Functional Studies Of C-type Lectins From Crustacean

Invertebrates lack an adaptive immunity, but have a rapid and effective innate immune system to defend against microorganisms. The recognition of pathogens by pattern recognition receptors (PRRs) triggers the first step of defense. Several kinds of PRRs have already been identified in invertebrate species. We used Chinese white shrimp, Fenneropenaeus chinensis and red swamp crayfish, Procambarus clarkii as experiment materials to research a kind of PRRs of crustacean, C-type lectin. We found three C-type lectins and research their biological functions in innate immune system.1. Expression and functional study of Fc-Lec2 in Chinese white shrimp.A novel C-type lectin (Fc-Lec2) was cloned from the hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The cDNA of Fc-Lec2 is 1219 bp with an open reading frame (ORF) of 1002 bp that encodes a protein of 333 amino acids. Fc-Lec2 contains a signal peptide and two different carbohydrate recognition domains (CRDs) arranged in tandem. The first CRD contains a Gln-Pro-Asp (QPD) motif that has a predicted binding specificity for galactose and the second CRD contains a Glu-Pro-Asn (EPN) motif for mannose. Fc-Lec2 was constitutively expressed in the hepatopancreas of normal shrimp, and its expression was up-regulated in the hepatopancreas of shrimp challenged with bacteria or viruses. Recombinant mature Fc-Lec2 and its two individual CRDs (CRD 1 and 2) did not have hemagglutinating activity against animal red blood cells, but agglutinated some Gram-positive and Gram-negative bacteria in a calcium-dependent manner. The three recombinant proteins also bound to bacteria in the absence of calcium. Fc-Lec2 seems to have a broader specificity and higher affinity for bacteria and some polysaccharides than each of the two individual CRDs. These data suggest that the two CRDs have synergistic effect, and the intact lectin may be more effective in response to bacterial infection, the Fc-Lec2 performs its pattern recognition function by binding to polysaccharides of pathogen cells.2. Expression and functional study of PcLecl in red swamp crayfish.C-type lectin (PcLecl) is reported in freshwater crayfish Procambarus clarkii. PcLecl encodes a protein of 163 amino acids with a putative signal peptide and a single carbohydrate recognition domain (CRD). It was constitutively expressed in various tissues of a normal crayfish, especially in the hepatopancreas and gills. Expressions of PcLecl were up-regulated in the hepatopancreas and gills of crayfish challenged with Vibrio anguillarum, Staphylococcus aureus, or the white spot syndrome virus (WSSV). Recombinant mature PcLecl bound bacteria and polysaccharides (peptidoglycan, lipoteichoic acid, and lipopolysaccharide) but did not agglutinate bacteria. PcLec1 enhanced hemocyte encapsulation of the sepharose beads in vitro, and the blocking of beads by a polyclonal antibody inhibited encapsulation. PcLecl promoted clearance of Vibrio anguillarum in vivo. These results suggest that PcLec1 is a pattern recognition receptor and participates in cellular immune response. PcLec1 performs its function as an opsonin by enhancing the encapsulation or clearance of pathogenic bacteria.3. Expression and functional study of PcLec3 in red swamp crayfish.We reported a novel C-type lectin (PcLec3) in freshwater crayfish Procambarus clarkii. PcLec3 encodes a protein of 252 amino acids that containing a signal peptide, an Ig domain (IG) and a C-type lectin domain (CTLD) in tandem. The mRNA of PcLec3 is expressed in hemocytes and hapatopancreas. Its mRNA level increased after bacteria V.anguillarum challenge in hepatopancreas.The recombinant PcLec3, IG and CTLD were used to perform the function research. All the three proteins have carbohydrates and bacterial binding capacities. As a member of Ig superfamily, PcLec3 is a hemocytes adhesion molecule and promotes hemocytes phagacytosis of invaded V.anguillarum.