Characterization and partial purification of cystatins from Malian medicinal plants

Abstract

Cysteine proteases (CPs) expressed by Schistosoma mansoni (Sm) participate in the hydrolysis of host hemoglobin [1]. Cystatins are proteinaceous CP inhibitors (CPIs) present in humans as well as in plants. Two Malian medicinal plants used against schistosomiasis, Securidaca longepedunculata Fres (root: SR and leaf: SL) and Stylosanthes erecta Beauv. (aerial part: SE), were investigated for presence of cystatins. Cystatins were purified by extraction of powdered plant material with Tris-HCl, followed by affinity chromatography, gel filtration (GF) and anion exchange chromatography (AEC). S. mansoni CPs activity and protease inhibitory assays were performed using fluorogenic substrates according to [2]. High papain inhibition observed in all crude extracts indicated presence of CPIs. The papain inhibitory activity in the three extracts eluted into one single peak each, after affinity chromatography. These fractions were resolved by GF into papain inhibitory activities consistent with the presence of cystatins. SmCP activities were also inhibited by these cystatins. The strongest papain and SmCPs inhibitory activities were observed in SR. After AEC, one papain inhibitory peak which weakly inhibited SmCP was obtained from SR. On SDS-PAGE (under reduction), this peak appeared as a single 88kDa- band. The purified cystatins were characterized with respect to their papain and SmCPs inhibitory activities and by Mr. Cystatins with strong papain and moderate SmCPs inhibitory activity are isolated from the extracts and could participate into the antischistosomal activity of the studied plants.