[Genetic variation and clustal analysis of Trichomonas vaginalis cysteine proteases]

To clone the genes coding for cysteine proteases (CPs, TvCPs) from Trichomonas vaginalis and to analyze their genetic variations with the related sequences from NCBI database (GenBank) and T. vaginalis Genome Project database from The Institute for Genomic Research (TIGR).

TvCP genes were amplified using PCR, and inserted into vector pET28b or pBS-T. The recombinant plasmids were then transformed to Escherichia coli BL21 or Topl0 strain. The recombinant plasmids were used for sequencing. Homologous TvCP genes were blasted based on NCBI GenBank and TIGR T. vaginalis Genome Project database. The sequences of cloned TvCP genes were aligned and clustered by Clustal X (1.83 version) with retrieved sequences. Comparisons of amino acids among cathepsin L-like TvCPs, human L-like cathepsins and papaya papain were performed using DNAstar software, and their phylogenic tree was constructed based on neighbor-joining method using Clustal X.

Two TvCP3 clones and one TvCP2 had a high identity of more than 99% with their responding TvCPs. Three clones of TvCP4 genes, GZ-CP4-clone 1-3, belonged to two members of a family showing a high percentage identity of more than 97.5% with the sequences of TvCP4 genes from databases (GenBank and TIGR) both at amino acid and nucleotide levels. Nine homologous TvCP4 pro-enzymes with 304 amino acids and other two members with deletions of N-terminal sequence existed in T. vaginalis sharing a similarity of 62.3-96.7% amino acids, which may evolve by means of gene replication and deletion. TvCP1-4, TvCP12, TvCP25 and CP65 had an identity of 61-88.2% at amino acid levels. So far, all reported sequences of C1 family from T. vaginalis belonged to capanthesin L-like subfamily with the same enzymatic active sites, conserved cysteine residues and similar structural features such as ERFNIN-like motif in pro-enzyme region, suggesting that they might result from gene duplication and mutations.

TvCPs belong to cathepsin L-like family with genetic diversity, but they have the same active amino acid residues, cysteine residues and similar structural characteristics, suggesting that they may derive from one ancestor.