Abstract
Our group has regularly investigated the recognition of glycans on host cells by pathogen lectins; we have also studied the biological responses triggered by such recognition. Our team has focused on particular lectins: (i) TgMIC1 and TgMIC4 - proteins secreted by micronemes of the protozoan Toxoplasma gondii that recognize glycans bearing sialic acid and beta-galactose as terminal residues, respectively; (ii) paracoccin, proteins of the surface of Paracoccidioides brasiliensis yeasts that recognize N-acetylglucosamine. In these cases, we have isolated the proteins by affinity chromatography on columns of their specific sugar; we have also characterized the structure and biological properties of the native forms of each of these lectins. The relevance of the activities performed by these proteins have motivated us to clone the gene codifying each lectin and express it heterologously. We have characterized the resulting recombinant proteins in terms of their ability to reproduce the properties of the corresponding native lectin. Functional studies revealed that the lectins derived from T. gondii and P. Brasiliensis play relevant roles in pathogen biology and affect host cell immunity. We have examined the recognition involved in the induction of these effects, the identification of molecules containing the recognition target, the cell signaling triggered by this recognition, the cellular responses stemming from cell activation, and the consequent production of immunological modulators as well as the resistance conferred by these processes. Concerning the Toxoplasma gondii lectins, we currently aim to (i) characterize their interaction with the N-glycans associated with TLR4, in the same way that we have proceeded in the case of TLR2; (ii) establish the structure of TLRs N-glycans identified as recognition targets of T. gondii and implicated in cell activation; (iii) conduct molecular modeling of TLR2 glycans and their interaction with TgMIC1; (iv) express TgMIC1 and TgMIC4 in Pichia pastoris, instead of Escherichia coli, to obtain a soluble LPS-free recombinant protein. As for the lectin paracoccin, we aim to (i) determine its expression in mycelia and mycellium-yeast transition forms of P. brasiliensis, and compare it with its expression in yeasts; (ii) characterize the interaction of paracoccin with innate immunity cell receptors, especially TLR2, targeting the requirement for receptor heterodimerization and the participation of accessory proteins; (iii) find out whether the interaction between paracoccin and TRL2 depends on carbohydrate recognition and, if this is the case, (iv) identify whether the ectodomain N-glycan(s) is(are) the target(s) of this recognition, (v) investigate paracoccin-modulation of the expression of these receptors in innate immunity cells and of the citokies production by these same cells; (vi) determine the effect of paracoccin administration to mice with respect to the pattern of citokines production and PRRs expression in phagocytes; (vii) characterize the effect of N-glycans on the activities of glucanase and amylase of P. brasiliensis yeasts as well as systematically analyze the role of AMPc on N-acetylglucosaminidase expression; (viii) determine the part played by N-glycans in the activity of P. brasiliensis enzymes and identify non-N-glycosilated proteins by DIGE - Difference gel electrophoresis), .These aims will be sought by our local group in collaboration with other research groups abroad, more specifically the teams led by Professor Nicholas Gay (University of Cambridge, UK), Prof. Igor de Almeida (University of Texas in El Paso, USA), Prof. Stephen Mathews (Imperial College, London, UK), and Prof. Ten Feizi (Imperial College, London, UK). (AU)
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