Cellular and biochemical characterization of two closely related triosephosphate isomerases fromTrichomonas vaginalis

Abstract

The glycolytic enzyme triosephosphate isomerase catalyses the isomerization between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Here we report that Trichomonas vaginalis contains 2 fully functional tpi genes. Both genes are located in separated chromosomal context with different promoter regulatory elements and encode ORFs of 254 amino acids; the only differences between them are the character of 4 amino acids located in α-helices 1, 2 and 8. Semi-quantitative RT-PCR assays showed that tpi2 transcript is approximately 3·3-fold more abundant than tpi1. Using an anti-TvTIM2 polyclonal antibody it was demonstrated that TIM proteins have a cytoplasmic localization and both enzymes are able to complement an Escherichia coli strain carrying a deletion of its endogenous tpi gene. Both TIM proteins assemble as dimers and their secondary structure assessment is essentially identical to TIM from Saccharomyces cerevisiae. The kinetic catalytic constants of the recombinant enzymes using glyceraldehyde-3-phosphate as substrate are similar to the catalytic constants of TIMs from other organisms including parasitic protozoa. As T. vaginalis depends on glycolysis for ATP production, we speculate 2 possible reasons to maintain a duplicated tpi copy on its genome: an increase in gene dosage or an early event of neofunctionalization of TIM as a moonlighting protein.

The Glycolytic Enzyme Triosephosphate Isomerase of Trichomonas vaginalis Is a Surface-Associated Protein Induced by Glucose That Functions as a Laminin- and Fibronectin-Binding Protein

“…TvTIM is encoded by two genes (tvtim1 and tvtim2) (23), a feature associated with expression divergence in other organisms (36,37). Although the nucleotide sequences of the two tvtim genes are 97.6% identical, our results showed that they were differentially expressed in response to glucose (tvtim1 was negatively regulated, whereas tvtim2 was positively regulated) (Fig.…”

“…To obtain cDNA, 5 g of total RNA was treated with DNase I (3 U) and reverse transcribed using a RevertAid first-strand cDNA synthesis kit (Thermo Scientific-Pierce, Rockford, IL) and oligo(dT) primers, as recommended by the manufacturer. For PCR amplification, 500 ng of cDNA, Taq DNA polymerase (Invitrogen), and primers specific for the reported sequences in TRICHDB for the tvtim1 (TVAG_497370) and tvtim2 (TVAG_096350) genes were used, as previously described (23). The tvtim1 primers were 5=-ACATTCTTTGTCGGAGGC-3= (sense) and 5=-AATGTTGATGAAA CCTGG-3= (antisense), and the tvtim2 primers were 5=-ACATTCTTCG TCGGTGGT-3= (sense) and 5=-AATGTTGATGAAGCCTGG-3= (antisense).…”

“…Images were captured with a ChemiDoc XRS system (Bio-Rad) and analyzed with Quantity One software (Bio-Rad). A recombinant alpha-enolase protein (TvENOr) previously shown to interact with Plg (12) was obtained by cloning the TVAG_329460 gene into the pET19b plasmid, expressed in the E. coli BL21(DE3) Star Rosetta II strain, affinity purified by immobilized-metal affinity chromatography (IMAC) and used as an FWB positive control (23). The unrelated recombinant protein ppTvCP4r was used as an FWB negative control (32).…”

“…The two proteins were recently identified and characterized as recombinant proteins, and their crystal structure and stability were determined (23,24). TvTIM1 and TvTIM2 are dimeric proteins of 254 amino acids in length.…”

“…Despite a difference of only four amino acids between these two proteins, the difference significantly affects their structural stability. The TvTIM1r dimer is more stable and less dissociable than the TvTIM2r dimer, which is easily dissociable, suggesting that these changes could directly affect protein function (23,24). TvTIMs exhibit dual cellular localizations (in the cytoplasm and on the surface of T. vaginalis) under regular growth conditions, suggesting that these proteins could have alternative novel functions when they are localized to the parasite surface (24).…”