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Analysis and expression of Leishmania surface proteinase and glycoproteins

University of British Columbia

Leishmania is a protozoan parasite transmitted by a sandfly vector to humans and other mammalian hosts causing chronic infection. Leishmania exists in two distinct life stages: the promastigote which replicates in the insect gut, and the amastigote which replicates in macrophage cells of the mammalian host. There is considerable evidence that Leishmania cell surface molecules play critical roles in the initiation and establishment of macrophage infection, evasion of non-specific immune defenses, and in stimulation of a cellular and humoral immune response. Relative to promastigotes, the amastigote cell surface is largely uncharacterized. The major surface glycoprotein of Leishmania promastigotes, referred to as GP63, is a zinc metalloproteinase of 63,000 Mr containing a glycosyiphosphatidylinositol (GPI) membrane anchor. GP63 has been predicted to be synthesized as a 602 amino acid precursor protein containing an NH₂-terminal leader sequence of 39 amino acids, a putative regulatory pro region of 61 amino acids, the mature protein of 477 amino acids, and a C00H-terminal GPI attachment signal sequence of 25 amino acids. Recent studies demonstrated that recombinant GP63 (rGP63) expressed by the baculovirus insect cell system was secreted as a glycosylated latent proteinase that required activation for full proteinase activity (Button,L.L. et al. Gene 134:75-81, 1993). The overall objectives of this study were to determine the activation mechanism of recombinant GP63 (rGP63), using both secreted and cell surface expression systems, and to identify novel glycoproteins expressed on the surface of L. mexicana amastigotes. The activation of secreted latent rGP63 (rGP63s) was found to be consistent with the cysteine switch mechanism described for mammalian matrix metalloproteinases. NH₂- terminal sequence analysis revealed that activation of rGP63s with HgCl₂ resulted in progressive autolytic removal of the predicted pro region, ultimately generating the mature NH₂-terminus. This processing included the removal of a conserved Cys residue (Cys-48) and occurred by a cis mechanism since the addition of previously activated rGP63s did not lead to an enhancement of latent rGP63s proteinase activation. rGP63s expressed in COS- 7 cells was also secreted as a latent proteinase and appeared to be activated by the same mechanism. rGP63 expressed on the surface of COS-7 cells as either a GPI-linked protein (rGP63gpi) or as a transmembrane fusion protein (rGP63tm) was produced as an active proteinase without the requirement for activation with HgCl₂ Expression of GP63 as an active proteinase, therefore, is not linked specifically to the GPI pathway and does not require a factor or microenvironment unique to Leishmania. The potential of glycoprotein purification to enrich for cell surface proteins was exploited in a search to identify novel amastigote surface molecules. The strategy was to isolate candidate glycoproteins, by lectin affinity chromatography and SDS-PAGE, for NH₂-terminal amino acid sequence analysis in order to design oligonucleotide probes to isolate a corresponding cDNA clone. The most abundant glycoproteins were identified by NH₂-terminal sequence analysis as Cys proteinases, GP63, and fragments thereof. Of 12 sequences obtained, 5 appeared to be unique and provide a basis for further studies to isolate the corresponding genes.

Thesis/Dissertation

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2009-06-09

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http://hdl.handle.net/2429/8874

Microbiology and Immunology

University of British Columbia

UBCV

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