NATA Koji Tohoku University, School of Medicine, Department of Biochemistry, Research Asso, 医学部, 助手 (90202233)
YONEKURA Hideto Tohoku University, School of Medicine, Department of Biochemistry, Lecturer, 医学部, 講師 (80240373)
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
(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