Function and regulation of glycosylphosphatidylinosito phospholipase C from trypanosoma brucei
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Trypanosoma brucei is the etiological agent of human African trypanosomiasis (or sleeping sickness). Over 300,000 people are infected with this disease and an estimated five million are at risk of contracting the disease. Glycosylphosphatidylinositol phospholipase C (GPI-PLC) is a virulence factor in T. brucei. However no in vivo activity has been demonstrated for the enzyme. The present study was aimed to: (i) determine the possible role of GPI-PLC in T. brucei response to cell stress; (ii) investigate the contribution of GPI-PLCp to endocytosis in T. brucei; (iii) determine if cysteine residues of GPI-PLC affect targeting of the enzyme to glycosomes in T. brucei; and (iv) study the activity profile of GPI-PLC during differentiation of blood stream form to procyclic T. brucei. The effects of alkali and hypo-osmotic buffer as agents of cell stress on T. brucei were studied. These cell stress signals induced GPI digestion by GPI-PLCp. Furthermore, we determined that enzyme activity and movement of GPI-PLCp from glycosomes to the endoplasmic reticulum (ER) was required for digestion of GPIs as response to cell stress. Potential contribution of GPI-PLC to endocytosis in T. brucei was considered. Using transferrin and dextran as endocytic markers, we found that GPI-PLCp expression increased rates of endocytosis in trypanosomatids. A product of enzyme action (i.e., diacylglycerol; DAG) also was capable of enhancing endocytosis. Addition of phorbol esters (phorbol 12-myristate 13-acetate) or exogenous DAG (oleyl-acetyl-sn-glycerol and dimyristyl-sn-glycerol) increased endocytosis in T. brucei implying that DAG binds to an intracellular receptor that affects endocytosis. Thus, GPI-PLCp signals to the endocytic pathway by producing DAG that binds to receptors, which to influence endocytosis. These observations were confirmed with studies in Leishmania major, a related trypanosomatid. An endosome targeting signal in L. major has been identified using GPI-PLCp as a reporter protein. Cysteines residues 80 and 269,270,273 are part of the peptide motif that is essential for targeting of GPI-PLCp in L. major, but not in T. brucei. Mutations of these cysteine residues, when expressed in L. major, mistargets the protein to glycosmes. However localization is unaltered when the Cys mutants are expressed in T. brucei. Thus, there is lack of conservation of endosome targeting signal among trypanosomatids. GPI-PLC is expressed by the blood stream form but not the procyclic form of T. brucei. Mechanism of loss of GPI-PLC activity during differentiation was investigated. We found that reduction of GPI-PLCp from blood stream forms is linked to cell replication post-differentiation. Translational repression coupled with protein dilution during cell replication might be employed by the parasites for developmentally regulated control of proteins that are not actively degraded during differentiation.