Differential expression of two plant-like enolases with distinct enzymatic and antigenic properties during stage conversion of the protozoan parasite Toxoplasma gondii

J Mol Biol. 2001 Jun 22;309(5):1017-27. doi: 10.1006/jmbi.2001.4730.

Abstract

The precise molecular mechanisms underlying the switch between the two developmental stages of Toxoplasma gondii, and the metabolic adaptations occurring during this stage conversion are poorly understood. Because inhibitors of mitochondrial respiration are known to trigger differentiation from tachyzoite into bradyzoite stages, we believe that some of the switch components may be sought in the regulation of central carbohydrate metabolism. We have previously described a cDNA encoding a bradyzoite-specific enolase, ENO1. We now report the isolation and characterization of another enolase-encoding cDNA (ENO2) that is expressed preferentially in the tachyzoite stage. The deduced amino acid sequences of ENO1 and ENO2 share 73.65 % identity. They both display significant homologies to plant enolases with the presence of two plant-like peptide insertions, a pentapeptide EWGW(Y)C(S) and a dipeptide EK (or DK). We demonstrate that deletions of the ENO1 pentapeptide motif on its own or together with the dipeptide reduce drastically the affinity for the 2PGA substrate, suggesting that the evolutionary acquisition of these peptides in enolases of land plants and apicomplexan parasites contribute a specific function to their enzymatic activities. T. gondii ENO1 and ENO2 were also expressed as active recombinant enzymes in Escherichia coli. While ENO1 and ENO2 display similar K(m) values, the pure tachyzoite-specific enzyme (ENO2) has a threefold specific activity at V(max) compared with that of the bradyzoite-specific enolase (ENO1). Moreover, immunoblot analyses performed using polyclonal antibodies raised against the recombinant enzymes revealed that the native enolase in tachyzoite and bradyzoite are also antigenically distinct. Taken together, our results indicate that the differences witnessed between the two activities may be instrumental in maintaining glycolysis in pace with the distinct stage-specific requirements of carbohydrate metabolism.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Antigens, Protozoan / chemistry
  • Antigens, Protozoan / genetics
  • Antigens, Protozoan / immunology*
  • Antigens, Protozoan / metabolism
  • Catalysis
  • Cloning, Molecular
  • DNA, Complementary / genetics
  • Enzyme Stability
  • Gene Expression Regulation, Developmental
  • Gene Expression Regulation, Enzymologic*
  • Genes, Protozoan / genetics
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis / genetics
  • Phosphopyruvate Hydratase / chemistry
  • Phosphopyruvate Hydratase / genetics
  • Phosphopyruvate Hydratase / immunology*
  • Phosphopyruvate Hydratase / metabolism*
  • Plants / enzymology*
  • RNA, Protozoan / genetics
  • RNA, Protozoan / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Temperature
  • Toxoplasma / enzymology*
  • Toxoplasma / genetics
  • Toxoplasma / growth & development*
  • Toxoplasma / immunology

Substances

  • Antigens, Protozoan
  • DNA, Complementary
  • RNA, Protozoan
  • Phosphopyruvate Hydratase

Associated data

  • GENBANK/AF051910
  • GENBANK/AF123457