Cloning, characterization and functional expression of a cyclophilin of Entamoeba histolytica.
Full-length Entamoeba histolytica cyclophilin gene (EhCyp) was isolated, characterized and recombinantly expressed in bacterial cells. The deduced amino acid sequence of EhCyp shows 60-70% identity with cyclophilins from other organisms and has conserved the cyclophilin signature motifs and residues involved in cyclosporin A binding. Upstream of the 501 bp open reading frame of EhCyp, sequences resembling the putative consensus E. histolytica CE1, CE2 and CE3 regulatory elements were found. Northern blot assays revealed a single transcript of 0.63 kb. The transcription start was determined by primer extension at position -13 relative to the initial ATG codon. Cyclosporin A binding and peptidyl-proplyl cis-trans isomerase activities characteristic of cyclophilin were detected in soluble extracts of E. histolytica trophozoites and in the recombinant protein. In both cases, the isomerase activity was inhibited by nanomolar concentrations of cyclosporin A. Treatment of cultured trophozoites with cyclosporin A decreased their proliferation with a 50% inhibition value of 1 microg/ml and was lethal in doses over 50 microg/ml.
Ostoa-Saloma
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P
Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Apartado Postal 70228, C.P. 04510, Mexico, Mexico.
César Carrero
J
J
Petrossian
P
P
Hérión
P
P
Landa
A
A
Pedro Laclette
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016572:Cyclosporine / Q000378:metabolism / Q000494:pharmacology; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics; D004748:Entamoeba histolytica / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D016054:DNA, Protozoan; D012333:RNA, Messenger; D016053:RNA, Protozoan; D011994:Recombinant Proteins; D016572:Cyclosporine; D019696:Peptidylprolyl Isomerase