BIOSYNTHESIS OF BLOOD CLOTTING PROTEINS

凝血蛋白的生物合成

• 批准号: 2218751
• 负责人: Bruce Furie
• 金额: $ 21.41万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2218751
• 关键词: antithrombin III; blood coagulation; blood proteins; carboxylation; coagulation factor IX; complementary DNA; genetic manipulation; genetic translation; glutamates; molecular genetics; nucleic acid sequence; protein biosynthesis; protein sequence; protein signal sequence; protein structure; prothrombin; vitamin K

During protein biosynthesis, the vitamin K-dependent blood coagulation proteins undergo post-translational processing that includes gamma-carboxylation. The mechanism by which these proteins are identified for gamma-carboxylation and specific glutamic acid residues are gamma-carboxylated is not known. These proteins are synthesized in a precursor form that includes a signal peptide and propeptide extension on the amino terminus of the mature protein. Based upon the marked sequence homology of the propeptide of the vitamin K-dependent proteins and our observation that a point mutation at residue -1 in Factor IX impairs gamma-carboxylation, we believe that the propeptide is the recognition site for the proteins associated with gamma- carboxylation. To test this hypothesis, we are developing an efficient expression system for recombinant prothrombin and Factor IX. The prothrombin and Factor IX cDNA have been introduced into mammalian host cells, including Chinese hamster ovary and HepG2 cells. The recombinant prothrombin and Factor IX are gamma-carboxylated and can be isolated by immunoaffinity chromatography. This expression system will be optimized by testing various host cells, vitamin K delivery systems, and methods of introducing the DNA coding sequences into the cells. The purified recombinant prothrombin and Factor IX will be structurally and functionally characterized. The specific role of the propeptide will be evaluated by altering specific amino acids in the propeptide by site-specific mutagenesis and by deletion of the propeptide to determine the effect of these structural changes on the gamma-carboxylation of these proteins. The prothrombin signal peptide/propeptide leader will be spliced to non-carboxylated proteins by linking the prothrombin cDNA coding for the signal peptide/propeptide with the cDNA coding for the mature non-carboxylated protein of interest, tissue plasminogen activator and antithrombin III. Carboxylation of glutamic acids in these proteins will prove that the propeptide directs gamma-carboxylation. The protein in the liver responsible for recognizing the propeptide, the K-propeptide recognition protein, will be detected using a binding assay based on a synthetic peptide analog of the propeptide, -18 to -1. This protein will be purified from liver microsomes and characterized. The relationship of this recognition protein and the gamma- carboxylase will be determined. These studies will further our basic knowledge of vitamin K-dependent gamma-carboxylation during protein synthesis and may facilitate the development of recombinant gamma-carboxylated proteins for therapeutic use.

在蛋白质生物合成期间，维生素K依赖性血液 凝结蛋白会经历翻译后的加工 包括γ-羧化。 这些机制 确定蛋白质用于伽马羧化和特定的蛋白质 谷氨酸残基是γ-羧化，尚不清楚。 这些蛋白质是以包括A的前体形式合成的 信号肽和丙肽延伸的氨基末端 成熟的蛋白质。 基于明显的序列同源 维生素K依赖性蛋白和我们 观察到因子IX中残基-1处的点突变 损害伽马羧化，我们认为丙肽是 与伽马相关的蛋白质的识别位点 羧化。 为了检验这一假设，我们正在开发 重组凝结蛋白的有效表达系统和 因子IX。 凝血酶原和IX因子cDNA已经 引入哺乳动物宿主细胞，包括中国仓鼠 卵巢和HEPG2细胞。 重组凝血酶原和因子 ix是伽玛羧化的，可以通过 免疫亲和力色谱。 这个表达系统将是 通过测试各种宿主细胞进行优化，维生素K递送 系统以及引入DNA编码序列的方法 进入细胞。 纯化的重组凝血酶原和因子 IX将在结构和功能上表征。 这 将通过更改来评估丙肽的具体作用 特定于位点特异性的丙肽中的特异性氨基酸 诱变和通过缺失丙肽来确定 这些结构变化对伽马羧化的影响 这些蛋白质。 凝血酶原信号肽/丙肽领导者 通过将 针对信号肽/晚肽编码的凝血酶蛋白cDNA与 编码成熟的非羧化蛋白的cDNA 兴趣，组织纤溶酶原激活剂和抗凝血酶III。 这些蛋白质中谷氨酸的羧化将证明 该前肽指导伽马羧化。 蛋白质中的蛋白 负责识别丙肽的肝脏，k-炎症 识别蛋白将使用基于结合测定的 在丙肽的合成肽类似物上，-18至-1。 这 蛋白质将从肝微粒体中纯化并表征。 这种识别蛋白与伽马的关系 将确定羧化酶。 这些研究将进一步 维生素K依赖性γ-羧化的基本知识 在蛋白质合成期间，可能有助于发展 重组γ-羧化蛋白用于治疗。