RESUMO
An Entamoeba invadens gene encoding a homologue of BiP/GRP78, a 70-kDa heat shock protein or chaperonin was cloned. The predicted E. invadens BiP contained an ATP-binding site, a substrate-recognition domain, and a carboxy-terminal KDEL-peptide. Messenger RNAs of E. invadens for BiP, for a 70-kDa heat shock cognate, for a cyst wall glycoprotein (Jacob), and for chitinase were all induced by heat shock and by encystation medium. The presence of Jacob in heat-shocked amebae was confirmed by confocal microscopy and suggests that heat shock and encystation responses in E. invadens are related.
Assuntos
Proteínas de Transporte/genética , Entamoeba/genética , Proteínas de Choque Térmico , Resposta ao Choque Térmico/genética , Chaperonas Moleculares/genética , Adaptação Biológica , Sequência de Aminoácidos , Animais , Chaperona BiP do Retículo Endoplasmático , Genes de Protozoários , Dados de Sequência Molecular , RNA Mensageiro/isolamento & purificação , RNA de Protozoário/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
The infectious stage of amebae is the chitin-walled cyst, which is resistant to stomach acids. In this study an extraordinarily abundant, encystation-specific glycoprotein (Jacob) was identified on two-dimensional protein gels of cyst walls purified from Entamoeba invadens. Jacob, which was acidic and had an apparent molecular mass of approximately 100 kDa, contained sugars that bound to concanavalin A and ricin. The jacob gene encoded a 45-kDa protein with a ladder-like series of five Cys-rich domains. These Cys-rich domains were reminiscent of but not homologous to the Cys-rich chitin-binding domains of insect chitinases and peritrophic matrix proteins that surround the food bolus in the insect gut. Jacob bound purified chitin and chitin remaining in sodium dodecyl sulfate-treated cyst walls. Conversely, the E. histolytica plasma membrane Gal/GalNAc lectin bound sugars of intact cyst walls and purified Jacob. In the presence of galactose, E. invadens formed wall-less cysts, which were quadranucleate and contained Jacob and chitinase (another encystation-specific protein) in secretory vesicles. A galactose lectin was found to be present on the surface of wall-less cysts, which phagocytosed bacteria and mucin-coated beads. These results suggest that the E. invadens cyst wall forms when the plasma membrane galactose lectin binds sugars on Jacob, which in turn binds chitin via its five chitin-binding domains.
