Biochemical analysis of type II Metacaspase ( mcII - Pa )

Abstract: Programmed Cell Death (PCD) is a physiological process of cell death to remove unwanted cells and damaged cells. This physiological process is a cascade of biochemical reactions triggered and completed by Caspase, a cysteine protease, in animals. After discovering PCD in plants scientists tried to find caspase or similar enzyme in plants. However, caspase is not present in plant but a distant homologue metacaspase were found in the end of last decade. Conversely the exact function of metacaspase is not known. The main objective of this study was to assess the biochemistry of type II metacaspase from Norway spruce (mcII-Pa). mcII-Pa is the member of cysteine protease family and contains Cys/His catalytic diad which is conserved in all metacaspases and caspase. In this study we have overexpressed metacaspase in E. coli and purified by affinity, ion exchange and gel filtration chromatography. Having several rare codons, mcII-Pa can only express in cells which are engineered with additional tRNAs for translation of rare codons. Reducing agents are required during purification and storage to minimize the chance of formation of disulphide bonds. The activation pH is in the range (7.4) of physiological condition. We found that mcII-Pa is stabilized by Ca ions. Activation of mcII-Pa optimally requires 50mM Ca++. In contrast, Zn++ inhibited mcII-Pa as it was also shown for caspase. Mg++ had no effect on the activation of mcII-Pa. We found that mutation of Lys269 to Gly inactivate mcII-Pa, suggesting that it may play role in the mcII-Pa activation. mcII-Pa was most active with 300mM NaCl which is in a range of physiological range. We found that mcII-Pa is active at its monomer form, whereas the mcII-Pa dimer is not active. Stabbilization experiments of mcII-Pa by CD showed that Ca++ can stabilized the protein. Understanding the mechanism of the mcII-Pa activation and proteolytic activity requires high resolution structural studies. Our finding that Ca++ inhibits aggregation of mcII-Pa will help in preparing suitable sample of the protein for crystallization and subsequent X-ray crystallography analysis.