Molecular & Biochemical Parasitology 165 (2009) 95–102 Contents lists available at ScienceDirect Molecular & Biochemical Parasitology Characterization and binding affinities of SmLANP: A new Schistosoma mansoni member of the ANP32 family of regulatory proteins C.S. Pinheiro a,d , O. Staub b , J. Mulvenna c , A. Loukas c , M.K. Jones c,e , E.M. Rabelo d,∗ a Programa de Pós-Graduac ¸ão e Pesquisa, Santa Casa de Belo Horizonte, Av. Francisco Sales 1111, 9-andar, Ala C, Belo Horizonte, MG, 30150-221, Brazil b Département de Pharmacologie et de Toxocologie, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland c Queensland Institute of Medical Research, 300 Herston Road, Queensland, Brisbane 4006, Australia d Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Presidente Antônio Carlos 6627, Campus Pampulha, CEP 31270-901 Belo Horizonte, Minas Gerais, Brazil e School of Veterinary Sciences, The University of Queensland, Brisbane, Qld 4072, Australia article info Article history: Received 1 December 2008 Received in revised form 15 January 2009 Accepted 16 January 2009 Available online 30 January 2009 Keywords: Schistosoma mansoni Leucine-rich repeat Immunolocalization Gene characterization Yeast two hybrid Pull down abstract Members of the leucine-rich repeat protein family are involved in diverse functions including protein phosphatase 2-inhibition, cell cycle regulation, gene regulation and signalling pathways. A novel Schis- tosoma mansoni gene, called SmLANP, presenting homology to various genes coding for proteins that belong to the super family of leucine-rich repeat proteins, was characterized here. SmLANP was 1184bp in length as determined from cDNA and genomic sequences and encoded a 296 amino acid open reading frame that spanning from 6 to 894 bp. The predicted amino acid sequence had a calculated molecular weight of 32 kDa. Analysis of the predicted sequence indicated the presence of 3 leucine-rich domains (LRR) located in the N-terminal region and an aspartic acid rich region in the C-terminal end. SmLANP transcript is expressed in all stages of the S. mansoni life cycle analyzed, exhibiting the highest expression level in males. The SmLANP protein was expressed in a GST expression system and antibodies raised in mice against the recombinant protein. By immunolocalization assay, using adult worms, it was shown that the protein is mainly present in the cell nucleus through the whole body and strongly expressed along the tegument cell body nuclei of adult worms. As members of this family are usually involved in protein–protein interaction, a yeast two hybrid assay was conducted to identify putative binding part- ners for SmLANP. Thirty-six possible partners were identified, and a protein ATP synthase subunit  was confirmed by pull down assays, as a binding partner of the SmLANP protein. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Genes encoding proteins involved in gene regulation and development are of great interest in understanding the complex multi-generational life cycle and intra-mammalian differentiation of the digenean parasite Schistosoma mansoni. This trematode is the etiologic agent of hepato-splenic schistosomiasis in Africa and South America and is responsible for significant human disease burden. Large scale sequencing of S. mansoni employing an EST (expressed sequence tag) approach has led to the discovery of many new genes, which were unlikely to be identified using classical pro- cedures based on phenotype [1–3]. Genes encoding proteins that in other organisms function in gene regulation, cell cycle regulation and signalling pathways are represented in the S. mansoni tran- scriptome [4]. Here, we characterise a gene isolated from an adult ∗ Corresponding author. Tel.: +55 31 34992851; fax: +55 31 34992970. E-mail address: rabelo@icb.ufmg.br (E.M. Rabelo). S. mansoni cDNA library that encodes a putative leucine-rich repeat acidic nuclear protein (Anp32a/LANP), which in other organisms functions in a range of intracellular signalling pathways. The Schis- tosome cDNA is named SmLANP due to the putative ORF with three Leucine-rich Repeat domains (LRR). LRRs are 20–29-residue sequence motifs present in a num- ber of proteins with diverse functions. The primary function of these motifs appears to be to provide a versatile structural frame- work for the formation of protein–protein interactions [5]. Proteins with LRRs belong to a family of acidic leucine-rich nuclear phos- phoproteins (ANP32 Family) associated with different activities, including cell proliferation (pp32) [6–10], mRNA stabilization in the pathogenesis of spinocerebellar ataxia type 1 (LANP) [9,11–14], intracellular signalling events (PHAPI and PHAPII) [9,15,16] and his- tone acetyltransferase inhibition activity (INHAT) [9,11,17]. ANP32 proteins are reported to bind microtubule associated proteins (MAPs) [9,11,18–20]. Other genes encoding proteins possessing the LRR domain have been described for S. mansoni, including SmLRR, an antigen with no defined function that shares cross-reactive 0166-6851/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2009.01.009