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

许多肽激素和神经递质具有c端α -酰胺结构，这些α -酰胺肽是由它们的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 -羟甘氨酸作为中间体进行两步反应，这两步反应由两种不同的酶（肽基α -甘氨酸羟化酶和肽基氨基乙酸水解酶）催化。本课题旨在通过基因工程的方法制备两种肽类α -酰胺化酶，并在体外重建肽类α -酰胺化体系。在本项目中，(1)通过对大鼠“肽酰甘氨酸α -酰胺化酶”cDNA的功能表达，我们发现肽酰甘氨酸羟化酶和肽酰甘氨酸水解酶是由一个mRNA合成的。(2)我们发现肽酰甘氨酸羟化酶和肽酰氨基乙酸水解酶分别编码在mRNA的5'-一半和3'-一半，分别是…(3)构建了携带5′-一半或3′-一半cDNA的表达载体，引导肽酰甘氨酸羟化酶或肽酰氨基甘油酯裂解酶分泌到培养基中，并将表达载体转染到COS-7细胞中产生这两种酶。(4)通过^<18>O_2的实验，证明了肽基甘氨酸α -羟基化反应是单加氧酶反应。(5)通过定点诱变，我们证实了位于107、108、172、242和244位的5个组氨酸残基对肽基甘氨酸羟化酶的活性至关重要。(6)我们发现肽酰氨基乙酸水解酶的最佳pH值为5.5，酶活性需要二价阳离子，而不需要O_2和抗坏血酸。(7)我们证明，在肽酰甘氨酸羟化酶和肽酰氨基甘油酯裂解酶的联合作用下，在体外成功地从甘氨酸延伸前体生成成熟的α -酰胺化肽。(8)在胰岛素启动子控制下，我们培育了携带人Vosoactive Intestinal Peptide (VIP)/PHM-27基因的转基因小鼠，发现由转基因胰腺β细胞产生的VIP和PHM-27能有效地增强葡萄糖诱导的胰岛素分泌，说明转基因胰腺β细胞中的VIP和PHM-27能有效地被肽酰甘氨酸羟化酶和肽酰氨基甘油酯裂解酶α -修饰。少

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