RESUMO
The protozoan responsible for Chagas' disease, Trypanosoma cruzi, expresses on its surface an unusual trans-sialidase enzyme thought to play an important role in host-parasite interactions. Trans-sialidase is the product of a multigene family encoding both active and inactive proteins. We have demonstrated that despite lacking enzymatic activity due to a single mutation, Tyr342-His, inactive trans-sialidase displays sialic acid binding activity, with identical specificity to that of its active analogue. In this work we demonstrate that binding of a recombinant inactive trans-sialidase to molecules containing alpha2,3-linked sialic acid on endothelial cell surface triggers NF-kappaB activation, expression of adhesion molecules and upregulation of parasite entry into host cells. Furthermore, inactive recombinant trans-sialidase blocks endothelial cell apoptosis induced by growth factor deprivation. These results suggest that inactive members of the trans-sialidase family play a role in endothelial cell responses to T. cruzi infection.
Assuntos
Células Endoteliais/parasitologia , Glicoproteínas/metabolismo , Neuraminidase/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/fisiologia , Substituição de Aminoácidos/genética , Animais , Apoptose/imunologia , Moléculas de Adesão Celular/biossíntese , Linhagem Celular , Glicoproteínas/genética , Glicoproteínas/imunologia , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Ácido N-Acetilneuramínico/metabolismo , NF-kappa B/metabolismo , Neuraminidase/genética , Neuraminidase/imunologia , Mutação Puntual , Ligação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Trypanosoma cruzi/genéticaRESUMO
Host/parasite interaction mediated by carbohydrate/lectin recognition results in the attachment to and invasion of host cells and immunoregulation, enabling parasite replication and establishment of infection. Trypanosoma cruzi, the protozoan responsible for Chagas disease, expresses on its surface a family of enzymatically active and inactive trans-sialidases. The parasite uses the active trans-sialidase for glycoprotein sialylation in an unusual trans-glycosylation reaction. Inactive trans-sialidase is a sialic acid-binding lectin that costimulates host T cells through leucosialin (CD43) engagement. The co-mitogenic effect of trans-sialidase can be selectively abrogated by N-acetyllactosamine, suggesting the presence of an additional carbohydrate binding domain for galactosides, in addition to that for sialic acid. Here we investigated the interaction of inactive trans-sialidase in the presence of beta-galactosides. By using NMR spectroscopy, we demonstrate that inactive trans-sialidase has a beta-galactoside recognition site formed following a conformational switch induced by sialoside binding. Thus prior positioning of a sialyl residue is required for the beta-galactoside interaction. When an appropriate sialic acid-containing molecule is available, both sialoside and beta-galactoside are simultaneously accommodated in the inactive trans-sialidase binding pocket. This is the first report of a lectin recognizing two distinct ligands by a sequential ordered mechanism. This uncommon binding behavior may play an important role in several biological aspects of T. cruzi/host cell interaction and could shed more light into the catalytic mechanism of the sialic acid transfer reaction of enzymatically active trans-sialidase.
Assuntos
Antígenos CD , Galactose/química , Neuraminidase/química , Trypanosoma cruzi/enzimologia , Animais , Sítios de Ligação , Sequência de Carboidratos , Carboidratos/química , Cromatografia , Galactosídeos/química , Glicoproteínas , Lactose/química , Leucossialina , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Ácido N-Acetilneuramínico/química , Ligação Proteica , Estrutura Terciária de Proteína , Sefarose/química , Sialoglicoproteínas/metabolismo , Linfócitos T/metabolismoRESUMO
Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, expresses on its surface an uncommon membrane-bound sialidase, known as trans-sialidase. trans-Sialidase is the product of a multigene family encoding both active and inactive proteins. We report here that an inactive mutant of trans-sialidase physically interacts with CD4(+) T cells. Using a combination of flow cytometry and immunoprecipitation techniques, we identified the sialomucin CD43 as a counterreceptor for trans-sialidase on CD4(+) T cells. Using biochemical, immunological, and spectroscopic approaches, we demonstrated that the inactive trans-sialidase is a sialic acid-binding protein displaying the same specificity required by active trans-sialidase. Taken together, these results suggest that inactive members of the trans-sialidase family can physically interact with sialic acid-containing molecules on host cells and could play a role in host cell/T. cruzi interaction.
