Abstract：
Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the highly conserved amino acid sequences among stefins A from different vertebrates, MdCSTA presents a Methionine-2 residue at the N-terminal region and the second binding loop (pos 73-79) that differs from human stefin A (HsCSTA) and might be related to the lower inhibition constant (Ki) value presented by this inhibitor in comparison to human stefin A inhibition to cathepsin B. Therefore, to investigate the importance of these variable regions in cathepsin B inhibition, recombinant stefins A MdCSTA and HsCSTA containing mutations at the second amino acid residue and second binding loop were expressed and evaluated in kinetic assays. Enzymatic inhibition assays with cathepsin B revealed that switching the amino acid residues at position 2 and second binding loop region between bat and human CSTAs improved the HsCSTA\'s and reduced MdCSTA\'s inhibitory activity. Additionally, molecular docking analysis estimated lower energy values for the complex between MdCSTA-cathepsin B, in comparison to human CSTA-cathepsin B, while the mutants presented intermediate values, suggesting that other regions might contribute to the higher inhibitory activity against cathepsin B by MdCSTA. In conclusion, MdCSTA, the first bat\'s stefin A-like inhibitor to be functionally characterized, presented a higher inhibitory activity against cathepsin B in comparison to the human inhibitor, which is partially related to the glutamine-rich second binding loop and Met-2. Further structural analysis should be performed to elucidate potential inhibitor effects on cysteine proteinases.
摘要：
Myotisdavidii胱抑素A（MdCSTA），中国本土蝙蝠M.davidii的StefinA样，表达为重组蛋白，功能上表征为半胱氨酸蛋白酶木瓜蛋白酶的强抑制剂，人组织蛋白酶L和B以及蜱类组织蛋白酶L样BmCL1。尽管来自不同脊椎动物的甜心A的氨基酸序列高度保守，MdCSTA在N末端区域和第二结合环（pos73-79）上存在甲硫氨酸-2残基，这与人StefinA（HsCSTA）不同，并且可能与该抑制剂呈现的较低抑制常数（Ki）值有关，与人StefinA对组织蛋白酶B的抑制相比，因此，为了研究这些可变区在组织蛋白酶B抑制中的重要性，重组StefinsA，MdCSTA和HsCSTA，在第二氨基酸残基和第二结合环处包含突变的表达和在动力学测定中进行评估。用组织蛋白酶B进行的酶抑制试验表明，在蝙蝠和人CSTA之间的第2位和第二结合环区的氨基酸残基的转换提高了HsCSTA的抑制活性，并降低了MdCSTA的抑制活性。此外,分子对接分析估计MdCSTA-组织蛋白酶B之间的复合物的能量值较低，与人CSTA-组织蛋白酶B相比，虽然突变体呈现中间值，表明其他区域可能有助于MdCSTA对组织蛋白酶B的更高抑制活性。总之,MdCSTA,第一个具有功能特征的蝙蝠stefinA样抑制剂，与人抑制剂相比，对组织蛋白酶B具有更高的抑制活性，这与富含谷氨酰胺的第二结合环和Met-2部分相关。应进行进一步的结构分析以阐明对半胱氨酸蛋白酶的潜在抑制剂作用。
