IN-VIVO EXPRESSION OF AN ALTERNATIVELY SPLICED HUMAN TUMOR MESSAGE THAT ENCODES A TRUNCATED FORM OF CATHEPSIN-B - SUBCELLULAR-DISTRIBUTION OF THE TRUNCATED ENZYME IN COS CELLS

Cathepsin B is a lysosomal cysteine protease whose increased expressio n is believed to be linked to the malignant progression of tumors. Alt ernative splicing and the use of alternative transcription initiation sites in humans produce cathepsin B mRNAs that differ in their 5'- and 3'-untranslated ends. Some human tumors also contain cathepsin B-rela ted transcripts that lack exon 3 which encodes the N-terminal signal p eptide and 34 of the 62-amino acid inhibitory propeptide. In this stud y we show that one such transcript, CB(-2,3), which is missing exons 2 and 3, is likely to be a functional message in tumors. Thus, CB(-2,3) was found to be otherwise complete, containing the remainder of the c athepsin B coding sequence and the part of the 3'-untranslated region that is common to all previously characterized cathepsin B mRNAs in hu mans. Its in vitro translation product can be folded to produce enzyma tic activity against the cathepsin B-specific substrate, a-benzyloxyca rbonyl-L-Arg-L-Arg-4-methycoumaryl-7- amide, Endogenous CB(-2,3) from the metastatic human melanoma cell line, A375M, co-sediments with poly somes, indicating that it engages the eukaryotic translation machinery in these cells. Epitope-tagged forms of the truncated cathepsin B fro m CB(-2,3) are produced in amounts comparable to the normal protein af ter transient transfection into COS cells. Immunofluorescence microsco py and subcellular fractionation show this novel tumor form of catheps in B to be associated with nuclei and other membranous organelles, whe re it is likely to be bound to the cytoplasmic face of the membranes. This subcellular distribution was different from the lysosomal pattern shown by the epitope-tagged, full-length cathepsin B in COS cells. Th ese results indicate that the message missing exons 2 and 3 is likely to be translated into a catalytically active enzyme, and that alternat ive splicing (exon skipping) could contribute to the aberrant intracel lular trafficking of cathepsin B that is observed in some human cancer s.