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

Sapna Mehtani, Qiuming Gong, Jeffrey Panella, Subbalakshmi Subbiah, Dennis M. Peffley, Allen Frankfater

Research output: Contribution to journalArticlepeer-review

63 Scopus citations

Abstract

Cathepsin B is a lysosomal cysteine protease whose increased expression is believed to be linked to the malignant progression of tumors. Alternative 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-related transcripts that lack exon 3 which encodes the N-terminal signal peptide and 34 of the 62-amino acid inhibitory propeptide. In this study 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 cathepsin B coding sequence and the part of the 3'- untranslated region that is common to all previously characterized cathepsin B mRNAs in humans. Its in vitro translation product can be folded to produce enzymatic activity against the cathepsin B-specific substrate, N(α)- benzyloxycarbonyl-L-Arg-L-Arg-4-methylcoumaryl-7-amide. Endogenous CB(-2,3) from the metastatic human melanoma cell line, A375M, co-sediments with polysomes, indicating that it engages the eukaryotic translation machinery in these cells. Epitope-tagged forms of the truncated cathepsin B from CB(-2,3) are produced in amounts comparable to the normal protein after transient transfection into COS cells. Immunofluorescence microscopy and subcellular fractionation show this novel tumor form of cathepsin B to be associated with nuclei and other membranous organelles, where 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. These results indicate that the message missing exons 2 and 3 is likely to be translated into a catalytically active enzyme, and that alternative splicing (exon skipping) could contribute to the aberrant intracellular trafficking of cathepsin B that is observed in some human cancers.

Original languageEnglish (US)
Pages (from-to)13236-13244
Number of pages9
JournalJournal of Biological Chemistry
Volume273
Issue number21
DOIs
StatePublished - May 22 1998
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of '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'. Together they form a unique fingerprint.

Cite this