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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: The C1q domain containing (C1qDC) proteins refer to a family of proteins containing the versatile charge pattern recognition globular C1q domain in the C-terminus, which could bind various ligands including PAMPs and trigger a serial of immune response. In this study, a novel C1qDC protein was identified from Argopecten irradians (designated as AiC1qDC-2). Its full-length cDNA was of 1062 bp with an open reading frame of 720 bp encoding a polypeptide of 240 amino acids containing a typical gC1q domain. This gC1q domain possessed the typical 10-stranded β-sandwich fold with a jelly-roll topology common to all C1q family members, and shared high homology with most of the other identified gC1q domains. The mRNA transcripts of AiC1qDC-2 were mainly detected in hepatopancreas, and also marginally detectable in mantle, gonad, adductor, gill and hemocytes. Its relative expression level in hemocytes was significantly up-regulated after challenges of fungi Pichia pastoris GS115 (P < 0.05), Gram-positive bacteria Micrococcus luteus (P < 0.05) and Gram-negative bacteria Vibrio anguillarum (P < 0.05). The recombinant protein of AiC1qDC-2 (rAiC1qDC-2) could bind various PAMPs, including LPS, PGN, polyI:C, mannan, β-1,3-glucan as well as Yeast-glucan, and displayed agglutinating activity to fungi P. pastoris GS115, Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Escherichia coli TOP10F′ as well as V. anguillarum. All these results indicated that AiC1qDC-2 could function as a pattern recognition receptor to recognize various PAMPs on different pathogens in the innate immune responses of scallop, and provided new clues to understand the role of invertebrate C1qDC proteins in the ancient complement system. [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]