The Repetitive Cytoskeletal Protein H49 of Trypanosoma cruzi Is a Calpain-Like Protein Located at the Flagellum Attachment Zone

Abstract

Background: Trypanosoma cruzi has a single flagellum attached to the cell body by a network of specialized cytoskeletal and membranous connections called the flagellum attachment zone. Previously, we isolated a DNA fragment (clone H49) which encodes tandemly arranged repeats of 68 amino acids associated with a high molecular weight cytoskeletal protein. in the current study, the genomic complexity of H49 and its relationships to the T. cruzi calpain-like cysteine peptidase family, comprising active calpains and calpain-like proteins, is addressed. Immunofluorescence analysis and biochemical fractionation were used to demonstrate the cellular location of H49 proteins.Methods and Findings: All of H49 repeats are associated with calpain-like sequences. Sequence analysis demonstrated that this protein, now termed H49/calpain, consists of an amino-terminal catalytic cysteine protease domain II, followed by a large region of 68-amino acid repeats tandemly arranged and a carboxy-terminal segment carrying the protease domains II and III. the H49/calpains can be classified as calpain-like proteins as the cysteine protease catalytic triad has been partially conserved in these proteins. the H49/calpains repeats share less than 60% identity with other calpain-like proteins in Leishmania and T. brucei, and there is no immunological cross reaction among them. It is suggested that the expansion of H49/calpain repeats only occurred in T. cruzi after separation of a T. cruzi ancestor from other trypanosomatid lineages. Immunofluorescence and immunoblotting experiments demonstrated that H49/calpain is located along the flagellum attachment zone adjacent to the cell body.Conclusions: H49/calpain contains large central region composed of 68-amino acid repeats tandemly arranged. They can be classified as calpain-like proteins as the cysteine protease catalytic triad is partially conserved in these proteins. H49/calpains could have a structural role, namely that of ensuring that the cell body remains attached to the flagellum by connecting the subpellicular microtubule array to it.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Beca Presidente de la Republica-ChileUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilUniv Antofagasta, Lab Bioquim, Dept Biomed, Antofagasta, ChileUniv Bandeirante São Paulo, São Paulo, BrazilUniv Brasilia, Dept Biol Celular, Inst Biol, Brasilia, DF, BrazilFiocruz MS, Ctr Pesquisa Rene Rachou CPqRR, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilWeb of Scienc