Abstract
BackgroundCysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity.Methodology/Principal FindingsThe data analysis revealed that the presence of heparin affects all steps of the enzyme reaction: (i) it decreases 3.5-fold the k 1 and 4.0-fold the k −1, (ii) it affects the acyl-enzyme accumulation with pronounced decrease in k 2 (2.7-fold), and also decrease in k 3 (3.5-fold). The large values of ΔG = 12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys25)-S−/(His163)-Im+ H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme.Conclusions/SignificanceTaken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface.
Highlights
Papain-like cysteine proteases have been identified in parasitic organisms, such as T. cruzi, T. brucei and different Leishmania spp. (CPA, CPB, CPC) [1]
Based on the temperature dependence upon the kinetics parameters kcat/KM and kcat for hydrolysis of Z-FR-MCA by the rCPB2.8 enzyme, we evaluated the individual constants k1, k21, k2 and k3 of the hydrolytic reactions in the absence or in the presence of 40 mM heparin using the procedure reported by Ayala and Di Cera [42] and Judice et al [43,44] which was based on the assumption that the hydrolytic process of a cysteine protease occurs as described below: The values of the kinetic parameters kcat and KM for rCPB2.8 was determined using 100 mM sodium acetate buffer, 20% glycerol, 5 mM DTT, pH 5.5 activating the enzyme for 10 min in the temperature range 10uC to 35uC
Activity The effect of heparin upon the rCPB2.8 endopeptidase activity was monitored with the aid of its fluorogenic substrate Z-FRMCA, covering the rCPB2.8 subsites from S2 to S91 [51,52]
Summary
Papain-like cysteine proteases have been identified in parasitic organisms, such as T. cruzi (cruzain), T. brucei (trypanopain, TbCatB) and different Leishmania spp. (CPA, CPB, CPC) [1]. Papain-like cysteine proteases have been identified in parasitic organisms, such as T. cruzi (cruzain), T. brucei (trypanopain, TbCatB) and different Leishmania spp. The cysteine proteinase (CP) activity of Leishmania mexicana is considerably greater in the mammalian amastigote form than in the promastigote forms [2]. These classes of CPs exist as multiple isoenzymes [3,4,5,6,7], which are encoded by a tandem array of 19 similar CPB genes [7,8,9,10,11]. Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. We have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
