HUMAN CATHEPSIN-F - MOLECULAR-CLONING, FUNCTIONAL EXPRESSION, TISSUE LOCALIZATION, AND ENZYMATIC CHARACTERIZATION

A cDNA for a novel human papain-like cysteine protease, designated cat hepsin F, has been cloned from a lambda gt10-skeletal muscle cDNA libr ary. The nucleotide sequence encoded a polypeptide of 302 amino acids composed of an 88-residue propeptide and a 214-residue mature protein. Protein sequence comparisons revealed 58% homology with cathepsin W; about 42-43% with cathepsins L, K S, H and O; and 38% with cathepsin B . Sequence comparisons of the propeptides indicated that cathepsin F a nd cathepsin W may form a new cathepsin subgroup. Northern blot analys is showed high expression levels in heart, skeletal muscle, brain, tes tis, and ovary; moderate levels in prostate, placenta, Liver, and colo n; and no detectable expression in peripheral leukocytes and thymus. T he precursor polypeptide of human recombinant cathepsin F, produced in Pichia pastoris, was processed to its active mature form autocatalyti cally or by incubation with pepsin. Mature cathepsin F was highly acti ve with comparable specific activities toward synthetic substrates as reported for cathepsin L. The protease had a broad pH optimum between 5.2 and 6.8. Similar to cathepsin L, its pH stability at cytosolic pH (7.2) was short, with a half-Life of approximately 2 min. This may sug gest a function in an acidic cellular compartment. Transient expressio n of T7-tagged cathepsin F in COS-7 cells revealed a vesicular distrib ution of the gene product in the juxtanuclear region of the cells. How ever, contrary to all known cathepsins, the open reading frame of the cathepsin F cDNA did not encode a signal sequence, thus suggesting tha t the protease is targeted to the lysosomal compartment via an N-termi nal signal peptide-independent lysosomal targeting pathway.