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 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.
Olsson
S L
SL
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala. sigrid-lisa.olsson@vmk.slu.se
Ek
B
B
Wilm
M
M
Broberg
S
S
Rask
L
L
Björk
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D003001:Cloning, Molecular; D055316:Cystatin C; D015891:Cystatins / Q000134:cerebrospinal fluid / Q000737:chemistry* / Q000235:genetics; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
C525030:CST3 protein, human; D055316:Cystatin C; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D011498:Protein Precursors; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase