| Co-Investigator(Kenkyū-buntansha) |
IUCHI Ichiro Sophia Univ., Dep.Sci.and Tech., Prof., 理工学部, 教授 (10011694)
TANOKURA Masaru The Univ.of Tokyo, Dep.of Applied Biol.Chem.Graduate School of Agr., Prof., 大学院・農学生命科学研究科, 教授 (60136786)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2003: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
Egg envelope (chorion) digestion occurring at the medaka hatching is brought about by the combined action of two distinct metalloproteases, HCE and LCE. HCE partially digests the chorion by its limited proteolytic action, resulting in the formation of swollen chorion. LCE is not able to digest intact chorion, but it efficiently solubilizes the swollen chorion. We have elucidated the three-dimensional structure of HCE and LCE. In addition, to analyze the molecular mode of action of HCE, we produced the recombinant HCE (RecHCE) using an E. coli expression system. RecHCE23 had high choriolytic activity as well as proteolytic (caseinolytic) activity. RecHCE21, a recombinant of another isoform of HCEs (95%-identical to HCE23), had the same caseinolytic activity as that of RecHCE23. However, it exhibited the remarkably lower choriolytic activity than RecHCE23. Replacement of two amino acid residues (29E/Q and 32G/R) in the RecHCE21 molecule restored its choriolytic activity at the same level as that of RecHCE23. Substitutions of the two residues didn't affect the substrate specificity of RecHCE21 that was estimated with MCA-substrates. Referring to the three-dimensional structure of HCE, a region containing the two amino acid residues was located at a surface loop structure, and it was far from the catalytic cleft. The results suggest that, independent of catalytic site, another functional region is present on the HCE molecule, and the region plays a significant role in HCE's recognizing a tough and solid substrate, the chorion. The region may function as "chorion binding site" as we have previously proposed based on immunological analyses. We also determined the cleaving sites of HCE and LCE on the subunit proteins of egg envelope. HCE cleaved mainly Pro-X-Y repeat domain in ZI1,2 protein and released small peptides from the chorion, whereas LCE cut the center of ZP-domain of ZI1,2 and solubilized swollen egg envelope completely.
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