Three dimensional structures of HCE and LCE and its mechanism of egg envelope digestion

卵包膜（绒毛膜）的消化是由两种不同的金属蛋白酶HCE和LCE共同作用的结果。HCE通过其有限的蛋白水解作用部分消化绒毛膜，导致绒毛膜肿胀形成。LCE不能消化完整的绒毛膜，但它能有效地溶解肿胀的绒毛膜。我们已经阐明了HCE和LCE的三维结构。此外，为了分析HCE的分子作用模式，我们利用大肠杆菌表达系统构建了重组HCE （RecHCE）。RecHCE23具有较高的绒毛膜溶解活性和蛋白溶解（酪蛋白溶解）活性。RecHCE21是hce的另一种异构体的重组（与HCE23相同95%），具有与RecHCE23相同的溶酪蛋白活性。但其溶绒毛膜活性明显低于RecHCE23。在RecHCE21分子中替换两个氨基酸残基（29E/Q和32G/R）使其绒毛膜溶解活性恢复到与RecHCE23相同的水平。这两个残基的替换不影响用mca底物估计的RecHCE21的底物特异性。参考HCE的三维结构，含有两个氨基酸残基的区域位于表面环状结构，远离催化裂口。结果表明，HCE分子上存在独立于催化位点的另一个功能区域，该区域在HCE识别坚硬固体底物绒毛膜中起着重要作用。正如我们先前基于免疫学分析提出的那样，该区域可能具有“绒毛膜结合位点”的功能。我们还测定了HCE和LCE在卵包膜亚基蛋白上的切割位点。HCE主要切割zi1,2蛋白的Pro-X-Y重复结构域，并从绒毛膜上释放小肽，而LCE则切割zi1,2蛋白的zp结构域中心，完全溶解肿胀的卵包膜。

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.