title: Differentiation of Leishmania major is impaired by over-expression of pyroglutamyl peptidase I.
abstract: Pyroglutamyl peptidases I (PPI) are cysteine peptidases of the clan CF, family C15, which hydrolyse N-terminal l-pyroglutamyl residues (l-pGlu). The l-pGlu modification is a post-transcriptional modification that confers relative aminopeptidase resistance and, in some cases, is essential to the modified peptides' biological activity. PPIs have been identified in a variety of organisms, although definitive biological functions have yet to be attributed to them. The L. major PPI was expressed in Escherichia coli as active recombinant enzyme, and shown to have biochemical properties more similar to mammalian than bacterial PPIs. The LmPPI active site catalytic triad of E101, C210, and H234 was confirmed by mutagenesis. PPI activity was detected in L. major promastigotes, and the enzyme localised to the parasite cytosol. No detectable phenotype could be observed for L. major PPI-deficient mutants, which retained infectivity to macrophages in vitro and mice. However, over-expression of the active PPI, but not inactive PPI(C210A), in L. major impaired differentiation from the procyclic promastigote to the infective metacyclic promastigote. Susceptibility to a natural l-pGlu-modified antimicrobial peptide, gomesin, was tested using the different cell lines, which were all equally susceptible. Whilst PPI is widespread through the eukaryotic kingdom, this study now suggests that the enzyme is not essential for normal eukaryotic cell function. However, PPI could be involved in regulating the action of l-pGlu-modified peptides required for differentiation of L. major.
authors.affiliation: Division of Infection & Immunity, Institute of Biomedical and Life Sciences and Wellcome Centre for Molecular Parasitology, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow G12 8TA, Scotland, UK.
authors.forename: Marie
authors.initials: M
authors.lastname: Schaeffer
authors.forename: Antonio
authors.initials: A
authors.lastname: de Miranda
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Mottram
authors.forename: Graham H
authors.initials: GH
authors.lastname: Coombs
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology; D002469:Cell Separation; D018320:Leishmania major / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000254:growth & development*; D008018:Life Cycle Stages; D017737:Macrophages, Peritoneal / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D010802:Phylogeny; D011751:Pyroglutamyl-Peptidase I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D023181:Antimicrobial Cationic Peptides; D011994:Recombinant Proteins; C415987:gomesin; D011751:Pyroglutamyl-Peptidase I