Production of the two enzymes concerned in peptide C-terminal alpha-amidation by genetic engineering and in vitro reconstitution of peptide C-terminal alpha-amidation reaction

肽C端α-酰胺化涉及的两种酶的基因工程生产及肽C端α-酰胺化反应的体外重构

基本信息

批准号：
03557016
负责人：
OKAMOTO Hiroshi
金额：
$ 10.37万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

Many peptide hormones and neurotransmitters have the C-terminal alpha-amide structure and these alpha-amidated peptides are enzymatically synthesized from their glycine-extended precursors. The C-terminal alpha-amidation reaction proceeds via a two step reaction through a peptidyl alpha-hydroxylglycine as an intermediate and these steps of the peptide alpha-amidation reaction are catalyzed by two distinct enzymes (peptidylglycine hydroxylase and peptidylamidoglycolate lyase). The aim of this project was to produce the two enzymes concerned in peptide alpha-amidation by genetic engineering and reconstitute the peptide alpha-amidation system in vitro.In this project,(1) By functional expression of rat "peptidylglycine alpha-amidating enzyme" cDNA, we found that peptidylglycine hydroxylase and peptidylamidoglycolate lyase are synthesized from a single mRNA.(2) We found that peptidylglycine hydroxylase and peptidylamidoglycolate lyase are encoded in the 5'-half and 3'-half of the mRNA, res … More pectively.(3) We constructed expression vectors carrying the 5'-half or 3'-half of the cDNA, which directed peptidylglycine hydroxylase or peptidylamidoglycolate lyase to secrete into the culture medium, and produced the two enzymes by transfection of the expression vectors into COS-7 cells.(4) By experiments using ^<18>O_2, we demonstrated that the peptidylglycine alpha-hydroxylation is a monooxygenase reaction.(5) By site-directed mutagenesis, we demonstrated that the five histidine residues at position 107, 108, 172, 242 and 244 are essential for the peptidylglycine hydroxylase activity.(6) We found that the peptidylamidoglycolate lyase exhibited a pH optimum at 5.5 and required a divalent cation but neither O_2 nor ascorbate for the enzyme activity.(7) We demonstrated that, in the combined presence of peptidylglycine hydroxylase and peptidylamidoglycolate lyase, the mature alpha-amidated peptide was successfully produced from the glycine-extended precursor in vitro.(8) We produced transgenic mice carrying human Vosoactive Intestinal Peptide (VIP)/PHM-27 gene under the control of insulin promoter and found that VIP and PHM-27 produced from the transgenic pancreatic beta-cells efficiently enhanced the glucose-induced insulin secretion, indicating that VIP and PHM-27 from the transgene were efficiently alpha-amidated by peptidylglycine hydroxylase and peptidylamidoglycolate lyase in the beta-cell. Less

许多肽激素和神经递质具有 C 端 α-酰胺结构，这些 α-酰胺化肽是由其甘氨酸延伸前体酶促合成的。C 端 α-酰胺化反应通过肽基 α-羟基甘氨酸通过两步反应进行。作为中间体，肽 α-酰胺化反应的这些步骤由两种不同的酶（肽基甘氨酸羟化酶和本项目的目的是通过基因工程生产与肽α-酰胺化有关的两种酶，并在体外重建肽α-酰胺化系统。在本项目中，(1)通过大鼠“肽酰甘氨酸α”的功能表达。 -酰胺化酶”cDNA，我们发现肽酰甘氨酸羟化酶和肽酰氨基乙醇酸裂合酶是由单个合成的mRNA。(2)我们发现肽基甘氨酸羟化酶和肽基酰胺乙醇酸裂合酶分别编码在mRNA的5'-半和3'-半中，具体而言。(3)我们构建了携带5'-半或3'-半的表达载体。 '-cDNA的一半，引导肽基甘氨酸羟化酶或肽基酰胺乙醇酸裂合酶分泌到培养基中，并将表达载体转染至COS-7细胞中产生这两种酶。(4)通过^<18>O_2实验，我们证明肽基甘氨酸α-羟基化是单加氧酶反应。(5)通过定点诱变，我们证明了位置 107、108、172、242 和 244 的五个组氨酸残基对于肽酰甘氨酸羟化酶活性。(6)我们发现肽酰甘氨酸羟化酶裂解酶的最适pH值为5.5，酶活性不需要二价阳离子，但不需要O_2或抗坏血酸。(7)我们证明，在肽酰甘氨酸羟化酶和肽酰氨基乙醇酸裂合酶，从甘氨酸延伸的前体成功生产出成熟的α-酰胺化肽(8)我们在胰岛素启动子的控制下制备了携带人VIP/PHM-27基因的转基因小鼠，发现转基因胰腺β细胞产生的VIP和PHM-27有效地增强了葡萄糖-胰岛素诱导分泌，表明来自转基因的 VIP 和 PHM-27 在β细胞较少。