Mutation of Schizophyllum commune Type Ia Metacaspase for Cleavage Site Identification

Metacaspases are cysteine proteases that are present in all non-metazoans, evenly distributed through the 3 domains of life. Metacaspases contain a conserved catalytic domain which houses the cysteine-histidine dyad involved in the hydrolysis of peptide bonds found after lysine and arginine residues that are recognized as cleavage locations. Many metacaspases require calcium for activation and undergo autoproteolysis in the presence of calcium ions. Three distinct types of metacaspases have been discovered, and our focus is on the Type Ia metacaspase from the fungus Schizophyllum commune (ScMCA-Ia). The study of metacaspases is challenging because the introduction of calcium ions induces autoproteolysis, leaving small fragments of the original protein structure. It is essential to identify specific lysine and/or arginine residues of ScMCA-Ia as potential cleavage sites and examine their role in the autoproteolytic process. Mutation of identified residues from lysine/arginine to alanine was hypothesized to prevent autoproteolytic activity at that site. Mutation of the cysteine-histidine dyad is performed in conjunction with specific lysine/arginine mutations to decrease autoproteolytic activity during protein purification. Point mutations were created via site-directed mutagenesis. Mutant DNA was transformed into chemically competent E. coli cells for use in nickel affinity chromatography protein purification. Mutant protein was run in an SDS-PAGE assay to observe the difference in proteolytic cleavage of mutant proteins in comparison to the wild-type. Activity assays and enzyme kinetic assays were performed on the wild type and mutant protein to determine how mutation affects activity and substrate binding relative to the wild-type. Activity assay data concludes mutation of the cysteine-histidine dyad reduces activity of ScMCA-Ia ten-fold relative to the wild-type. Enzyme kinetics data concludes mutation of the cysteine-histidine dyad reduces catalysis but does not affect substrate binding. Gel data concludes that mutation of potential cleavage sites to alanine does not affect the autoproteolytic process of ScMCA-Ia. Future research will focus on alternative methods to prevent cleavage of ScMCA-Ia.