Investigation of Autoproteolytic Cleavage in Schizophyllum commune Type Ia Metacaspases

Metacaspase are cysteine-dependent proteases that are found in fungi, plants, and protozoa. They play a critical role in programmed cell death and innate immune response in these organisms. Metacaspases cleave specifically after arginine or lysine residues during proteolytic events, and the bond cleavage is catalyzed by the highly conserved cysteine-histidine dyad in the metacaspase active site.  In order to exhibit enzymatic activity, metacaspases typically require the presence of calcium ions. The activation of metacaspases by calcium is also associated with autoproteolytic activity in the enzyme, although the functional role of autoproteolysis is not fully understood. This is true of the metacaspase studied in our lab, Schizophyllum commune Metacaspase Type Ia (ScMCIa).  The structure of ScMC1 has not been determined due to its high autoproteolytic activity, which limits our ability to understand the functional nature of the enzyme.  It is predicted that identification and subsequent mutation of the residues involved in autoproteolysis will halt the autoproteolytic activity of ScMC1.  This exploration is two fold.  First, liquid chromatography-mass spectrometry(LC-MS) can be utilized to analyze a sample of metacaspase protein digested by chymotrypsin, which will effectively separate the peptide fragments and determine the mass of these peptides. The FindPept software can then be used to determine the specific identity of the protein fragments within the ScMC1 sequence in order to identify the autoproteolytic cleavage sites in ScMC1.  Additionally, the cysteine at position 199 has been identified as a possible alternate catalytic cysteine specifically implicated in autoproteolysis.  We used site directed mutagenesis to generate point mutations at the three potential catalytic sites, as well as at the suspected cleavage sites to abolish autoproteolytic activity.  Using the mutated DNA we  expressed metacaspase proteins with these specific mutations, and determined their autoproteolytic activity using SDS polyacrylamide gel electrophoresis.