HUMAN CYSTATIN-D - CDNA CLONING, CHARACTERIZATION OF THE ESCHERICHIA-COLI EXPRESSED INHIBITOR, AND IDENTIFICATION OF THE NATIVE PROTEIN IN SALIVA

A cDNA coding for cystatin D, a human member of the cystatin protein f amily, has been cloned after specific amplification of reverse-transcr ibed parotid gland RNA. After replacing the segment encoding the putat ive 20-residue signal peptide with one encoding the Escherichia coli O mpA leader sequence, the cDNA was expressed in E. coli. The isolated r ecombinant protein exhibited K(i) values of 1.2 nM and >1 muM for papa in and cathepsin B, respectively. An antiserum raised against recombin ant cystatin D recognized a protein in human saliva with electrophoret ical mobility identical to that of the recombinant protein. Immunoenzy matic analysis revealed that this cysteine proteinase inhibitor is pre sent in human saliva and tears at concentrations of 3.8 and 0.5 mg/lit er, respectively, while it was not detected in seminal plasma, blood p lasma, milk, or cerebrospinal fluid. Cystatin D purified from human sa liva by immunosorption displayed a heterogeneous N-terminal end, with sequences starting at residues 5, 7, 9, and 11 of the predicted N-term inal portion of the mature protein. On the basis of structural and fun ctional properties, cystatin D represents a novel cysteine proteinase inhibitor possibly playing a protective role against proteinases prese nt in the oral cavity.