Legumains or vacuolar processing enzymes (VPE) are cysteine proteases that are present in all organisms. They are also called asparaginyl endopeptidases, because they cleave peptide bonds at the C-terminal side of asparagine or aspartic acid residues. In plant cells, they are located in the vacuole, and in animal cells, they are located in the lysosome. Their main function is the processing of storage proteins, hydrolytic enzymes and stress proteins. However, they also have an important role in plant programmed cell death, because they are involved in the vacuolar collapse and the release of hydrolytic enzymes into the cytosol. In this respect, their function equals that of caspases, which cause programmed cell death in animal cells.
There are four VPE isoforms in plants: αVPE, βVPE, γVPE in δVPE. The roles of these isoforms are already known. On the other hand, algal VPEs are poorly understood. There is one gene coding for a vacuolar processing enzyme in the genome of the model green alga Chlamydomonas reinhardtii, but its function remains unknown.
By performing pairwise alignments and constructing phylogenetic trees we determined the evolutionary relationship of VPE from Chlamydomonas reinhardtii (CrVPE) with VPE isoforms from higher plants. Based on these we concluded that CrVPE is most closely related to the βVPE isoform. We compared the structure model of CrVPE to the known structure of γVPE from Arabidopsis thaliana, up to now the only determined structure of a plant VPE. By comparing known active sites and substrate-binding pockets of VPEs from higher plants we determined the residues that form the substrate-binding pocket and active site in CrVPE. We examined the VPE gene expression patterns in Chlamydomonas reinhardtii under different conditions and found its upregulation during stress conditions.
We also carried out the first steps of cloning CrVPE in the bacterium Escherichia coli. Due to the presence of an unwanted insertion in the coding sequence, we removed it by several consecutive PCR reactions.