Trypanosoma evansi Sialidase: Surface Localization, Properties and Hydrolysis of Ghost Red Blood Cells and Brain Cells-Implications in Trypanosomiasis

Abstract

A membrane-bound sialidase was isolated from blood stream (BS) Trypanosoma evansi partially purified and characterized. The enzyme is a glycosyl phosphatidyl inositol (GPI) membrane anchored protein. It was solubilized from T.evansi cells recovered from infected camel blood by detergent treatment with Triton CF 54 and partially purified by a series of chromatography steps. The enzyme was optimally active at pH 5.5 and 37 °C. It had a Kᴍ and V_max values of 4.8 x 10^-6ᴍ and 3.75 x 10^-6 mol/min.mg protein with Neu5Acα2, 3lac as substrate respectively. The Kᴍ and V_max values with fetuin (4-nitrophenyl-oxamic acid) as substrate were 2.9 x 10^-2ᴍ and 4.2 \ 10^-3 mol/min.mg protein in the same respect. Kinetic analysis with methly umbelliferyl sialate (MU-Neu5Ac) gave Kᴍ and V_max values of 0.17 mᴍ and 0.84 mmol/min.mg protein respectively. The T. evansi SD could hydrolyse internally linked sialic acid residues of the ganglioside GM₂ , but was inactive towards colomic acid, and Neu5Ac2, 6. lac. When ghost red blood cell (RBC) was used as substrate, it desialylated the RBC in the following order of efficiency; mouse, rat, camel, goat, and dog. Similarly, cerebral cells isolated from BalbC mouse was desialylated by the T. evansi SD.

Inhibition studies using 2-deoxy-2, 3 didehydro-N-acetyl neuraminic acid (NeuAc2, 3en) against MU-Neu5Ac revealed a competitive inhibition pattern with K_i of 5.8 μm. The enzyme was also inhibited non-competitively by parahydroxy oxamic acid (pHOA), and competitively by N-ethylmaleimide and N-bromosuccinate with K_i values of 25, 42, and 53 μm, respectively. It was activated by Mg²⁺ ion and inhibited by Cu²⁺ and Zn²⁺.