Subtilisin-like Plant Protease Cleaving at Multiple Acidic Amino Acid Residues

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9722984 Tan-Wilson Protease C1 catalyzes the initial limited proteolysis of the major soy storage globulins during early growth. Its partial cDNA sequence suggests that it belongs to the subtilisin family of serine proteases. This merits further study since only seven of the enzymes in this large family of proteases are from plants. Moreover, sequences at protease C1's cleavage sites show a unique requirement for glutamate or aspartate residues at the P1, Pl', and P4' positions. The cleavage specificity of protease C1 mirrors the specificity of the subtilisin-related Kex2-1ike protein-processing proteases, except that the latter require multiple basic instead of multiple acidic amino acid residues. In this project, the cleavage specificity of protease C1 will be further delineated using protein and synthetic peptide substrates gene expression will be studied by examining different parts of the plant at different developmental stages for activity and by hybridization of oligonucleotide probes to northern blots. The project would also determine whether other legume species that store seed proteins bearing strings of Glu and Asp residues have protease C1-like enzymes to initiate proteolysis during germination. Hybridization of oligonucleotide probes to Southern blots would reveal whether protease Cl-like genes exist in these species. If positive, maturing seeds and seedling cotyledons of these plants would be studied for protease Cl-like activity and hybridization of oligonucleotide probes to northern blots. Information on this proteolytic enzyme would be of interest to research groups attempting to clone commercially important products in legume storage proteins, and would contribute to our understanding of enzyme evolution. Legumes constitute important sources of protein and edible oil. When genetic transformation of these plants becomes easier, the storage proteins are likely targets for manipulation to improve nutritional quality or to produce therapeutic peptides. In the latter case, it will be vital to know the cleavage specificities of the proteolytic enzymes that these proteins would encounter since these enzymes cleave proteins, thus destroying the intended gene product. This project is a study of protease C1, a proteolytic enzyme in soybean that initiates the breakdown of the stored proteins during soybean germination and early seedling growth. The aims of the project are to determine what features the enzyme requires in a cleavage site and where in the plant and when during the plant's growth cycle this enzyme is present in active form so that cloning strategies to avoid its action can be designed. Alternatively, proteolytic enzymes such as soybean protease C1 that is very discriminating with regard to clevage site may be put to good use in processing protein products for biotechnology.