Location of the cell-binding domain of CP65, a 65kDa cysteine proteinase involved in Trichomonas vaginalis cytotoxicity.
The cysteine proteinase (CP) of 65kDa, CP65, binds to the surface of HeLa cells and is involved in Trichomonas vaginalis cellular damage. To identify and locate the CP65 cellular-binding domain, we enriched the CP65 protein band by ammonium sulfate fractionation and ion-exchange chromatography and the N-terminal sequence was obtained. A 618bp gene fragment was obtained by PCR using genomic DNA as template and primers derived from the N-terminal sequence of CP65 and the Asn papain-catalytic conserved region. This gene fragment encodes for 206 amino acid (aa) residues corresponding to the N-terminal region of a mature CP with 67-76% identity to the reported trichomonad cathepsin-L-like CPs. This gene fragment was expressed in a bacterial system for antibody production and functional analysis. Antibodies against the native trichomonad CP65 recognized the recombinant protein, referred to as rCP65, confirming its relationship with the CP65 gene. The rCP65 protein was bound to the surface of HeLa cells and competed with the native CP65 for binding. Antibodies to the rCP65 (alpha-rCP65) reacted with the trichomonad CP65 located on the parasite surface, and inhibited trichomonal cytotoxicity in a concentration-dependent manner. These data strongly suggest that this gene fragment encodes for the putative cell-binding domain (CBD) of CP65 located at its N-terminal region.
Solano-González
Eduardo
E
Department of Biotechnology and Bioengineering, Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Av. IPN 2508, Col. San Pedro Zacatenco, CP 07360, Mexico City, Mexico.
Alvarez-Sánchez
María Elizbeth
ME
Avila-González
Leticia
L
Rodríguez-Vargas
Victor Hugo
VH
Arroyo
Rossana
R
Ortega-López
Jaime
J
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000378:metabolism; D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D020029:Base Pairing / Q000235:genetics; D016923:Cell Death; D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017125:Genes, Protozoan / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010271:Parasites / Q000201:enzymology; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D014246:Trichomonas vaginalis / Q000201:enzymology*
D000913:Antibodies, Protozoan; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; D000645:Ammonium Sulfate