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

许多多肽激素和神经递质都有C-末端的α-酰胺结构，这些α-酰胺化的多肽是由它们的甘氨酸延伸的前体酶促合成的。C-末端的α-酰胺化反应通过两步反应进行，以肽基α-羟基甘氨酸为中间体，这些步骤由两种不同的酶(肽基甘氨酸羟基酶和肽氨基乙醇酸裂解酶)催化。本课题的目的是利用基因工程技术生产与多肽α-酰胺化有关的两种酶，并在体外重建多肽-α-酰胺化系统。在本课题中，(1)通过对大鼠肽甘氨酸α-酰胺化酶基因的功能表达，我们发现由单个基因合成的肽基甘氨酸羟基酶和肽氨基乙醇酸裂解酶。(2)我们发现，肽基甘氨酸羟基酶和肽氨酸乙醇酸裂解酶编码在…的5‘-半边和3’-半边。(3)构建了携带cDNA5‘-半段或3’-半段的表达载体，引导肽甘氨酸羟基酶或肽酰甘氨酸裂解酶分泌到培养基中，并将表达载体导入COS-7细胞中表达。(5)通过定点突变，我们证明了位于107、108、172、242和244位的5个组氨酸残基是该酶活性所必需的。(6)我们发现，该酶的最适pH为5.5，需要二价阳离子，但不需要O_2或抗坏血酸。(7)我们证明了在肽甘氨酸羟基酶和抗坏血酸裂解酶的共同存在下，该酶的活性是最适的。(8)在胰岛素启动子的控制下，我们建立了携带人血管活性肠肽(VIP)/PHM-27基因的转基因小鼠，发现转基因胰岛细胞产生的VIP和PHM-27有效地促进了葡萄糖诱导的胰岛素分泌，表明转基因的VIP和PHM-27可以被胰岛细胞中的肽甘氨酸羟基酶和肽酰甘氨酸裂解酶有效地α-酰胺化。较少

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