Cysteine proteinases are proteolytic enzymes with a relatively broad substrate specificity. They play a major role in intracellular protein degradation and have been widely implicated in prohormone processing, tumor metastasis, joint disease and cellular aging. Despite the vital nature of their catalytic activity, very little is known about the biosynthesis of these enzymes as they each comprise only a very small percentage of the total cellular protein. Recently a complete gene encoding a mouse macrophage Cys proteinase has been isolated and sequenced. This is presently the only complete gene available for a mammalian Cys proteinase. The enzyme encoded by this mouse gene will serve as a model for the study of Cys proteinase biosynthesis. Cell fractionation and immunofluorescence will be used to determine the cellular localization of this enzyme, revealing whether the protein is a secreted Cys proteinase like the plant enzyme papain or a lysosomal Cys proteinase like the mammalian enzyme cathepsin B. Using antibodies raised both against peptides and against a fusion protein generated by molecular biology technology, the biosynthesis of this mouse protein will be studied by in vitro protein synthesis, pulse-chase analysis in tissue culture cells, and amino-terminal sequence analysis. The mouse Cys proteinase will also be used as a model system for the study of the mechanism by which secretory and lysosomal enzymes are distinguished and sorted to their respective cellular locations. Mutant genes will be constructed and expressed in mouse macrophage cell lines recently shown to be missing the mouse Cys proteinase gene. Pulse-chase studies and cell fractionation will be used to assay the effects of each mutation on the cellular localization of the gene product, in the hopes of identifying a specific sorting sequence for this protein. Finally, large fragments of the mouse Cys proteinase gene shown by detailed sequence analysis to be highly conserved across superfamily members will be used as probes to isolate other Cys proteinase genes. New Cys proteinases sorted to cellular compartments other than lysosomes, such as the nucleus and the cytosol, may be isolated by this technique. In addition, the gene encoding the cathepsin B-like enzyme widely reported to be secreted by tumor cells may be cloned.
