Role of purified β-1, 3 glucan binding protein (β-GBP) fromParatelphusa hydrodromusand their anti-inflammatory, antioxidant and antibiofilm properties

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Abstract

β- 1, 3-glucan binding protein (β-GBP), a pattern recognition protein (PRP), plays a critical role in triggering the innate immune response by detecting β-glucan found on the surface of microbes. In the present study, β-GBP was purified from the haemolymph of rice field crab Paratelphusa hydrodromus by affinity column chromatography. The monomeric protein Ph-β-GBP appeared as a single band with a molecular weight of approximately 95 kDa in SDS-PAGE analysis and its purity was determined to be 89% by HPLC. MALDI-TOF/TOF analysis revealed that, the purified 95 kDa protein display 36% similarity with β-GBP of crayfish Astacus lepidodactylus. Purified Ph-β-GBP exhibited increased agglutination, phagocytic activity and encapsulation in a dose-dependent manner, indicating the involvement of Ph-β-GBP in cellular immune response against pathogens in crustaceans. Moreover, addition of Ph-β-GBP increased the prophenoloxidase (proPO) and serine protease activity, possibly contributing to the clearance of pathogens. The antioxidant activity of Ph-β-GBP was determined by DPPH radical scavenging activity demonstrates maximum scavenging activity of 78.4%. In addition, RBC membrane stabilization and inhibition of protein (albumin) denaturation proved anti-inflammatory property of Ph-β-GBP. Furthermore, light microscopic and confocal laser scanning microscopic analysis revealed that the reactive compound (laminarin and Ph-β-GBP) reduced the biofilm thickness of Gram-positive (Enterococcus faecalis) and Gram-negative (Vibrio parahaemolyticus) bacteria at the concentration of 25 μg/ml. Taken together, our results demonstrate that, the β-GBP triggers proPO activating system in rice field crab P. hydrodromus and plays a vital role in innate defense mechanism against invading pathogens.

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