应性肽底物。 这 抗体还将用于研究该酶在中的生物合成 输卵管细胞。 将开发抑制酶的肽的肽 并研究了许多细胞中糖基化的体内抑制剂 类型。 其次，将努力更好地理解为什么确定 -ASN-X-SER/THR-蛋白质中的位点没有作用于 体内的寡糖胆碱转移酶。 使用卵蛋白作为模型 确定空间因子是否防止第二受体的糖基化 当体内合成椭圆蛋白时，位于椭圆形的位置。 另外， 卵蛋白第二位点糖基化的时间因素 将研究体外合成的。 第三，控制因素 将寡糖链转移到蛋白质后的加工 将被研究。 众所周知，天然牛的多曼糖链 胰腺膜未通过牛的高尔基膜处理胰腺RNase B 胰腺，但由大鼠肝膜处理。 可能性将是 调查这是由于特性的差异 来自这两个来源的GlcNAC转移酶I。 此外，努力将是 为了识别牛多肽链中的结构特征 防止其寡糖链的胰腺RNase B是 由胰腺酶加工。 最后，这些处理研究将 从中扩展到其他3种高甘露糖糖蛋白和高尔基膜 相对于 糖蛋白的来源。