Olsson S L, Ek B, Wilm M, Broberg S, Rask L, Björk I
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala.
Biochim Biophys Acta. 1997 Dec 5;1343(2):203-10. doi: 10.1016/s0167-4838(97)00110-6.
The N-terminal region of human cystatin C has been shown to be of crucial importance for the interaction of the inhibitor with cysteine proteinases. However, several studies have been unable to identify the corresponding region in bovine cystatin C, indicating that the binding of proteinases to the bovine inhibitor may not be dependent on this region. With the aim to resolve this apparent discrepancy and to elucidate the relation of bovine cystatin C to other cystatins, we have isolated a cDNA clone encoding bovine precystatin C. The sequence of this cDNA was similar to that of the human inhibitor and showed a putative signal peptidase cleavage site consistent with the N-terminal regions of the bovine and human inhibitors being of comparable size. This suggestion was verified by determination of the relative molecular mass of the mature bovine inhibitor isolated from cerebrospinal fluid under conditions minimising proteolysis. The N-terminal of the purified inhibitor was blocked, but the sequence of the N-terminal peptide produced by digestion with endopeptidase LysC could be unequivocally determined by tandem mass spectroscopy. Together, these results show that bovine cystatin C has 118 residues, in contrast with 110-112 residues reported previously, and has an N-terminal region analogous to that of human cystatin C. This region presumably is of similar importance for tight binding of target proteinases as in the human inhibitor.
人胱抑素C的N端区域已被证明对于该抑制剂与半胱氨酸蛋白酶的相互作用至关重要。然而，多项研究未能在牛胱抑素C中鉴定出相应区域，这表明蛋白酶与牛抑制剂的结合可能不依赖于该区域。为了解决这一明显差异并阐明牛胱抑素C与其他胱抑素的关系，我们分离出了一个编码牛前胱抑素C的cDNA克隆。该cDNA的序列与人类抑制剂的序列相似，并显示出一个推定的信号肽酶切割位点，这与牛和人类抑制剂的N端区域大小相当一致。通过在最小化蛋白水解的条件下测定从脑脊液中分离出的成熟牛抑制剂的相对分子质量，这一推测得到了验证。纯化抑制剂的N端被封闭，但通过串联质谱法可以明确确定用内肽酶LysC消化产生的N端肽的序列。这些结果共同表明，牛胱抑素C有118个残基，与之前报道的110 - 112个残基不同，并且其N端区域与人类胱抑素C的类似。该区域对于与靶蛋白酶紧密结合的重要性可能与人类抑制剂中的情况相似。
