Physicochemical characterization of an aspin (rBm-33) from a filarial parasiteBrugia malayiagainst the important human aspartic proteases

Abstract

The aspartic protease inhibitory efficiency of rBm-33, an aspin from a filarial parasite Brugia malayi was investigated. rBm-33 was found to be thermostable up to 90°C and it forms a stable 'enzyme-product' complex with human pepsin. Aspartic protease inhibitory activity was investigated using UV spectroscopy and isothermal titration calorimetry. Our results suggest that rBm-33 inhibits the activity of important human aspartic proteases that were examined with binding constants (Kb) values between 10.23 × 10(3) and 6.52 × 10(3) M(-1). The binding reactions were enthalpy driven with ΔHb values between -50.99 and -46.07 kJ mol(-1). From kinetic studies, pepsin inhibition by rBm-33 was found to be linear competitive with an inhibition constant (Ki) of 2.5 (±0.8) nM. Because of the inhibitory efficacy of Bm-33 against important human aspartic proteases which play a vital role in immune-regulation along with other functions, Bm-33 can be projected as a drug target for the filariasis.

A Structural Biology Approach to Understand Human Lymphatic Filarial Infection

“…This correlates with our previous iso-thermal calorimetric titration experiment results indicating that the preference of protease inhibition by Bm-Aspin is in the following order: pepsin>rennin>cathepsin-E>cathepsin-D [20]. Such mode of inhibition was comparable to that of aspartic protease inhibition by pepstatin, suggesting that the protease inhibition of Bm-Aspin is similar to that of pepstatin mode of inhibition [37].…”

“…Our data clearly indicates that the protease inhibition by Bm-Aspin was found to be competitive for pepsin and cathepsin-E, non-competitive for renin and mixed for cathepsin-D. Similar trend of linear competitive inhibition was observed with Pepsin when Casein was used as a substrate [20]. Thus, this finding confirms the reproducibility of pepsin inhibition kinetics by Bm-Aspin and also the ability to use Casein as a substrate to determine the inhibition constant.…”

“…Though the functional characterizations of serpins and cystatins have been reported [11], [12], the studies on aspins and its inhibition properties are not fully understood. Our previous studies reported that the Brugia malayi Aspin inhibits the important human aspartic proteases [20]. In continuation to our earlier work, the present paper reports the inhibition kinetics using a specific substrate and also reports the feasibility of structure determination of Bm-Aspin by NMR.…”

“…Determination of inhibition constant for pepsin inhibition activity of Bm-Aspin was reported with Casein as a substrate in our previous studies [20]. Formation of macroscopic aggregates, hazy zones, and high background with poor readability might sometimes limits the usage of casein as a substrate in monitoring the aspartic protease activity [32]–[34].…”

“…In addition to this, we have recently reported the pepsin inhibition activity and physicochemical characterization of Bm-Aspin [19]–[20]. Interestingly, this inhibitor has been reported to inhibit the important human aspartic proteases such as pepsin, renin, cathepsin-E and cathepsin-D.…”