Is Na, K-ATPase involved in the invasion of cardiomyocytes by Trypanosoma cruzi?.

SA, PAULO; FESSEL, M; PEREIRA, CA; ALVES, MARIA JULIA MANSO

Caxambu

Congreso; XXI Annual Meeting of the Brazilian Society of Protozoology; 2005

Almost all nucleated cells can be invaded by T. cruzi. Our laboratory has showed that peptide J, containing a conserved motif of the gp85/trans-sialidase superfamily (FLY domain), binds to the host epithelial cell and enhances T. cruzi inva- sion (Magdesian et al., 2001). In order to identify putative molecules from heart myocytes that interact with FLY do- main, membrane extracts of rat cardiomyocytes were submit- ted to a±nity chromatography on J-matrix. More than one polypeptide was eluted with 1 M NaCl or 4-8 M urea, what agrees with the hypothesis that peptide J contains an ex- tremely adhesive motif. After SDS-PAGE, the major bands were removed from the gel, digested, and the fragments se- quenced after HPLC puri¯cation. With special interest to our receptor-ligand recognition study, the ¯3 subunit of the Na+,K+-ATPase complex, a 35 kDa surface protein was iden- ti¯ed. To further characterize the ¯3 subunit as possible lig- and of the FLY domain, we ampli¯ed the portion of the pro- tein corresponding to the extracellular segment by using rat heart mRNA and RT-PCR and cloning into pRSET vector. After expression and puri¯cation of a 30 kDa molecule, the binding of the protein was tested by overlay assay against dots containing peptide J or J-Ala (negative control). A strong response was obtained only with peptide J. Working is in progress to establish the importance of the FLY domain-T. cruzi. Our laboratory has showed that peptide J, containing a conserved motif of the gp85/trans-sialidase superfamily (FLY domain), binds to the host epithelial cell and enhances T. cruzi inva- sion (Magdesian et al., 2001). In order to identify putative molecules from heart myocytes that interact with FLY do- main, membrane extracts of rat cardiomyocytes were submit- ted to a±nity chromatography on J-matrix. More than one polypeptide was eluted with 1 M NaCl or 4-8 M urea, what agrees with the hypothesis that peptide J contains an ex- tremely adhesive motif. After SDS-PAGE, the major bands were removed from the gel, digested, and the fragments se- quenced after HPLC puri¯cation. With special interest to our receptor-ligand recognition study, the ¯3 subunit of the Na+,K+-ATPase complex, a 35 kDa surface protein was iden- ti¯ed. To further characterize the ¯3 subunit as possible lig- and of the FLY domain, we ampli¯ed the portion of the pro- tein corresponding to the extracellular segment by using rat heart mRNA and RT-PCR and cloning into pRSET vector. After expression and puri¯cation of a 30 kDa molecule, the binding of the protein was tested by overlay assay against dots containing peptide J or J-Ala (negative control). A strong response was obtained only with peptide J. Working is in progress to establish the importance of the FLY domain-¯3 subunit of the Na+,K+-ATPase complex, a 35 kDa surface protein was iden- ti¯ed. To further characterize the ¯3 subunit as possible lig- and of the FLY domain, we ampli¯ed the portion of the pro- tein corresponding to the extracellular segment by using rat heart mRNA and RT-PCR and cloning into pRSET vector. After expression and puri¯cation of a 30 kDa molecule, the binding of the protein was tested by overlay assay against dots containing peptide J or J-Ala (negative control). A strong response was obtained only with peptide J. Working is in progress to establish the importance of the FLY domain-+,K+-ATPase complex, a 35 kDa surface protein was iden- ti¯ed. To further characterize the ¯3 subunit as possible lig- and of the FLY domain, we ampli¯ed the portion of the pro- tein corresponding to the extracellular segment by using rat heart mRNA and RT-PCR and cloning into pRSET vector. After expression and puri¯cation of a 30 kDa molecule, the binding of the protein was tested by overlay assay against dots containing peptide J or J-Ala (negative control). A strong response was obtained only with peptide J. Working is in progress to establish the importance of the FLY domain-¯3 subunit as possible lig- and of the FLY domain, we ampli¯ed the portion of the pro- tein corresponding to the extracellular segment by using rat heart mRNA and RT-PCR and cloning into pRSET vector. After expression and puri¯cation of a 30 kDa molecule, the binding of the protein was tested by overlay assay against dots containing peptide J or J-Ala (negative control). A strong response was obtained only with peptide J. Working is in progress to establish the importance of the FLY domain- ¯3 interaction in the invasion of cardiomyocyte by T. cruzi.3 interaction in the invasion of cardiomyocyte by T. cruzi.