MOLECULAR-CLONING AND N-TERMINAL ANALYSIS OF BOVINE CYSTATIN-C IDENTIFICATION OF A FULL-LENGTH N-TERMINAL REGION

The N-terminal region of human cystatin C has been shown to be of cruc ial importance for the interaction of the inhibitor with cysteine prot einases. However, several studies have been unable to identify the cor responding region in bovine cystatin C, indicating that the binding of proteinases to the bovine inhibitor may not be dependent on this regi on. With the aim to resolve this apparent discrepancy and to elucidate the relation of bovine cystatin C to other cystatins, we have isolate d a cDNA clone encoding bovine precystatin C. The sequence of this cDN A was similar to that of the human inhibitor and showed a putative sig nal peptidase cleavage site consistent with the N-terminal regions of the bovine and human inhibitors being of comparable size. This suggest ion was verified by determination of the relative molecular mass of th e mature bovine inhibitor isolated from cerebrospinal fluid under cond itions minimising proteolysis. The N-terminal of the purified inhibito r 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 cy statin C has 118 residues, in contrast with 110-112 residues reported previously, and has an N-terminal region analogous to that of human cy statin C. This region presumably is of similar importance for tight bi nding of target proteinases as in the human inhibitor. (C) 1997 Elsevi er Science B.V.