Structure-function analysis of plasmodium falciparum chloroquine resistance transporter in chloroquine resistance

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Structure-function analysis of plasmodium falciparum chloroquine resistance transporter in chloroquine resistance


Author: Choy, Kit Ying
Title: Structure-function analysis of plasmodium falciparum chloroquine resistance transporter in chloroquine resistance
Degree: M.Phil.
Year: 2007
Subject: Hong Kong Polytechnic University -- Dissertations.
Plasmodium falciparum.
Department: Dept. of Applied Biology and Chemical Technology
Pages: xii, 85 leaves : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record:
Abstract: Malaria is a common and serious tropical disease spread by mosquitoes affecting more than 100 countries in the world. There are approximately 270 million clinical cases and 2 million deaths due to malaria every year, especially amongst children and pregnant women. Chloroquine (CQ) was an effective drug against malaria but its intensive use has led to the emergence of chloroquine resistance (CQR) since 1970s', resulting in a dramatic increase in mortality and morbidity of malaria. Recent studies have identified a gene, Plasmodium falciparum chloroquine resistance transporter (pfcrf) gene, as the determinant gene for CQR. The 48.6kDa PfCRT protein has 424 amino acids and is encoded by pfcrt on chromosome 7. PfCRT is localized to the parasitic digestive vacuole (DV) membrane and belongs to a previously uncharacterized family of putative transporters with 10 transmembrane domains. 8 point mutations at different positions of PfCRT have been found to be associated with CQR. Among all, the point mutation at position 76 is regarded as the major determinant of CQR while the rest are believed to enhance CQR. In this research, a systemic approach is used to study the effect of mutations at the 76th position of PfCRT on its function, including (1) the activity to transport radiolabeled CQ and different fluorescent substrates and (2) pH regulation across membrane. Nine mutants have been made at the 76th position of PfCRT, including K76, K76T, K76I, K76N, K76L, K76E, K76R, K76D and K76S. These mutations are duplicated on two PfCRT backgrounds, one from CQR strain (Dd2) and the other from chloroquine sensitive (CQS) strain (3D7). These PfCRT mutants have been expressed in a yeast expression system, Pichia pastoris and the microsomes derived are used in Hoechst 33342 Dye transport, pH and [3H]-CQ-accumulation assay. Dd2 K76T PfCRT has the highest [3H]-CQ transport activity and most acidic internal pH. Comparable [3H]-CQ transport abilities are observed in PfCRT mutants with similar structure, K76T and K76S or K76I and K76L. However, PfCRT mutants with different charges, negatively charged K76E and K76D and positively charged K76R, shared similar [3H]-CQ transport abilities, suggesting that the current loss of positive charge model of PfCRT requires modification.

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