title: 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.
  
  
  RESULTS
  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.
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, São Paulo, Brasil.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Galetović
authors.forename: Renata T
authors.initials: RT
authors.lastname: Souza
authors.forename: Marcia R M
authors.initials: MR
authors.lastname: Santos
authors.forename: Esteban M
authors.initials: EM
authors.lastname: Cordero
authors.forename: Izabela M D
authors.initials: IM
authors.lastname: Bastos
authors.forename: Jaime M
authors.initials: JM
authors.lastname: Santana
authors.forename: Jeronimo C
authors.initials: JC
authors.lastname: Ruiz
authors.forename: Fabio M
authors.initials: FM
authors.lastname: Lima
authors.forename: Marjorie M
authors.initials: MM
authors.lastname: Marini
authors.forename: Renato A
authors.initials: RA
authors.lastname: Mortara
authors.forename: José Franco
authors.initials: JF
authors.lastname: da Silveira
mesh_terms: D015139:Blotting, Southern; D015153:Blotting, Western; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D016521:Electrophoresis, Gel, Pulsed-Field; D004591:Electrophoresis, Polyacrylamide Gel; D005407:Flagella / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000235:genetics / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D002154:Calpain
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