IDENTIFICATION OF TARGET PROTEIN OF ALPHA-2-MACROGLOBULIN FROM WHITE SHRIMP Litopenaeus vannamei BY BIOCHEMICAL AND MOLECULAR MODELING TECHNIQUES

Abstract

Alpha-2-macroglobulin (A2M) is an evolutionarily conserved protease inhibitor. In invertebrates including shrimp, A2M has been shown to be crucially involved in immune responses. Herein, A2M from the Pacific white shrimp Litopenaeus vannamei named LvA2M was characterized for its immune function against bacterial proteases. The recombinant LvA2M protein (rLvA2M) with the predicted molecular mass and pI of 166.9 kDa and 5.77, respectively, was produced in Escherichia coli but was not successfully purified by Ni-NTA column chromatography. Then, the native LvA2M was partially purified from the plasma of P. vannamei and exhibited inhibitory activity against trypsin in a dose-dependent manner. The cleavage site on bait region of LvA2M by trypsin was confirmed by molecular docking. Using agar disc diffusion method, the inhibitory activity against Vibrio harveyi proteases of native LvA2M was found to be much higher than rLvA2M. Moreover, native LvA2M also inhibited other bacterial secreted proteases from Staphylococcus aurues, Micrococcus luteus, Escherichia coli and Vibrio parahaemolyticus. Unfortunately, the specific protease of LvA2M could not be identified by co-immunoprecipitation and zymography techniques. Consequently, the full length (PAP6F) and the partial sequence (PAP6P) of PAP6 gene encoding a metalloprotease from V. harveyi were cloned and expressed in E. coli system to investigate whether PAP6 is the target protease of LvA2M. The recombinant proteins of PAP6F and PAP6P did not exhibit protease activity but both bound to native LvA2M with the dissociation constant (Kd) of 8.1×10-7M and 1.1×10-6M, respectively. Since A2M has been reported to bind toxin produced from pathogens, so we tested if LvA2M could neutralize Pir toxins secreted from V. parahaemolyticus, the causative agent of the deadly disease Early mortality Syndrome (EMS). The binding assay showed that LvA2M could interact with PirA and PirB toxins in vitro with the Kd of 3.6×10-7M and 5.8×10-7M, respectively. This binding was confirmed by molecular docking. Furthermore, LvA2M also neutralized toxic effect of the crude Pir toxins resulting in a decrease of cumulative mortality of shrimp. Taken together, our results suggested that LvA2M might participate in many immune responses to inhibit different target proteases and toxins of invading bacteria as well as controlling the host immune reactions.