Identification of a Novel Prostaglandin F2α Synthase in Trypanosoma brucei

Abstract

Members of the genus Trypanosoma cause African trypanosomiasis in humans and animals in Africa. Infection of mammals by African trypanosomes is characterized by an upregulation of prostaglandin (PG) production in the plasma and cerebrospinal fluid. These metabolites of arachidonic acid (AA) may, in part, be responsible for symptoms such as fever, headache, immunosuppression, deep muscle hyperaesthesia, miscarriage, ovarian dysfunction, sleepiness, and other symptoms observed in patients with chronic African trypanosomiasis. Here, we show that the protozoan parasite T. brucei is involved in PG production and that it produces PGs enzymatically from AA and its metabolite, PGH2. Among all PGs synthesized, PGF2α was the major prostanoid produced by trypanosome lysates. We have purified a novel T. brucei PGF2α synthase (TbPGFS) and cloned its cDNA. Phylogenetic analysis and molecular properties revealed that TbPGFS is completely distinct from mammalian PGF synthases. We also found that TbPGFS mRNA expression and TbPGFS activity were high in the early logarithmic growth phase and low during the stationary phase. The characterization of TbPGFS and its gene in T. brucei provides a basis for the molecular analysis of the role of parasite-derived PGF2α in the physiology of the parasite and the pathogenesis of African trypanosomiasis.

Structural and Mutational Analysis of Trypanosoma brucei Prostaglandin H2 Reductase Provides Insight into the Catalytic Mechanism of Aldo-ketoreductases

“…The rationale used for substrates. This finding is consistent with our site-directed mutagenesis data and explains our previous results showing that the reduction of PGH 2 by TbPGFS was not inhibited by 9,10-PQ when both substrates were incubated simultaneously in excess (31). A profile of the k cat versus pH for the tetrad mutants revealed that mutants Y52F, K77L, H110A, and H110F eliminated the titratable group for 9,10-PQ reduction.…”

“…Enzyme Assay and Kinetic Measurements-PGH 2 reductase activity was measured by incubating at 37°C for 2 min a reaction mixture containing an appropriate enzyme aliquot, 5 M [1-14 C]PGH 2 (final concentration), 100 l of 100 mM sodium phosphate (pH 7.0), and 500 M NADPH (31). After the reaction had been terminated by the addition of 250 l of stop solution (30:4:1 (v/v/v) diethyl ether/methanol/2 M citric acid), aliquots were analyzed by thin-layer chromatography as described previously (31).…”

“…After the reaction had been terminated by the addition of 250 l of stop solution (30:4:1 (v/v/v) diethyl ether/methanol/2 M citric acid), aliquots were analyzed by thin-layer chromatography as described previously (31). For the reduction of 9,10-PQ, the reaction mixture consisted of 100 mM sodium phosphate (pH 7.0), the enzyme, 100 M NADPH, and 40 M 9,10-PQ, in a total volume of 500 l. The reaction was initiated by the addition of the substrate, and then the decrease in absorbance at 340 nm (⑀ ϭ 6270 M Ϫ1 cm Ϫ1 ) was monitored at 37°C.…”

“…We have identified a unique PGF 2␣ synthase from the protozoan parasite Trypanosoma brucei (31), the etiological agent of African trypanosomiasis in humans and animals. T. brucei PGF 2␣ synthase belongs to 5A subfamily of the AKRs (16,31).…”

“…T. brucei PGF 2␣ synthase belongs to 5A subfamily of the AKRs (16,31). The uniqueness of this synthase stems from the discovery that it exhibits a high specificity toward PGH 2 and shows no enzymatic activity toward substrates such as PGD 2 , PGE 2 , or PGF 2␣ .…”