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

在青鳉孵化时发生的卵被膜（绒毛膜）消化是由两种不同的金属蛋白酶HCE和LCE的联合作用引起的。HCE通过其有限的蛋白水解作用部分破坏绒毛膜，导致绒毛膜肿胀的形成。LCE不能消化完整的绒毛膜，但它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，我们使用E. coli表达系统。RecHCE 23具有高的绒毛膜溶解活性以及蛋白水解（酪蛋白溶解）活性。RecHCE 21是HCEs的另一种同种型的重组体（与HCE 23具有95%的同一性），具有与RecHCE 23相同的酪蛋白溶解活性。然而，它表现出比RecHCE 23显著更低的绒毛膜溶解活性。在RecHCE 21分子中替换两个氨基酸残基（29 E/Q和32 G/R）使其绒毛膜溶解活性恢复到与RecHCE 23相同的水平。这两个残基的取代并不影响RecHCE 21的底物特异性，这是估计与MCA底物。从HCE的三维结构来看，含有两个氨基酸残基的区域位于表面环状结构中，并且远离催化裂。结果表明，独立于催化位点，另一个功能区存在于HCE分子上，该区域在HCE识别坚韧和固体底物-绒毛膜中起着重要作用。该区域可能作为“绒毛膜结合位点”，因为我们以前提出的免疫学分析的基础上。我们还确定了HCE和LCE在卵膜亚基蛋白上的切割位点。HCE主要切割ZI 1，2蛋白的Pro-X-Y重复结构域，从绒毛膜释放小肽，而LCE切割ZI 1，2蛋白的ZP结构域中心，完全溶解肿胀的卵膜。

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.