br Materials and methods br Results br Discussion Enolase
Materials and methods
Discussion Enolase (2-phospho-D-glycerate hydrolase) is a glycolytic enzyme that is also involved with various important roles in the intracellular and extracellular space (Copley, 2003; S. Zhang et al., 2015) such as transcriptional regulation, apoptosis, growth, and motility (Kim and Dang, 2005; Sriram et al., 2005; S. Zhang et al., 2015). While previous studies reported on recombinant enolases from T. pisiformis (S. Zhang et al., 2015), T. multiceps (Li et al., 2015), and T. asiatica (Du et al., 2009), an enolase from T. solium was conspicuously absent. Based on the amino Doxofylline sale sequence, T. solium enolase appeared in a monophyletic group of flatworms (T. asiatica, E. granulosus, F. hepática, C. sinensis, S. japonicum and S. bovis), with high identity with enolases from other cestodes like T. asiatica and E. granulosus. The T. solium enolase shows conserved amino acids that includes the enolase signature (LLLKVNQIGSVTES), the active site residues and the catalytic motif (SHRSGETED). Recombinant rEnoTs not only exhibited enzymatic activity by hydrolyzing the specific substrate 2-PGA into PEP, but its specific activity (60 U/mg) proved to be noticeably higher than those reported for other recombinantly expressed helminth enolases such as those from C. sinensis (36.5 ± 0.31 U/mg; (X. Wang et al., 2011)), S. japonicum (35.8 ± 2 U/mg; (Yang et al., 2010)), T. multiceps (46.91 U/mg protein; (Li et al., 2015)) and T. pisiformis (30.71 ± 2.15 U/mg; (S. Zhang et al., 2015)). Moreover, in comparison with enolases of other worms, the affinity of rEnoTs for 2-PGA (Km = 0.091 mM) was higher than the enolase from S. japonicum (Km = 0.53 mM, pH: 6.8–7.1; (Yang et al., 2010)) but lower than that of C. sinensis (Km = 0.05 mM, pH: 7.0–8.0; (X. Wang et al., 2011)). Because post-translation modifications can affect subcellular localization and various other properties of enolase, such as immunogenicity (Gan et al., 2010; Sotillo et al., 2008), we recombinantly expressed T. solium enolase in Sf9 insect cells. As it happens, rEnoTsBac turned out to be 2.2 kDa heavier than rEnoTs, with this discrepancy in molecular weights likely attributable to the presence of various glycans on the former, as 5 glycosylation sites were predicted to exist in the T. solium enolase. Such glycosylation of an enolase was previously demonstrated in E. caproni (Sotillo et al., 2008). We analyzed the localization of native cysticercus T. solium enolase by evaluating recognition of fluid and extracts with anti-rEnoTs hyperimmune sera from rabbits. This experiment confirmed that native T. solium enolase is present in these fractions, implying that it is expressed for use both at the cellular level (i.e. in the cytosol and cell membrane) and as an excreted/secreted product (i.e. in the vesicular fluid) – an expression profile similar to what was previously reported in an enolase from T. pisiformis metacestode (S. Zhang et al., 2015) and recently confirmed in T. solium cysticercus tissues by immunohistochemistry (Ayon-Nunez et al., 2018). This suggests that enolase is widely distributed throughout T. solium cysticercus, being its position at tegument level remarkable, which has been confirmed in different flatworm studies by immunofluorescence (Du et al., 2009; Gan et al., 2010; Mulvenna et al., 2010; Ramajo-Hernandez et al., 2007). This finding is also consistent with both the putative transmembrane domain predicted in the T. solium enolase sequence and with previous studies, as other enolases have been shown to be vital to other cellular processes via their functionality as excreted/secreted proteins in different flatworms (Perez-Sanchez et al., 2006; Ramajo-Hernandez et al., 2007; Sotillo et al., 2008) in addition to their important enzymatic roles in the cytoplasm. For example, excreted/secreted enolases on the plasma membranes of T. pisiformis and T. multiceps (Li et al., 2015; S. Zhang et al., 2015) may be implicated in the binding of plasminogen and its activation to plasmin, thereby aiding in the degradation and proteolysis of the host\'s extracellular-matrix and facilitating the subsequent establishment of the cyst in the tissue. Moreover, we speculate that excreted/secreted enolase in the vesicular fluid of T. solium function as targets for permeable IgG antibodies, which could eventually be digested into individual amino acids for use by the parasite. If true, this would establish enolase as being involved with both nutrient acquisition as well as evasion of the host immune response (Plow and Das, 2009; Ramajo-Hernandez et al., 2007; White et al., 1997).