Molecular cloning and characterization of the lipopolysaccharide and β-1,3-glucan binding protein from red claw crayfish, Cherax quadricarinatus

Abstract

Abstract A pattern recognition protein (PRP), lipopolysaccharide and β-1,3-glucan binding protein (LGBP), cDNA was cloned from the hemocytes of red claw crayfish, Cherax quadricarinatus by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1456 bp has an open reading frame of 1095 bp encoding a protein of 364 amino acids including a 15 amino acid signal peptide. The calculated molecular mass of the mature protein (349 aa) was 39.92 kDa with an estimated pI of 4.50. Sequence comparison of the deduced amino acid sequence of C. quadricarinatus LGBP (named as CqLGBP) showed a high identity of 82%, 77%, 76% and 74% with Procambarus clarkii LGBP, Homarus gammanus BGBP, Penaeus monodon BGBP and Litopenaeus stylirostlis LGBP, respectively. The CqLGBP sequence contains: (1) two putative integrin-binding motifs, (2) a glucanase motif, (3) two putative N-glycosylation sites, (4) one protein kinase C phosphorylation site, and (5) a putative recognition motif for β-1,3-linkage of polysaccharides. The recombinant CqLGBP were expressed in Escherichia coli BL21 with pGEX-6P-2 expression vector. The titer of rabbit anti-CqLGBP serum was above 1:12,800. Microorganism binding assay showed CqLGBP binds two kinds of crustacean pathogens Spiroplasma eriocheiris and Aeromonas hydrophila. Quantitative RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with S. eriocheiris and A. hydrophila. A significant enhancement of CqLGBP transcriptions was apparent post-injection in response to bacterial infection compared to the control group. These data should be helpful to better understand the function of CqLGBP in the crayfish immune system.