Breast and Colorectal Carcinomas Cathepsin L 2 , a Novel Human Cysteine Proteinase Produced by Updated

We have identified and cloned a new member of the papain family of cysteine proteinases from a human brain eDNA library. The isolated cDNA codes for a polypeptide of 334 amino acids that exhibits all of the structural features characteristic of cysteine proteinases, including the active site cysteine residue essential for peptide hydrolysis. PairWise corn parisons of this amino acid sequence with the remaining human cysteine proteinases identified to date showed a high percentage of identity (78%) with cathepsin L; the percentage of identity with all other members of the family was much lower (<40%). On the basis of these structural charac teristics, we have tentatively called this novel protein cathepsin L2. The eDNA encoding the mature cathepsin L2 was expressed In Escherichia coli, and after purification, the recombinant protein was able to degrade the synthetic peptide benzyloxycarbonyl-L-phenylalanyl-L-arginine-7amido-4-rnethylcoumarln, which is commonly used as a substrate for cysteine proteinases. Cathepsin L2 proteolytic activity on this substrate wasabolishedby trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cysteine proteinases, thus providing additional evidence that the isolated eDNA encodes a functional cysteine proteinase. Northern blot analysis of polyadenylated RNAs isolated from a variety of human tissues demonstrated that cathepsin L2 is predominantly expressed in the thymus and testis. This finding is in marked contrast with the wide tissue distribution of most cysteine protelnases characterized to date, including cathepsin L, and suggests that cathepsin L2 may play a specialized role in the thymus and testis. Expression analysis of cathepsin L2 in human tumors revealed a widespread expression in colorectal and breast carci nomas but not in normal colon or mammary gland or in peritumoral tissues. Cathepsin L2 was also expressed by colorectal and breast cancer cell lines as well as by some tumors of diverse origin, including ovarian and renal carcinomas. These results open the possibility that this novel enzyme may be involved in tumor processes, as already reported for other cysteine proteinases, including cathepsin L.