Leishmania Carbonic Anhydrases : Preliminary Structural and Biochemical Studies

Baroi, Bappa Shona (2013) Leishmania Carbonic Anhydrases : Preliminary Structural and Biochemical Studies. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS Dissertation of Bappa Shona Baroi (08MS16)) Bappa_Shona_Baroi-08MS16-MSThesis.pdf - Submitted Version Restricted to Repository staff only Download (1MB)

Abstract

Carbonic Anhydrases are enzymes that are involved in a wide range of physiological activities like pH balance, CO2 carrier, gluconeogenesis, lipogenesis, ureagenesis, tumorigenicity and the growth and virulence of various pathogens. The key reaction involved is the reversible conversion of CO2 to bicarbonate. Across organisms, till date, five distinct classes of CAs have been discovered: α, β, γ, δ and ε CAs. The fact that Leishmania parasites survive and proliferate in the highly acidic conditions of the lysosome led us to hypothesize that the Leishmania genome might encode for atleast one putative form of Carbonic Anhydrase. Analysing the genome gave us two putative CA/CA-like sequences. Bioinformatics studies deduced one as beta CA(LmCA1) and the other as an alpha CA(LmCA2). Modelling of LmCA1 was done to know more about the Zinc-binding catalytic site of the protein which came out starkly different from human alpha CAs: two Cys and one His in LmCA1 vs three His in human alpha CAs. This is a positive result because we can exploit this differenc to design drugs specific only to LmCA1. LmCA2 due to low sequence similarity with any template, wasn’t taken up further. For biochemical properties, We amplified the LmCA1 gene by PCR from L. major genomic DNA and were able to clone it into the EcoR1 site of the bacterial cloning/expression plasmid vector, pET28a to produce LmCA1/pET28a clone. The clone was then transformed into E. coli BL21(DE3) cells and the 35kDa N-terminal histidine-tagged LmCA1 protein was expressed upon 0.5mM IPTG induction. Activity was measured using these as samples with the Maren’s CO2hydration essay and we came up with significant activity. As such the presence of a biochemically active form of beta CA was confirmed. Localization of the protein is also a key to know about its role in Leishmania physiology. So we cloned LmCA1 in pXG-GFP and right now, waiting to transfect these clones in Leishmania. Since inhibition of LmCA1 even in neutral pH also inhibits growth, we were led to hypothesise that LmCA1 is involved in other key physiological processes too. Right now, the lab is working on extracting a pure form of the protein, localization and the role of LmCA1 in three key physiological processes: gluconeogenesis,pyrimidine synthesis and fatty acid synthesis.

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