Investigation of S. commune Type-Ia Metacaspase Proteolysis Using LC-MS and Mutagenesis

Metacaspases are cysteine-dependent proteases found in fungi, plants, and protozoa. They play a significant role in regulating cell proliferation, stress responses, and apoptosis, also known as programmed cell death. Type Ia metacaspases from Schizophyllum commune (ScMCA-Ia) require calcium for activation and undergo autoproteolysis, where the metacaspase cleaves itself into smaller fragments. They cleave most efficiently after arginine (Arg) and lysine (Lys) residues. To investigate the autoproteolytic cleavage sites, liquid chromatography-mass spectroscopy (LC-MS) was used to characterize peptide fragments based on their mass-to-charge ratios. Samples containing specific cleaved peptide bands were extracted from SDS-PAGE and digested with chymotrypsin to yield smaller peptides within the optimal range for ESI-MS analysis. The MS data of the main peaks were analyzed using Expasy's FindPept tool to identify specific fragments based on the known protein sequence. Furthermore, two mutant variants were generated by site-directed mutagenesis to assess the functional importance of catalytic residues. The active site of ScMCA-Ia is made up of histidine 264 and cysteine 320, but cysteine 199 has been identified as a potential alternate catalytic cysteine. The first mutant contained the cysteine at position 199 substituted with alanine, while the second was a triple mutant containing the C199A substitution and mutations in the double-active-site residues. These mutant variants were used in comparative kinetic analysis with wild-type to determine the impact that the mutations had on the catalytic activity. Using LC-MS on mutants is another way to further explore cleavage sites by identifying whether a mutation eliminates a specific cleavage peptide or alters its cleavage specificity. These findings help define the biochemical features that prompt ScMCA-Ia autoproteolysis by mapping the cleavage patterns and better understanding the role of autoproteolysis in regulating enzymatic activity.