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Abstract: Fibrinogen-related proteins (FREPs) are a kind of pattern recognition receptors (PRRs) containing fibrinogen-like (FBG) domains, and they play curial roles in the innate immune response. In the present study, a new FREP protein was identified from bay scallop Argopecten irradians (designated as AiFREP-2). The full-length cDNA of AiFREP-2 was of 1299 bp with an open reading frame of 762 bp encoding a polypeptide of 253 amino acids, including a signal sequence and an FBG domain. The FBG domain in AiFREP-2 was highly similar to those of ficolins, tenascins and other FREPs. The mRNA expression of AiFREP-2 could be detected in all the examined tissues with the highest level in gill. The mRNA expression of AiFREP-2 in hemocytes was significantly up-regulated post the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN) and β-glucan (GLU) (P < 0.01). The recombinant AiFREP-2 (rAiFREP-2) could bind not only different PAMP ligands including LPS, PGN and GLU, but also various microbes including Gram-negative bacteria (Vibrio anguillarum), Gram-positive bacteria (Staphylococcus aureus) and fungus (Pichia pastoris and Yarrowia lipolytica). Additionally, rAiFREP-2 exhibited obvious agglutination activity towards Gram-negative bacteria V. anguillarum and Gram positive bacteria S. aureus. The results indicated that AiFREP-2 was involved in the immune response against Gram-negative bacteria, Gram-positive bacteria and fungus as a PRR in bay scallop, and the information was helpful to understand the innate immune defense mechanisms of mollusks. [Copyright &y& Elsevier]

Abstract: C-type lectins are a large family of Ca2+-dependent carbohydrate binding proteins which play crucial roles to recognize and eliminate pathogens in innate immunity. In the present study, a novel C-type lectin was identified from Eriocheir sinensis (designated as EsCTL). The full-length cDNA of EsCTL was of 789 bp with an open reading frame of 468 bp encoding a polypeptide of 156 amino acids with a signal sequence and single carbohydrate-recognition domain (CRD). The potential tertiary structure of the CRD adopted a typical double-loop structure with Ca2+-binding site 2 in the long loop region and two conserved disulfide bridges at the bases of the loops. An EPQ motif to determine carbohydrate binding specificity was identified in the CRD of EsCTL. The mRNA transcripts of EsCTL were mainly detected in hepatopancreas and its relative expression level in hemocytes was significantly up-regulated after the challenges of Vibrio anguillarum (P < 0.05) and Pichia pastoris (P < 0.05). The recombinant EsCTL protein (rEsCTL) could bind different PAMPs, including LPS, PGN, β-glucan, and polyI:C; and also bind various microorganisms including three Gram-positive bacteria, three Gram-negative bacteria and two yeasts. Moreover, rEsCTL could significantly enhance the in vitro encapsulation of crab hemocytes. All these results suggested that EsCTL functioned as an important PRR involved in immune defense against invading pathogen in crab. [Copyright &y& Elsevier]

Abstract: Peptidoglycan recognition protein (PGRP) is a pattern recognition receptor, playing important roles in the innate immune response against invasive pathogens. The single nucleotide polymorphism (SNP) loci in scallop PGRP gene (CfPGRP) were screened from Chlamys farreri to investigate their association with disease resistance of scallop against Listonella anguillarum. Thirteen SNP sites were identified in PGRP domain of CfPGRP, and two of them at positions 4407 and 4408 which are located in the same codon resulted in a nonsynonymous substitution. The genotype frequency of CG/CG in the resistant stock was significantly lower than that in susceptible stock (0% vs 32.4%), while that of CG/TA in the resistant stock was significantly higher than that in susceptible stock (P < 0.01). The pathogen-associated molecular patterns (PAMP) binding activity of two recombinant proteins, rCfPGRP-S1 (R) with CG variant in 4407–4408 site, rCfPGRP-S1 (Y) with TA variant in 4407–4408 site, were elucidated by examining their P/N value at 405 nm with ELISA assay. The in vitro binding activities of the two rCfPGRP-S1 variants to both lipopolysaccharide (LPS) and peptidoglycan (PGN) varied (P < 0.05) in a dose-dependent manner, and rCfPRPP-S1(Y) exhibited significantly higher affinity to PGN and LPS than that of rCfPGRP-S1(R) (P < 0.05). The growth inhibition assay was conducted to find the antibacterial activities of the two variants. Both rCfPGRP-S1(R) and rCfPGRP-S1 (Y) displayed obvious activity to suppress the growth of Escherichia coli, but there was no significant difference in suppression activity of two variants (P > 0.05). The results suggested that the polymorphism at locus 4407–4408 of CfPGRP-S1 considerably affected its PAMP binding activity, and the SNP locus 4407–4408 CG/TA was associated with disease resistance of scallop against L. anguillarum infection, which could be used as a candidate marker for future selection in zhikong scallop breeding program. [Copyright &y& Elsevier]

Abstract: Lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) is a pattern recognition receptor (PRR) recognizing and binding both LPS and β-1, 3-glucan, playing important roles in innate immunity. In the present study, the single nucleotide polymorphisms (SNPs) were assessed in LGBP gene from scallop Chlamys farreri (designated CfLGBP), and eight SNPs were found in its potential LPS and glucanase binding motif. The locus +7679 with the transition of G-A, which produced an amino acid substitution at codon 360 from a non polar Glycine to polar Serine, was selected to inspect their association with disease resistance/susceptibility to Listonella anguillarum. Three genotypes G/G, G/A and A/A, were revealed at locus +7679, and their frequencies were 89.7%, 7.7% and 2.6% in the resistant stock, while 63.2%, 34.2% and 2.6% in the susceptible stock, respectively. The frequency of genotypes G/G and G/A were significantly different (P < 0.05) between the two stocks. The pathogen-associated molecular patterns (PAMP) binding activity of two recombinant proteins, rCfLGBP (G) with G variant at locus +7679 and rCfLGBP (S) with A variant at locus +7679, were elucidated by ELISA assay. The binding affinities of both LPS and β-glucan binding affinity were varied in a dose-dependent manner, where the binding affinity of rCfLGBP (G) was significantly higher than that of rCfLGBP (S) (P < 0.05). The results collectively suggested that the polymorphism of +7679 G/G in CfLGBP possibly enhances the binding activity of LPS and β-glucan, and was associated to disease resistance of scallop against L. anguillarum, which could be a potential marker applied in future selection of scallop with enhanced resistance to L. anguillarum. [Copyright &y& Elsevier]

Abstract: C-type lectins are a superfamily of proteins that can bind pathogen-associated molecular patterns (PAMPs) and microorganisms through the recognition of carbohydrates, thus they are directly involved in innate defense mechanisms as part of the acute-phase response to infection. In this study, the cDNA of a novel C-type lectin (designated as AiCTL-7) was cloned from bay scallop Argopecten irradians by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of AiCTL-7 was of 651 bp containing a 525 bp open reading frame which encoded a signal peptide of 15 residues and a conserved carbohydrate-recognition domain (CRD) of 174 residues with the EPD and WSD motifs instead of the invariant EPN and WND motifs for determining the carbohydrate-binding specificity and constructing Ca2+-binding site 2 in vertebrates. The deduced amino acid sequence of AiCTL-7 CRD shared homology not only with the CRDs of C-type lectins in mollusks, but also with the fish lectin CRDs. The mRNA transcripts of AiCTL-7 were mainly detected in the tissue of hepatopancreas and also marginally detectable in kidney, gonad, hemocytes, heart and adductor of health scallop. After challenge with fungi Pichia pastoris GS115 and Gram-negative bacteria Listonella anguillarum, the relative expression level of AiCTL-7 was up-regulated significantly in hepatopancreas and hemocytes. The CRD of AiCTL-7 was recombined and expressed in Escherichia coli, and the recombinant protein (rAiCTL-7) aggregated P. pastoris remarkably in a Ca2+-dependent manner, and this agglutination could be inhibited by d-mannose, but not by d-galactose or β-1,3-glucan. However, rAiCTL-7 displayed no obvious agglutinating activity against L. anguillarum. These results collectively indicated that AiCTL-7 was involved in the primitive acute-phase response to microbial invasion as an important pattern recognition receptor (PRR) in the innate immune system of scallops. [Copyright &y& Elsevier]