Recombinant expression and functional characterization of a C-type lectin(Fclectin)from the Chinese shrimp(Fenneropenaeus chinensis)

Abstract

Chinese shrimp(Fenneropenaeus chinensis)is distributed mainly along Chinese inshore areas,and is one of the most important farmed shrimp in China.The studies on innate immune responses of shrimps,especially on immune defense against the main crustacean pathogens,will provide more knowledge of shrimp immunity to prevent infectious diseases.Invertebrates do not possess an adaptive immune system based on highly specific antibodies and antigen receptors.They must rely on efficient immune defenses capable of protecting them against invading microorganisms.The chief issue of crustacean immunity should concern non-self-recognition mechanisms.Proteins that specifically bind to certain carbohydrate components on the surface of microorganisms play an important role in non-self-recognition and cleaning up of the invading microorganisms.Such proteins are known as pattern recognition receptors(PRRs).Lectins exist in almost all living organisms.Due to their ability of binding to terminal sugars on glycoproteins and glycolipids,lectins are primary candidates for pattern recognition receptors in innate immunity.C type Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean.In the preliminary study,a novel C-type lectin was cloned from hemocytes of Chinese shrimp,(Fenneropenaeus chinensis).It contains two tandem carbohydrate recognition domains(CRDs)/C-type lectin-like domains.Both of the CRDs contain a QPD(Gln-Pro-Asp)motif that has a predicted binding specificity for galactose-type sugar.In this research,two recombinant target proteins(rFclectin-CRD1 and rFclectin-CRD2)were expressed by prokaryotic expression system.The result showed that fusion protein was expressed in the form of inclusion bodies.The LC-ESI-MS analysis showed that two peptide fragments of rFclectin-CRD1 and rFclectin-CRD2 were identical with the corresponding sequence of F.chinensis C-type lectin.Recombinant protein was purified by immobilized-metal affinity chromatography and Ni-NTA technology.The concentrations of purified target proteins were 0.4 g/L.rFclectin-CRD1 and rFclectin-CRD2 had agglutinating and antimicrobial activity against main pathogens in aquaculture in a calcium-dependent manner.The agglutinating activity can be inhibited by multiple carbohydrates,such as galactose,peptidoglycan and lipopolysaccharide.These results suggest that Fclectin,as a Ca2+dependent carbohydrate-recognition protein,is one of the important PRRs.It might play a crucial role in the innate immunity of the shrimp and is expected to be applied in disease control.