Papel da proteína LAMP-2 associada à membrana lisossomal na invasão celular do Trypanosoma cruzi mediada pela molécula de superfície gp82

• Ano de defesa: 2019
• Autor(a) principal: Rodrigues, Joao Paulo Ferreira [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• dARK ID: ark:/48912/0013000027gr6
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Invasion By Metacyclic Forms; Trypanosoma Cruzi; Lysosome-Associated Membrane Proteins; gp82; Invasão Celular Por Formas Metacíclicas; Proteínas Associadas A Membrana Lisossomal
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=8029572; https://repositorio.unifesp.br/handle/11600/59404

Cell invasion by Trypanosoma cruzi metacyclic trypomastigote forms is ediated by the surface glycoprotein gp82, which has the property to bind to the host cell in a receptor-dependent manner and to induce the spreading and exocytosis of lysosomes, events that are necessary for the parasitophorous vacuole formation. The objective of this work was to investigate the involvement of lysosome-associated membrane proteins 1 and 2 (LAMP1 and LAMP2) in the process of cell invasion by metacyclic forms, as well as to determine whether these proteins bind to gp82. By using HeLa cells and T. cruzi strain CL, invasion assays were performed initially in the presence of antibody against LAMP1 or LAMP2. In the presence of anti-LAMP2 antibody, the internalization of metacyclic forms was significantly reduced, whereas the anti-LAMP1 had no effect. HeLa cells deficient in LAMP1 ou LAMP2 were generated and tested for the susceptibility to invasion by metacyclic forms. The levels of invasion by metacyclic forms were significantly lower in LAMP2-deficient cells, as compared to wild type cells, in contrast to LAMP1-deficient cells, which displayed invasion rate similar to that of control cells. Cells deficient in LAMP1 or LAMP2 were used in binding assays of the recombinant protein containing the full gp82 sequence. It was found that the recombinant gp82 binding to LAMP2-deficient cells was significantly reduced, as compared to LAMP1-deficient and wild type cells. The possibility that LAMP2 could be the receptor for gp82 was examined in coimmunoprecipitation assays, in which extracts of HeLa cells and parasites were incubated with magnetic beads coupled to anti-LAMP1 or anti-LAMP2 antibody. Binding of gp82 to LAMP2, but not to LAMP1, was detected. In co-immunoprecipitation assay, in which beads coupled to the anti-gp82 monoclonal antibody were used, gp82