Expression, localization, and kinetic characterization of the phospholipid biosynthesis enzyme CTP: phosphocholine cytidylyltransferase from the protozoan parasite Leishmania major
Date of Award
Master of Science (MS)
School of Biological Sciences
Jon A. Friesen
The eukaryotic parasite Leishmania is the causative agent of the disease leishmaniasis. L. major is the most common of 21 species that causes visceral leishmaniasis in humans, and 30 that cause the same disease in other mammals. Visceral leishmaniasis causes fever, weight loss, and over a short amount of time, multiple organ failure, and has a 100% mortality rate within 2 years. This makes it the second largest parasitic killer in the world behind malaria. Over 90% of the worldâ??s cases of visceral leishmaniasis have been reported in underdeveloped countries of India, Bangladesh, Nepal, Sudan, Ethiopia and Brazil, with 500,000 new cases being diagnosed worldwide each year. Nonetheless, the most popular drugs of choice for leishmaniasis treatment remain toxic, expensive, or subject to refractory infections.
Phospholipids used to construct biological membranes account for approximately 70% of total cellular lipid in Leishmania, and of that 30-40% is phosphatidylcholine (PC). PC is formed via the Kennedy pathway in Leishmania, as well as in many higher eukaryotes, including mammals. The enzyme CTP: phosphocholine cytidylyltransferase (CCT) catalyzes the addition of cytidine triphosphate (CTP) to phosphocholine yielding CDP-choline, a second, critical, rate limiting step in the formation of PC by the Kennedy pathway that has been shown to be reversibly membrane associated. Amino acid sequence analysis of CCT shows a high level of homology in the CCT catalytic region but a lack of the known C-terminal membrane-binding region in L. infantum and L. major. Thus, the nuclear localization signal, along with the classical C-terminal membrane binding domain is absent in these organisms and has been recently unknown whether CCT localizes to the nucleus, endoplasmic reticulum, or cytoplasm.
In investigation of the enzyme, the gene for L. major CCT has been tagged with a red fluorescent protein gene (mCherry) and successfully cloned into non-pathogenic Leishmania tarentolae. Confocal microscopy has revealed that L. major CCT is primarily cytoplasmic, while most likely also able to reversibly associate with internal membranes such as the endoplasmic reticulum and plasma membrane. In addition, a truncated mutant of L. major CCT containing the catalytic domain has been expressed in Escherichia coli and purified. In vitro assay of the enzyme yielded a maximal velocity (Vmax) of approximately 350 nmol/min/mg and substrate constant (Km) values of 3 mM and 0.2 mM respectively for the substrates phosphocholine and CTP. The Mg2+ ion was used as a cofactor in the reaction, but the enzyme was also able to achieve maximal activity using Mn2+, Co2+, Ni2+, and possibly Fe2+. This summarizes investigation of CCT as a possible therapeutic target for more efficacious treatment of leishmaniasis.
Lange, Justin Daniel Theodore, "Expression, localization, and kinetic characterization of the phospholipid biosynthesis enzyme CTP: phosphocholine cytidylyltransferase from the protozoan parasite Leishmania major" (2015). Theses and Dissertations. 451.
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Imported from ProQuest Lange_ilstu_0092N_10580.pdf