RESUMEN
As part of our ongoing project of identification of actin-binding proteins implicated in the cell transition (flagellate to amoeboid/adherent) of Trichomonas vaginalis, we have characterized an alpha-actinin-related protein in this parasite. The protein (P100) has a molecular mass of 100 kDa and an isoelectric point of 5.5. A monoclonal antibody raised against this protein co-localizes with the actin network. P100 gene transcripts are co-expressed with actin throughout the cell cycle. Analysis of the deduced protein sequence reveals three domains: an N-terminal actin-binding region; a central region rich in alpha-helix; and a C-terminal domain with Ca(2+)-binding capacity. Whereas the N- and C-terminal regions are well-conserved as compared to other alpha-actinins, we observe in the central region an atypical distribution of residues in five repeats. The sequence of the repeats does not show any homology with the rod domain of the other alpha-actinins, except for the first repeat which shows some similarity. The four other repeats of T. vaginalis P100 appear to result from a duplication event which is not detectable in the other sequences.
Asunto(s)
Actinina/química , Trichomonas vaginalis/química , Regiones no Traducidas 5' , Actinina/genética , Actinina/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Calcio/metabolismo , Secuencia de Consenso , Citoesqueleto/química , ADN Complementario , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Alineación de Secuencia , Transcripción Genética , Trichomonas vaginalis/genética , Trichomonas vaginalis/crecimiento & desarrolloRESUMEN
Adherence to the vaginal epithelium by the sexually transmitted parasite Trichomonas vaginalis is mediated by four trichomonad surface proteins (AP65, AP51, AP33 and AP23). We recently showed that the 65-kDa adhesin is a member of a multigene family comprised of two similar but distinct proteins, AP65-1 and AP65-2, encoded by the genes ap65-1 and ap65-2, respectively. An additional immuno-crossreactive clone, the 1.2 kb F11.1 cDNA, was isolated from a phagemid expression library and encoded a fusion protein of approximately 46,000 daltons (46 kDa) that bound to HeLa cell surfaces. A significant portion of the 5' end was missing which, using the 5'-RACE method, was obtained and combined with the F11.1 clone to give a full-length cDNA. The ap65-3 gene encoded for a protein of 567 amino acids with a molecular mass of 63.1 kDa. The gene showed 88% and 96% identity at the DNA level with ap65-1 and ap65-2, respectively. Restriction mapping confirmed that the three AP65 genes are different. Southern analysis revealed that the ap65-3 gene is present in the T. vaginalis genome in multiple copies. Experiments with agar clones of trichomonads showed that each gene of the multigene family is present in all parasites, and Northern analysis showed that ap65-3 is expressed and transcriptionally regulated by iron. The ap65-3 gene had a leader sequence and, as with ap65-1 and ap65-2, showed significant homology to malic enzyme. Finally, analysis of the 3'-untranslated regions revealed that the transcript of ap65-3 had a long poly (A) tail in comparison to ap65-1 and ap65-2. Even more intriguing, sequences were found that may relate to differential degradation of select AP65 transcripts, such as the sequence motifs AUUUA for ap65-1 mRNA and UUAUUUAU for the ap65-2 mRNA, which were not found for ap65-3.