In agreement with the evidence on the Plg binding function

In agreement with the evidence on the Plg-binding function of tapeworm enolases, eukaryotic enolases have been shown to be multifunctional proteins, with other activities besides their role as glycolytic enzymes (Pancholi, 2001). In this respect, several flatworms have been proved to express enolase as a tegumentary protein; its exposure on the external surface has also been suggested (Jolodar et al., 2003; De la Torre-Escudero et al., 2010; Wang et al., 2011; Figueiredo et al., 2015; Zhang et al., 2015). Other reports have found enolase as an excretion/secretion (E/S) protein in several flatworms, including T. solium (Bernal et al., 2004; Hewitson et al., 2009; Virginio et al., 2012; Victor et al., 2012). In any case, either as E/S products or as surface proteins, enolases have been associated to Plg binding and activation during parasite invasion of the host tissues (Ghosh and Jacobs-Lorena, 2011; González-Miguel et al., 2012, González-Miguel et al., 2015). In this study, we confirmed that T. solium enolase shows a widespread distribution in cysticercus tissues (Fig. 4); in the TUG-770 clinical wall, enolase was detected in the tegument and in subtegumental cytons that are assumed to elaborate E/S products to be sent to the tegument; in the invaginated scolex, enolase was clearly present in the tegument of the spiral canal and other cellular compartments. This widespread tissue distribution is compatible with both an enzyme with a metabolic role and a protein involved in host-parasite interplay. The antibody used in immunolocalization studies clearly recognized TsEnoA, as demonstrated by mass spectrometry and Western blot assays. In contrast, it failed to recognize TsEno4, and this was then confirmed by Western blot. However, the possibility of cross-reaction at least with TsEnoC cannot be ruled out, considering that TsEnoB did not seem be expressed in cysticerci. Ligand blotting assays demonstrated that T. solium cysticerci expressed several proteins with Plg-binding properties (Fig. 1B). Plg ligands have been reported as involved in the pathogenesis of other parasites (Ghosh et al., 2011; González-Miguel et al., 2013). The role of T. solium enolase as a Plg-binding protein was initially suggested by its identification in Plg-binding spots in the 2D/SDS-PAGE, by MS (Table 2). The high coverage of the amino acid sequence determined by MS for spots 6 and 7 allowed identification of TsEnoA. In contrast, TsEnoB, TsEnoC, and TsEno4 were not detected. The other Plg-binding proteins found in T. solium cysticerci (fasciclins 1 and 2, MAPK, anexin, actin, and cytosolic malate dehydrogenase) require detailed characterization studies for their involvement on Plg activation. TsEnoA contains 433 amino acid residues in a sequence that shows high identity with enolases from other Taeniid parasites (T. multiceps: 99%, T. pisiformis: 96%, E. granulosus: 95%, and E. multilocularis: 94%); identity rates with other platyhelminths like Fasciola spp. and Schistosoma spp. ranged from 78 to 72%. This highly conserved amino acid sequence was expected, considering the crucial metabolic role of this and other glycolytic enzymes (Pancholi, 2001). Previous studies on enolase sequences, including helminth parasites, have proposed that Plg binding involves an internal motif such as 248FYDKERKVY256, described for bacteria (Bergmann et al., 2003; Ehinger et al., 2004; Bernal et al., 2004; Vanegas et al., 2007; Ghosh et al., 2011). A putative Plg-binding motif was also identified in TsEnoA: 251FYQDGKYNL259 (Fig. 3). Equivalent ligand blotting assays (Fig. 5A) and other determinations confirmed the Plg-binding ability of rTsEnoA (Fig. 5B). The competitive inhibitor εACA also apparently decreased Plg-enolase binding. rTsEnoA was also demonstrated to increase Plg activation by tPA more than twice, suggesting that enolase potentiates the action of this physiological plasminogen activator (Fig. 5C). Only a small decrease in Plg activation by tPA was observed when the competitive inhibitor εACA, that blocks Plg binding to exposed lysine residues, was added to the reaction mixture, suggesting that Plg binding and activation by rTsEnoA did not significantly involve lysine residues. Clearly, εACA is not an adequate Plg binding inhibitor for TsEnoA, in agreement with reports about enolase in other pathogenic organisms (Rojas et al., 2008; Floden et al., 2011; Zhang et al., 2015). Exploration through directed mutagenesis could shed some light on this possibility.