Hall A, Ekiel I, Mason R W, Kasprzykowski F, Grubb A, Abrahamson M
Department of Clinical Chemistry, Institute of Laboratory Medicine, University of Lund, University Hospital, Sweden.
Biochemistry. 1998 Mar 24;37(12):4071-9. doi: 10.1021/bi971197j.
Human cystatins C and D share almost identical primary structures of two out of the three segments proposed to be of importance for enzyme interactions but have markedly different profiles for inhibition of the target cysteine peptidases, cathepsins B, H, L, and S. To investigate if the N-terminal binding regions of the inhibitors are responsible for the different inhibition profiles, and thereby confer biological selectivity, two hybrid cystatins were produced in Escherichia coli expression systems. In one hybrid, the N-terminal segment of cystatin C was placed on the framework of cystatin D, and the second was engineered with the N-terminal segment of cystatin D on the cystatin C scaffold. Truncated cystatin C and D variants, devoid of their N-terminal segments, were obtained by incubation with glycyl endopeptidase and isolated, in a second approach to assess the importance of the N-terminal binding regions for cystatin function and specificity. The affinities of the four cystatin variants for cathepsins B, H, L, and S were measured. By comparison with corresponding results for wild-type cystatins C and D, it was concluded (1) that both the N-terminal and framework part of the molecules significantly contribute to the observed differences in inhibitory activities of cystatins C and D and (2) that the N-terminal segment of cystatin C increases the inhibitory activity of cystatin D against cathepsin S and cathepsin L but results in decreased activity against cathepsin H. These differences in specificity were explained by the residues interacting with the S2 subsite of peptidases (Val- and Ala-10 in cystatin C and D, respectively). Also, removal of the N-terminal segment results in total loss of enzyme affinity for cystatin D but not for cystatin C. Therefore, structural differences in the framework parts, as well as in the N-terminal segments, are critical for both inhibitory specificity and potency. Homology modeling was used to identify residues likely responsible for the generally reduced inhibitory potency of cystatin D.
人胱抑素C和D在被认为对酶相互作用重要的三个片段中的两个片段上具有几乎相同的一级结构，但对靶标半胱氨酸蛋白酶组织蛋白酶B、H、L和S的抑制谱却明显不同。为了研究抑制剂的N端结合区域是否导致了不同的抑制谱，从而赋予生物学选择性，在大肠杆菌表达系统中产生了两种杂合胱抑素。在一种杂合体中，胱抑素C的N端片段被置于胱抑素D的框架上，另一种则是将胱抑素D的N端片段构建在胱抑素C的支架上。通过与甘氨酰内肽酶孵育获得缺失N端片段的截短型胱抑素C和D变体，并进行分离，这是评估N端结合区域对胱抑素功能和特异性重要性的第二种方法。测量了这四种胱抑素变体对组织蛋白酶B、H、L和S的亲和力。通过与野生型胱抑素C和D的相应结果比较，得出以下结论：(1)分子的N端和框架部分均对胱抑素C和D观察到的抑制活性差异有显著贡献；(2)胱抑素C的N端片段增加了胱抑素D对组织蛋白酶S和组织蛋白酶L的抑制活性，但导致对组织蛋白酶H的活性降低。这些特异性差异是由与肽酶S2亚位点相互作用的残基（胱抑素C和D中分别为Val-10和Ala-10）所解释的。此外，去除N端片段会导致酶对胱抑素D的亲和力完全丧失，但对胱抑素C则不然。因此，框架部分以及N端片段的结构差异对于抑制特异性和效力都至关重要。同源建模用于鉴定可能导致胱抑素D总体抑制效力降低的残基。
