Echinostoma caproni: Identification of enolase in excretory/secretory products, molecular cloning, and functional expression Antonio Marcilla a, * , Ana Pe ´rez-Garcı ´a a , Ana Espert a , Dolores Bernal b , Carla Mun ˜ oz-Antolı ´ a , Jose ´ Guillermo Esteban a , Rafael Toledo a a Departamento de Biologı ´a Celular y Parasitologia, Facultat de Farma ` cia, Universitat de Valencia, Av. V.A. Estelle ´s, s/n, 46100 Burjassot, Valencia, Spain b Departamento de Bioquı ´mica y Biologı ´a Molecular, Facultat de Cie `ncies Biolo ` giques, C/Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain Received 5 February 2007; received in revised form 12 March 2007; accepted 14 March 2007 Available online 27 March 2007 Abstract In order to investigate molecules that could be involved in host-trematode relationships, we have analysed the excretory/secretory products (ESP) of Echinostoma caproni following a proteomic approach. Actin, Gluthathione S-transferase (GST) and enolase have been identified in the ESP. Enolase, observed to be one of the most abundant proteins, was further characterized. The molecular cloning and in vitro expression in Escherichia coli of E. caproni enolase allowed us to determine that the protein contains 431 amino acids and a the- oretical MW of 46272 Da. E. caproni enolase shows high homology to other trematode enolases. The recombinant protein binds specif- ically to human plasminogen in vitro, as observed for the native protein, confirming its properties as a host-interacting molecule. Ó 2007 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: ESP, Excretory/secretory products; 2DE, Two-dimension gel electrophoresis; ORF, Open reading frame; Echinostoma caproni; Enolase; ESP; Molecular cloning and expression 1. Introduction Echinostoma caproni is an intestinal trematode which does not migrate within the tissue of its definitive host (Fried and Huffman, 1996). The use of this parasite as a model to study intestinal helminthiasis is based on the observation that it develops chronic or acute infections depending on the rodent host used (hamster or rat, respec- tively) (Toledo et al., 2004a; Toledo and Fried, 2005). The host response to E. caproni has been analysed by determining immunoglobulin production and also by the analysis of the histopathological changes in the intestines from hamsters and rats (Toledo et al., 2004a, 2005, 2006). These studies confirmed the immunogenic nature of excretory/secretory products (ESP), as previously sug- gested by Toledo et al. (2004b). However, the parasite mol- ecules of ESP involved in host-trematode relationships have yet to be characterized on the molecular level. In the related trematode species Echinostoma friedi, sev- eral cytoskeletal, stress-responsive and glycolytic proteins have been identified in the ESP (Bernal et al., 2006). One of the most highly expressed proteins was the glycolytic enzyme phosphopyruvate hydratase (enolase, EC 4.2.1.11) (Bernal et al., 2006). This protein is potentially important in host–parasite relationships since it has been identified as the main plasminogen-binding molecule in vitro in Fasciola hepatica (Bernal et al., 2004). Enolase is a glycolytic enzyme which may play roles in several other cellular functions (Pancholi, 2001). In this context, it has also been identified in the tegument and ESP of trematodes like Schistosoma spp. (Knudsen et al., 2005; Pe ´rez-Sa ´nchez et al., 2006), and nematodes like 0014-4894/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.03.011 * Corresponding author. Fax: +34 963544769. E-mail address: Antonio.marcilla@uv.es (A. Marcilla). www.elsevier.com/locate/yexpr Experimental Parasitology 117 (2007) 57–64