CYSTATIN-F IS A GLYCOSYLATED HUMAN LOW-MOLECULAR-WEIGHT CYSTEINE PROTEINASE-INHIBITOR

A previously undescribed human member of the cystatin superfamily call ed cystatin F has been identified by expressed sequence tag sequencing in human cDNA libraries. A full-length cDNA clone was obtained from a library made from mRNA of CD34-depleted cord blood cells. The sequenc e of the cDNA contained an open reading frame encoding a putative 19-r esidue signal peptide and a mature protein of 126 amino acids with two disulfide bridges and enzyme-binding motifs homologous to those of Fa mily 2 cystatins. Unlike other human cystatins, cystatin F has 2 addit ional Cys residues, indicating the presence of an extra disulfide brid ge stabilizing the N-terminal region of the molecule. Recombinant cyst atin F was produced in a baculovirus expression system and characteriz ed. The mature recombinant protein processed by insect cells had an N- terminal segment 7 residues longer than that of cystatin C and display ed reversible inhibition of papain and cathepsin L (K-i = 1.1 and 0.31 nM, respectively), but not cathepsin B. Like cystatin E/M, cystatin F is a glycoprotein, carrying two N-linked carbohydrate chains at posit ions 36 and 88. An immunoassay for quantification of cystatin F showed that blood contains low levels of the inhibitor (0.9 ng/ml). Six B ce ll lines in culture secreted barely detectable amounts of cystatin F, but several T cell lines and especially one myeloid cell line secreted significant amounts of the inhibitor. Northern blot analysis revealed that the cystatin F gene is primarily expressed in peripheral blood c ells and spleen. Tissue expression clearly different fi-om that of the ubiquitous inhibitor, cystatin C, was also indicated by a high incide nce of cystatin F clones in cDNA libraries from dendritic and T cells, but no clones identified by expressed sequence tag sequencing in seve ral B cell libraries and in >600 libraries from other human tissues an d cells.