Characterization of sialidase from bloodstream forms ofTrypanosoma vivax

Abstract

Sialidase (EC: 3.2.1.18) from Trypanosoma vivax (Agari Strain) was isolated from bloodstream forms of the parasite and purified to apparent electrophoretic homogeneity. The enzyme was purified 77-fold with a yield of 32% and co-eluted as a 66-kDa protein from a Sephadex G 110 column. The T. vivax sialidase was optimally active at 37 degrees C with an activation energy (E(a)) of 26.2 kJ mole(-1). The pH activity profile was broad with optimal activity at 6.5. The enzyme was activated by dithiothreitol and strongly inhibited by para-hydroxy mercuricbenzoate thus implicating a sulfhydryl group as a possible active site residue of the enzyme. Theenzyme hydrolysed Neu5Ac2,3lac and fetuin. It was inactive towards Neu5Ac2,6lac, colomic acid and the gangliosides GM1, and GDI. Initial velocity studies, for the determination of kinetic constants with fetuin as substrate gave a V(max) of 142.86 micromol h(-1) mg(-1) and a K(M) of 0.45 mM. The K(M) and V(max) with Neu5Ac-2,3lac were 0.17 mM and 840 micromole h(-1) mg(-1) respectively. The T. vivax sialidase was inhibited competitively by both 2,3 dideoxy neuraminic acid (Neu5Ac2,3en) and para-hydroxy oxamic acid. When ghost RBCs were used as substrates, the enzyme desialylated the RBCs from camel, goat, and zebu bull. The RBCs from dog, mouse and ndama bull were resistant to hydrolysis.

Host-Parasite Interactions in Trypanosomiasis: on the Way to an Antidisease Strategy

“…Although the experimental demonstration of sialidase activity in T. congolense bloodstream forms was quite recent (116), a direct interaction of this trypanosome with RBC resulting in alteration of these cells had already been shown in earlier studies (13,14). T. vivax sialidase was shown to present some host specificity, desialating camel, goat, and zebu RBC, while those from mice, dogs, and NЈDama bulls were shown to be resistant (26). In T. evansi, an isolated sialidase was found to desialate ghost RBC in the following order of efficiency: mouse, rat, camel, goat, and dog (115).…”

“…RBC surface alterations have indeed been shown to be caused by bloodstream T. vivax, T. congolense, and T. evansi sialidases, thus leading to their subsequent phagocytosis (26,40,115,116). Although the experimental demonstration of sialidase activity in T. congolense bloodstream forms was quite recent (116), a direct interaction of this trypanosome with RBC resulting in alteration of these cells had already been shown in earlier studies (13,14).…”

“…Indeed, sialic acid was shown to contain less O-acetyl than glycolyl groups in zebu cattle, while the inverse situation occurred in trypanotolerant NЈDama cattle. Characterization of bloodstream T. congolense, T. evansi, and T. vivax sialidases showed pH optima of 6, 5.5, and 6.5, respectively, and a common optimum temperature of 37°C (26,115,116). Sialidases can, moreover, be considered in the context of alterations other than anemia and are known to desialate thrombocytes, leukocytes, and brain cells as well (115).…”

Production, purification and crystallization of atrans-sialidase fromTrypanosoma vivax

“…In other cases, trypanosomal (trans-) sialidases are produced in the so-called 'bloodstream form' (i.e. the mammalian host stage) and their actions are thought to be directly correlated with the induced pathology in the infected mammalian host (Schenkman et al, 1991;Nok & Balogun, 2003;Buratai et al, 2006). In T. cruzi, the only intracellular trypanosome, it has been shown that its transsialidases play a role in host-cell invasion, parasite virulence and immune suppression (Schenkman et al, 1992;Buschiazzo et al, 2002;Jacobs et al, 2010;Giorgi & de Lederkremer, 2011).…”

“…In T. cruzi, the only intracellular trypanosome, it has been shown that its transsialidases play a role in host-cell invasion, parasite virulence and immune suppression (Schenkman et al, 1992;Buschiazzo et al, 2002;Jacobs et al, 2010;Giorgi & de Lederkremer, 2011). In T. vivax infections, trans-sialidase activity has been proposed to be linked to the typically observed severe anaemia (Bratosin et al, 1998;Buratai et al, 2006;Guegan et al, 2013). Hence, for disease-associated trypanosomal transsialidases, unravelling their structure-function relationship might yield insights into the molecular mechanisms behind the onset of trypanosomosis-induced pathologies.…”