Molecular cloning, characterization and expression of a serine protease with clip-domain homologue from scallop Chlamys farreri

Serine proteases play critical roles in a variety of invertebrate immune defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization. The first mollusk serine protease with clip-domain (designated CFSP1) cDNA was obtained from the scallop Chlamys farreri challenged with Vibrio anguillarum by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the C. farreri serine protease was 1211 bp, consisting of a 5-terminal untranslated region (UTR) of 72 bp, a 3'-terminal UTR of 77 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1062 bp. The CFSP1 cDNA encoded a polypeptide of 354 amino acids with a putative signal peptide of 19 amino acids and a mature protein of 335 amino acids. The deduced amino acid sequence of CFSP1 contained an amino-terminal clip domain, a low complexity region, and a carboxyl-terminal serine protease domain. CFSP1 mRNA was mainly expressed constitutively in the hemocytes and was up-regulated and increased 2.9- and 1.9-fold at 16 h after injury and injection of bacteria. (c) 2006 Elsevier Ltd. All rights reserved.

Dissection and localization of the immunostimulating domain of Edwardsiella tarda FliC

Bacterial flagellin is known to induce potent immune response in vertebrate systems via the toll-like receptor (TLR) 5. As a result, flagellin has been studied extensively as a vaccine adjuvant. In a previous study, we examined the vaccine and adjuvant potentials of the flagellin (FliC) of the fish pathogen Edwardsiella tarda. We found that E. tarda FliC induced low protective immunity by itself but could function as a molecular adjuvant and potentiate the specific immune response induced by the E. tarda antigen Eta6. Since FliC is a large protein and organized into distinct structural domains, we wondered whether the immunostimulating effect observed with the full-length protein could be localized to a certain region. To investigate this question, we in the present study dissected the FliC protein into several segments according to its structural features: (i) N163, which consists of the conserved N-terminal 163 residues of FliC; (ii) M160, which consists of the variable middle 160 residues; (iii) C94, which consists of the conserved C-terminal 94 residues; (iv) NC257, which is an artificial fusion of N163 and C94. To examine the adjuvanticity of the FliC fragments, DNA vaccine plasmids expressing FliC fragments in fusion with Eta6 were constructed and used to immunize Japanese flounder. The results showed that N163 produced the best adjuvant effect, which, in respect to improvement in the relative percent survival of the vaccinated fish, was comparable to that of the full-length FliC. None of the other FliC fragments exhibited apparent immunopotentiating effect. Further analysis showed that N163 enhanced the production of serum specific antibodies and, like full-length FliC, significantly upregulated the expression of the genes that are possibly involved in innate and adaptive immunity. These results indicate that N163 is the immunodominant region of FliC and suggest that E. tarda FliC may induce immune responses in Japanese flounder via mechanisms alternative to that involving TLR5. (C) 2010 Elsevier Ltd. All rights reserved.

AiC1qDC-1, a novel gC1q-domain-containing protein from bay scallop Argopecten irradians with fungi agglutinating activity

The globular C1q-domain-containing (C1qDC) proteins are a family of versatile pattern recognition receptors via their globular C1q (gC1q) domain to bind various ligands including several PAMPs on pathogens. In this study, a new gC1q-domain-containing protein (AiC1qDC-1) gene was cloned from Argopecten irradians by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNA of AiC1qDC-1 was composed of 733 bp, encoding a signal peptide of 19 residues and a typical gC1q domain of 137 residues containing all eight invariant amino acids in human C1qDC proteins and seven aromatic residues essential for effective packing of the hydrophobic core of AiC1qDC-1. The gC1q domain of AiC1qDC-1, which possessed the typical 10-stranded beta-sandwich fold with a jelly-roll topology common to all C1q family members, showed high homology not only to those of Cl qDC proteins in mollusk but also to those of C1qDC proteins in human. The AiC1qDC-1 transcripts were mainly detected in the tissue of hepatopancreas and also marginally detectable in adductor, heart, mantle, gill and hemocytes by fluorescent quantitative real-time PCR. In the microbial challenge experiment, there was a significant up-regulation in the relative expression level of AiC1qDC-1 in hepatopancreas and hemocytes of the scallops challenged by fungi Pichia pastoris GS115, Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Listonella anguillarum. The recombinant AiC1qDC-1 (rAiC1qDC-1) protein displayed no obvious agglutination against M. luteus and L. anguillarum, but it aggregated P. pastoris remarkably. This agglutination could be inhibited by D-mannose and PGN but not by LPS, glucan or D-galactose. These results indicated that AiC1qDC-1 functioned as a pattern recognition receptor in the immune defense of scallops against pathogens and provided clues for illuminating the evolution of the complement classical pathway. (C) 2010 Elsevier Ltd. All rights reserved.

SmC2P1, a C2 domain protein from Scophthalmus maximus that binds bacterial pathogen-infected lymphocytes and reduces bacterial survival

C2 domains are protein structural modules found in many eukaryotic proteins involved in signal transduction, membrane trafficking, and immune defense. Most of the studied C2 domain-containing proteins are multi-domained in structure, in which the C2 domain is an independently folded motif and plays an essential role in calcium-dependent membrane-targeting. Although C2 domains isolated from intact proteins have been studied for biological functions, no study on natural proteins containing C2 domain only has been documented. In this study, we identified a Scophthalmus maximus protein SmC2P1 that is comprised of a single C2 domain and lacks any other apparent domain structures. The deduced amino acid sequence of SmC2P1 contains 129 residues and shares 36-38% identities with the C2 domains of the perforins of several fish species. Like typical C2 domains, SmC2P1 is predicted to organize into eight beta-strands with a Ca2+-binding site located in inter-strand loops. SmC2P1 expression was detected, in deceasing order, in liver, spleen, blood, brain, muscle, kidney, gill, and heart. Experimental challenge of turbot with a bacterial pathogen significantly upregulated SmC2P1 expression in kidney in a time-dependent manner. Recombinant SmC2P1 purified from yeast exhibits no hemolytic activity but binds to pathogen-infected kidney lymphocytes in the presence of calcium. Furthermore, interaction of recombinant SmC2P1 with bacterium-infected lymphocytes reduced bacterial survival. These results indicate that SmC2P1 is a functional protein that is involved in host immune defense against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.

Identification, expression, function and localization of a DUF985 domain-containing hypothetical gene from amphioxus Branchiostoma belcheri

The progress in genome sequencing has led to an increasing submission of uncharacterized hypothetical genes with the domain of unknown function, DUF985, in GenBank, and none of these genes is related to a known protein. We therefore underwent an experimental study to identify the function of a DUF985 domain-containing hypothetical gene BbDUF985 (GenBank Accession No. AY273818) isolated from amphioxus Branchiostoma belcheri (B. belcheri). BbDUF985 was successfully expressed in both prokaryotic and eukaryotic systems, and its recombinant proteins expressed in both systems definitely exhibited an activity of phosphoglucose isomerase (PGI). Both tissue-section in situ hybridization and immunohistochemistry demonstrated that BbDUF985 was expressed in a tissue-specific manner, with most abundant levels in the hepatic caecum and ovary. In CHO cells transfected with the expression plasmid pEGFP-N1/BbDUF985, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that BbDUF985 is a cytosolic protein. In contrast, Western blotting indicated that BbDUF985 was also present in amphioxus humoral fluids, suggesting that it exists as a secreted protein as well. Our study provided a framework for further understanding the biochemical properties and physiological function of DUF985-containing hypothetical proteins in other species. (c) 2008 Elsevier Inc. All rights reserved.

Isolation and characterization of novel marine Roseobacter clade members producing unique intracellular chromium-rich aggregates

The marine Roseobacter clade comprises one of the largest fractions of heterotrophic marine bacteria and accounts for about 16% of 16S rRNA gene clones retrieved from marine bacterioplankton. Their global distribution seems to be related to oceanic water masses and their environmental and biogeochemical properties. In this study, we report isolation and characterization of novel Roseobacter clade members from the Yellow Sea, China. Phylogenetic analysis of 16S rRNA gene sequences reveals that the new isolates (YSCB1, YSCB2, YSCB3 and YSCB4) are closely related to uncultured Arctic seawater bacterium R7967 (99.57-100% sequence identity) and to the cultured Roseobacter sp. DSS-1 (99.27-99.76% sequence identity) isolated from the southeastern coastal water of the USA. Interestingly, YSCB strains possess unique intracellular chromium-containing aggregates. Therefore, these novel Roseobacter clade members exhibit a peculiar property in mineral biogeneration. (c) 2006 Elsevier SAS. All rights reserved.

Intracellular copper/zinc superoxide dismutase from bay scallop Argopecten irradians: Its gene structure, mRNA expression and recombinant protein

Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Two unique and highly compartmentalized bay scallop Argopecten irradians superoxide dismutases: MnSOD and ecCuZnSOD, have been molecularly characterized in our previous study. To complete characterize the SOD family in A. irradians, a novel intracellular copper/zinc SOD from the A. irradians (Ai-icCuZnSOD) was obtained and characterized. The full-length cDNA of Ai-icCuZnSOD was 1047 bp with a 459 bp open reading frame encoding 152 amino acids. The genomic length of the Ai-icCuZnSOD gene was about 4279 bp containing 4 exons and 3 introns. The promoter region containing many putative transcription factor binding sites were analyzed. Furthermore, quantitative reverse transcriptase real-time PCR (qRT-PCR) analysis indicated that the highest expression of the Ai-icCuZnSOD was detected in gill and the expression profiles in hemocytes of bay scallops challenged with bacteria Vibrio anguillarum and lipopolysaccharide (LPS) were different. The result presented an increased expression after injection with LPS whereas no significant changes were observed after V. anguillarum injection. A fusion protein containing Ai-icCuZnSOD was produced in vitro. The rAi-icCuZnSOD is a stable enzyme, retaining more than 80% of its activity between 10 and 60 degrees C and keeping above 88% of its activity at pH values between 5.8 and 9. Ai-icCuZnSOD is more stable under alkaline than acidic conditions. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.