Molecular characterization of calcineurin B from the non-virulent Trypanosoma rangeli kinetoplastid indicates high gene conservation M. Montenegro • C. Cardenas • C. Cuervo • C. Bernal • E. C. Grisard • M. C. Thomas • M. C. Lopez • C. J. Puerta Received: 26 September 2012 / Accepted: 29 April 2013 / Published online: 16 May 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract Calcineurin B, the regulatory subunit of calci- neurin, a serine/threonine protein phosphatase, is highly conserved throughout the evolutionary scale including trypanosomatids such as Trypanosoma cruzi, and Leish- mania major. Thus, in these flagellates the protein is required for mammalian host cell invasion and virulence and stress responses. With the aim of determining the presence of calcineurin B in Trypanosoma rangeli, a non- virulent trypanosome for mammals, the respective gene was amplified by PCR, cloned and sequenced. Two sequences of 531 bp in length showing a nucleotide poly- morphism (314A [ C) were obtained in spite of a single- copy gene was revealed by Southern blot. These sequences, probably the alleles from the gene, showed a 79 % of identity with those from T. cruzi and clustered as the sister group of this trypanosome species in a Maximum Parsi- mony analysis. Deduced amino acid sequence comparison with trypanosomatids and other organisms through the phylogenetic scale as well as the obtained protein structural homology model suggested the presence of the four potential EF-hand regions and the corresponding calcium binding sites of the last three of these domains. Having assessed the expression of this protein in T. rangeli epimastigotes, and taking into account the following facts: (i) calcineurin inhibitors have inhibitory effect on the in vitro replication of T. cruzi, (ii) L. major promastigote growth is inhibited by chelating agents, and (iii) T. rangeli does not seem to productively infect mammalian cells, it is hypothesized herein that the function of this protein in T. rangeli is required for epimastigote growth. Keywords Calcineurin Á Calcineurin B Á Trypanosoma rangeli Á EF-hand Á Calcium binding Á Protein structural homology model Introduction Calcineurin (Cn) is a serine/threonine protein phosphatase, regulated by calcium and calmodulin (CaM), which par- ticipates in signal transduction pathways. This protein, highly conserved throughout the evolutionary scale, is a heterodimer formed by a catalytic subunit of approximately 60 kDa known as calcineurin A (CnA) and a regulatory subunit of approximately 19 kDa called calcineurin B (CnB). In addition to the catalytic domain, CnA includes three regulatory domains: the CnB binding domain, the Electronic supplementary material The online version of this article (doi:10.1007/s11033-013-2590-7) contains supplementary material, which is available to authorized users. M. Montenegro Á C. Cuervo Á C. Bernal Á C. J. Puerta (&) Laboratorio de Parasitologı ´a Molecular, Departamento de Microbiologı ´a, Facultad Ciencias, Pontificia Universidad Javeriana, Carrera 7 No. 43–82, Bogota ´, Colombia e-mail: cpuerta@javeriana.edu.co C. Cardenas Nu ´cleo de Biotecnologı ´a Curauma, Pontificia Universidad Cato ´lica de Valparaı ´so, Avenida Universidad 330, Valparaı ´so, Chile E. C. Grisard Laborato ´rios de Protozoologia e de Bioinforma ´tica, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Santa Catarina, Caixa Postal 476, Floriano ´polis, SC 88040-900, Brazil M. C. Thomas Á M. C. Lopez Instituto de Parasitologı ´a y Biomedicina Lo ´pez Neyra, Consejo Superior de Investigaciones Cientı ´ficas (IPBLN-CSIC), Parque Tecnolo ´gico de Ciencias de la Salud, Avda. del Conocimiento, s/n, 18100 Granada, Spain 123 Mol Biol Rep (2013) 40:4901–4912 DOI 10.1007/s11033-013-2590-7