A study on β-glucan binding protein (β-GBP) and its involvement in phenoloxidase cascade in Indian white shrimp...

The present study reports the purification of novel immune molecule β-1, 3 glucan binding protein from the heamolymph of the Indian white shrimp, Fenneropenaeus indicus (Fiβ-GBP). The purified Fiβ-GBP had 95kDa molecular weight in SDS-PAGE analysis. MALDI-TOF/TOF analysis revealed that the purified Fiβ-GBP showed similarity to various crustacean proteins; 48 and 46% similarity was observed for β-1, 3 glucan binding protein of Chinese white shrimp Fenneropenaeus chinensis and banana shrimp Fenneropenaeus merguiensis, with MOWSE score of 3.11e+12 and 2.05e+8, respectively. The phenoloxidase activity (PO) of Fiβ-GBP was evaluated and, in the presence of laminarin, PO activity increased significantly. Substrate specificity assay demonstrated that Fiβ-GBP had the specific binding site for soluble or insoluble β-glucan (laminarin), since the PO activity increased in the presence of laminarin when compared to other sugars. Enzymatic activities revealed that the optimum temperature and pH for Fiβ-GBP activating PO were 40°C and pH 7-8. Moreover, even at 100°C Fiβ-GBP enhanced PO activity highlighting that Fiβ-GBP was thermostable and thermophilic in nature. Among various divalent metallic ions, Fiβ-GBP significantly promoted the PO activity in presence of Mg²⁺ and Ca²⁺. The breakdown of para nitroanilide from N_α-Benzoyl-l-Arginine 4-Nitroanilide hydrochloride showed that serine protease activity was induced by Fiβ-GBP and also increased concentration of Fiβ-GBP evoked the activity. Furthermore, hemolytic activity tests revealed that PO reaction product induced RBC membrane damage and cell shrinkage. Lastly, Baclight bacterial viability assays showed maximum killing effect of PO reaction product on both Gram positive and Gram negative bacteria.