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.

细胞表面糖蛋白被认为在细胞识别中发挥作用 正常和异常生长和分化期间的事件。 一把钥匙 N-连接糖蛋白组装的步骤是共翻译 从多醇焦磷酸转移预组装的寡糖链 到生长的多肽链中适当的-Asn-X-Ser/Thr-位点。 如果成熟的N联糖蛋白注定是多甘露糖 类型，高尔基复合体中的后续处理非常有限。 在 相比之下，如果注定是复杂类型的粗放加工， 包括去除除三个之外的所有人类残留物并添加 发生“封顶糖”。 拟议的研究将重点关注三个事件 在这个整体过程中。 首先，催化转移的酶 寡糖链，寡糖基转移酶，将从 母鸡输卵管和特征。 酶抗体和活性位点 光反应性肽底物将用于分离。 这 抗体也将用于研究这种酶的生物合成 输卵管细胞。 将开发体外抑制该酶的肽 并在许多细胞中作为糖基化的体内抑制剂进行研究 类型。 其次，将努力更好地理解为什么某些 蛋白质中的 -Asn-X-Ser/Thr- 位点不受 体内寡糖转移酶。 使用卵清蛋白作为模型 确定空间因素是否阻止第二个受体的糖基化 体内合成卵清蛋白的位点。 此外，作用 卵清蛋白第二位点糖基化的时间因素 将研究体外合成。 三、控制因素 低聚糖链转移至蛋白质后的加工 将被研究。 众所周知，天然牛的多聚甘露糖链 胰腺 RNase B 不被牛高尔基膜加工 胰腺，但由大鼠肝膜处理。 可能性将是 经调查，这是由于属性差异造成的 GlcNAc 转移酶 I 来自这两个来源。 此外，还将努力 鉴定牛多肽链的结构特征 胰腺 RNase B 可以防止其寡糖链被 由胰酶加工。 最后，这些加工研究将 扩展到其他 3 种高甘露糖糖蛋白和高尔基体膜 相对于同源或异源的来源 糖蛋白的来源。