Studies on cathepsin B of Eimeria tenella and pyroglutamyl peptidase of Leishmania major.
PhD thesis, University of Glasgow.
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A sequence encoding a cathepsin B-like cysteine peptidase of the clan CA, family C1 was identified in the genome database of E. tenella. The sequence corresponded to a single copy gene, and did not carry any introns.
The E. tenella enzyme, sharing 42% identity with the Toxoplasma gondii toxopain-1, which is reportedly involved in the invasion of host cells, was expressed as a soluble inactive zymogen in E. coli. Recombinant cathepsin B of E. tenella was functionally expressed as a mature enzyme in the Pichia pastoris inducible system as a glycosylation protein, but the glycosylations did not apparently affect the enzyme’s activity. The biochemical characteristics of the recombinant enzyme were consistent with what has been reported in the literature for cathepsin Bs. The cathepsin B of E. tenella was detected in oocyst and sporozoite extracts, and, more specifically, in microneme preparations. In the sexual stages, the enzyme localised to mature macrogametes in discrete granules, the size and location of which suggest that they are wall-forming bodies, organelles involved in the oocyst wall formation in Eimeria. These data suggest that the cathepsin B may play roles in both cell invasion by sporozoites and the formation of the protective wall of the oocyst.
A single copy gene encoding a PPI was identified in the genome database of L. major. Active recombinant enzyme was successfully produced in E. coli and its biochemical properties coincided with those of mammalian PPIs. The active site catalytic triad E101, C210, and H234 (L. major PPI numbering) was confirmed by mutagenesis. The PPI activity was detected in L. major promastigotes, and the enzyme localised to the parasite cytoplasm. PPI knockout mutants were generated, and knock-out of the PPI activity did not seem to induce a phenotype in L. major. The parasites retained the properties in vivo and in vitro of L. major wild type cells. The over-expression of the active PPI in L. major promastigotes seemed to impair metacyclogenesis and in vivo infectivity, while the over-expression of the C210A mutant did not have any detrimental effect.
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