Purification and characterization of cysteine protease of Sarcocystis fusiformis from infected Egyptian water buffaloes

Abstract

AbstractSarcocystisspp. infects water buffaloes (Bubalus bubalis) causing sarcocystosis. In the present study,Sarcocystis fusiformiswas recognized in Egyptian water buffaloes based on histological observation and molecular analysis of internal transcribed spacer 1 (ITS1),18S ribosomalRNA (18S rRNA) and cytochrome c oxidase subunit I (COX-1) gene fragments. Chemotherapy and vaccines againstSarcocystisspp. could potentially target proteases because they may play a crucial role in the infection. Cysteine proteases are multifunctional enzymes involved in vital metabolic processes. However, the involvement of proteases inS. fusiforminfection has not yet been characterized. Here, the purification and study on some biochemical properties of protease isolated from cysts ofS. fusiformwere carried out. Protease with a molecular weight of 100 kDa was purified. LC–MS/MS analyzed the protein sequence of purified protease and the data suggested that the enzyme might be related to the cysteine protease. The purified protease exhibited maximum activity at pH 6 and a temperature of 50 °C. The Michaelis–Menten constant (Km), the maximum velocity (Vmax), and the turnover number (Kcat) were determined. The complete inhibition effect of cysteine inhibitors indicated that the purified enzyme is a cysteine protease. The results suggested thatS. fusiformproteolytic enzyme may be necessary for parasite survival in water buffaloes by digesting host tissues. Therefore, cysteine protease could be a suitable target for vaccinations.