Evidence of the presence of a calmodulin-sensitive plasma membrane Ca2+-ATPase in Trypanosoma equiperdum

• Published in: Molecular and biochemical parasitology 2017-04, Vol.213, p.1-11
• Main Authors: Pérez-Gordones, María Carolina, Ramírez-Iglesias, José Rubén, Cervino, Vincenza, Uzcanga, Graciela L., Benaim, Gustavo, Mendoza, Marta
• Format: Article
• Language: English
• Subjects: Animal trypanosomiasis; Ca2+-ATPase; Calmodulin; PMCA; Trypanosoma equiperdum
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/j.molbiopara.2017.02.001

[Display omitted] •Trypanosoma equiperdum presents a Calmodulin sensitive PMCA.•T. equiperdum PMCA contains P-type ATPase conserved characteristics.•T. equiperdum presents in its C-terminal a 1–18 motif within CaM-binding domain.•Plasma membrane fraction (PMF) of T. equiperdum presents Ca2+-ATPase activity.•PMF of T. equiperdum presents a 120kDa protein that is recognized by 5F10 antibody.•T. equiperdum PMCA is purified by CaM-sepharosa and binds CaM-Biotinylated. Trypanosoma equiperdum belongs to the subgenus Trypanozoon, which has a significant socio-economic impact by limiting animal protein productivity worldwide. Proteins involved in the intracellular Ca2+ regulation are prospective chemotherapeutic targets since several drugs used in experimental treatment against trypanosomatids exert their action through the disruption of the parasite intracellular Ca2+ homeostasis. Therefore, the plasma membrane Ca2+-ATPase (PMCA) is considered as a potential drug target. This is the first study revealing the presence of a PMCA in T. equiperdum (TePMCA) showing that it is calmodulin (CaM) sensitive, revealed by ATPase activity, western-blot analysis and immuno-absorption assays. The cloning sequence for TePMCA encodes a 1080 amino acid protein which contains domains conserved in all PMCAs so far studied. Molecular modeling predicted that the protein has 10 transmembrane and three cytoplasmic loops which include the ATP-binding site, the phosphorylation domain and Ca2+ translocation site. Like all PMCAs reported in other trypanosomatids, TePMCA lacks a classic CaM binding domain. Nevertheless, this enzyme presents in the C-terminal tail a region of 28 amino acids (TeC28), which most likely adopts a helical conformation within a 1–18 CaM binding motif. Molecular docking between Trypanosoma cruzi CaM (TcCaM) and TeC28 shows a significant similarity with the CaM-C28PMCA4b reference structure (2kne). TcCaM-TeC28 shows an anti-parallel interaction, the peptide wrapped by CaM and the anchor buried in the hydrophobic pocket, structural characteristic described for similar complexes. Our results allows to conclude that T. equiperdum possess a CaM-sensitive PMCA, which presents a non-canonical CaM binding domain that host a 1–18 motif.