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Sequence and expression of the cDNA for MEP (major excreted protein), a transformation-regulated secreted cathepsin
B R Troen1, S Gal, M M Gottesman
1Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892.
Abstract:
The major excreted protein (MEP) of malignantly transformed mouse fibroblasts is a secreted thiol proteinase. Sequencing of the MEP cDNA shows the coding region for the protein to be identical with the sequence for a mouse cysteine proteinase isolated from macrophages, but the MEP cDNA is polyadenylated at a different site in the 3' non-coding region. Strong homology of MEP with human cathepsin L suggests that MEP is the mouse analogue of cathepsin L. Amino acid sequencing of the N-terminus of the secreted form of MEP indicates that, during secretion, the polypeptide is cleaved between amino acids 17 and 18. We have placed the MEP cDNA in a eukaryotic expression vector and demonstrated the production of the 39 kDa polypeptide form of mouse MEP in monkey CV-1 cells.
Insights
The major excreted protein (MEP) is a secreted thiol proteinase from mouse fibroblasts, analogous to human cathepsin L. This study confirms its identity and demonstrates its production in cultured cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The major excreted protein (MEP) is a secreted thiol proteinase found in malignantly transformed mouse fibroblasts.
- MEP shares sequence identity with a mouse cysteine proteinase from macrophages.
Purpose of the Study:
- To characterize the major excreted protein (MEP) from mouse fibroblasts.
- To investigate the relationship between MEP and human cathepsin L.
- To demonstrate the production of MEP in a heterologous expression system.
Main Methods:
- cDNA sequencing of MEP.
- Amino acid sequencing of the N-terminus of secreted MEP.
- Eukaryotic expression of MEP cDNA in monkey CV-1 cells.
Main Results:
- MEP cDNA sequence is identical to a mouse cysteine proteinase but differs in 3' non-coding region polyadenylation.
- MEP shows strong homology to human cathepsin L, suggesting it is the mouse analogue.
- Secreted MEP is cleaved between amino acids 17 and 18.
- Recombinant mouse MEP (39 kDa form) was produced in CV-1 cells.
Conclusions:
- MEP is the mouse homologue of human cathepsin L.
- MEP undergoes specific cleavage during secretion.
- Heterologous expression of MEP is feasible.