Cloning and transcriptional analysis of two sialic acid-binding lectins (SABLs) from razor clam Solen grandis

Sialic acid-binding lectin (SABL) plays crucial role in both innate and adaptive immune responses benefiting from its predominant affinity toward glycan. In the present study, two SABLs from razor clam Solen grandis (designated as SgSABL-1 and SgSABL-2) were identified, and their expression patterns, both in tissues and towards microorganism glycan stimulation, were then characterized. The cDNA of SgSABL-1 and SgSABL-2 was 988 and 1281 bp, containing an open reading frame (ORF) of 744 and 570 bp, respectively, and deduced amino acid sequences showed high similarity to other invertebrates SABLs. Both SgSABL-1 and SgSABL-2 encoded a C1q domain. SgSABL-1 and SgSABL-2 were found to be constitutively expressed in a wide range of tissues with different levels, including mantle, gill, gonad, hemocyte, muscle, and hepatopancreas, and both of them were highly expressed in hepatopancreas. SgSABL-1 and SgSABL-2 could be significantly induced after razor clams were stimulated by acetylated subunits-containing glycan LPS and PGN, suggesting the two SgSABLs might perform potential function of glycan recognition. In addition, SgSABL-2 could also be induced by β-1,3-glucan. All these results indicated that SgSABL-1 and SgSABL-2 might be involved in the immune response against microbe infection and contributed to the pathogens recognition.     

Identification and transcriptional analysis of two types of lectins (SgCTL-1 and SgGal-1) from mollusk Solen grandis

C-type lectin and galectin are two types of animal carbohydrate-binding proteins which serve as pathogen recognition molecules and play crucial roles in the innate immunity of invertebrates. In the present study, a C-type lectin (designated as SgCTL-1) and galectin (designated as SgGal-1) were identified from mollusk Solen grandis, and their expression patterns, both in tissues and toward three pathogen-associated molecular patterns (PAMPs) stimulation were characterized. The full-length cDNA of SgCTL-1 and SgGal-1 was 1280 and 1466 bp, containing an open reading frame (ORF) of 519 and 1218 bp, respectively. Their deduced amino acid sequences showed high similarity to other members of C-type lectin and galectin superfamily, respectively. SgCTL-1 encoded a single carbohydrate-recognition domain (CRD), and the motif of Ca(2+)-binding site 2 was EPN (Glu(135)-Pro(136)-Asn(137)). While SgGal-1 encoded two CRDs, and the amino acid residues constituted the carbohydrate-binding motifs were well conserved in CRD1 but partially conserved in CRD2. Although SgCTL-1 and SgGal-1 exhibited different tissue expression pattern, they were both constitutively expressed in all tested tissues, including hemocytes, gonad, mantle, muscle, gill and hepatopancreas, and they were both highly expressed in hepatopancreas and gill. Furthermore, the mRNA expression of two lectins in hemocytes was significantly (P < 0.01) up-regulated with different levels after S. grandis were stimulated by lipopolysaccharide (LPS), peptidoglycan (PGN) or β-1,3-glucan. Our results suggested that SgCTL-1 and SgGal-1 from razor clam were two novel members of animal lectins, and they might function as pattern recognition receptors (PRRs) taking part in the process of pathogen recognition.     

Molecular cloning and thermal stress-induced expression of a pi-class glutathione S-transferase (GST) in the Antarctic bivalve Laternula elliptica

Glutathione S-transferases (GSTs) are multifunctional phase II detoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione. The pi-class GST cDNA (leGSTp) was cloned from the cold-adapted Antarctic bivalve Laternula elliptica. We used degenerated primers designed based on highly conserved regions of known mollusk GSTs to amplify the corresponding L. elliptica mRNA. Full-length cDNA was obtained by rapid amplification of cDNA ends (RACE). The full sequence of the GST cDNA was 1189 bp in length, with a 5' untranslated region (UTR) of 74 bp, a 3' UTR of 485 bp, and an open reading frame of 630 bp encoding 209 amino acid residues with an estimated molecular mass of 23.9 kDa and an estimated isoelectric point of 8.3. Quantitative RT-PCR confirmed basal expression of leGSTp, which was up-regulated upon heat treatment (10 degrees C for different time periods) by a factor of 2.3 (at 24 h) and 2.7 (at 48 h) in the digestive gland and gill tissues, respectively. The recombinant leGSTp expressed in Escherichia coli was purified by affinity chromatography and characterized. The purified leGSTp exhibited high activity towards the substrates ethacrynic acid (ECA) and 1-chloro-2,4-dinitrobenzene (CDNB). The recombinant leGSTp had a maximum activity at approximately pH 8.0, and its optimum temperature was 35 degrees C.     

Cloning and expression pattern analysis of a lipopolysaccharide -and beta-1,3-glucan-binding protein in Portunus trituberculatus

The lipopolysaccharide -and beta-1,3-glucan-binding protein (LGBP) is a pattern recognition receptor, which is fundamental for the innate immune response of crustaceans. A LGBP gene was cloned from the haemocytes of Portunus trituberculatus using SMART RACE methods. The full-length LGBP cDNA (1 378 bp) had a 1 095 bp open reading frame encoding a protein of 365 amino acid residues including a 16 amino acid residues signal peptide, a 138 bp 5' untranslated region (UTR) and a 144 bp untranslated region in the 3' UTR with a 29 bp polyA tail. The calculated molecular mass of the mature protein (349 amino acid residues) is 39,825.24 with an estimated pI of 4.49. The gene sequence and secondary structure of LGBP were analyzed by bio-informatics. Additionally, a Glyco hydro 16 domain was identified. The expression of P. trituberculatus in various tissues were detected through RT-PCR methods. The results showed that the LGBP gene expressed in all the tissues detected, including haemocytes, hepatopancreas, heart, gills and muscle. In response to the challenge of Staphyloccocus aureus and Vibrio alginolyticus, the LGBP gene expression in haemocytes of the group challenged with mixed bacteria were higher than the control group within 48 h. It suggested that the LGBP gene plays an active role in immunologic process against bacterial infection.     

Molecular characterization and expression analysis of an inhibitor of NF-κB (IκB) from Asiatic hard clam Meretrix meretrix

Inhibitor of NF-κB (IκB) is an important member of Rel/NF-κB signaling pathway, which is an important mediator of immune responses in innate immune system. In this study, the IκB cDNA of hard clam Meretrix meretrix (designated as Mm-IκB) was cloned and characterized. The full-length cDNA of Mm-IκB was of 2098 bp, containing a 5' untranslated region (UTR) of 123 bp, a 3' UTR of 810 bp with a poly (A) tail, and an open reading frame (ORF) of 1164 bp encoding a polypeptide of 387 amino acids. The high similarity of Mm-IκB with other IκBs from invertebrates indicated that Mm-IκB should be a member of IκB family. Similar to most IκBs, Mm-IκB possessed all conserved features critical for the fundamental structure and function of IκBs, such as five ankyrin repeats and a conserved degradation motif (DS(44)RYSS(48)). Two PEST domains and a phosphorylation site motif (S(367)EEE(370)) at the C-terminus of Mm-IκB were identified. By quantitative real-time RT-PCR analysis, mRNA level of Mm-IκB was found to be most abundantly expressed in the tissues of mantle, gill and hepatopancreas, weakly expressed in muscle, foot and haemocyte. The Mm-IκB gene expression was significantly up-regulated at 24 h in haemocyte and at 12 h in gill after Vibrio anguillarum challenge, respectively. The results suggested the involvement of Mm-IκB in response against bacterial infection and further highlighted its functional importance in the immune system of M. meretrix.     

Molecular characterization of a glutathione peroxidase gene and its expression in the selected Vibrio-resistant population of the clam Meretrix meretrix

Glutathione peroxidase (GPx) is an important member of cellular enzymatic antioxidant system, which may be involved in pathogen defense of host. In the present study, a selenium-dependent glutathione peroxidase (MmeGPx) gene from clam Meretrix meretrix was cloned and analyzed. The MmeGPx gene was composed of two introns of 723 bp and 238 bp and an open reading frame (ORF) of 711 bp. The ORF encodes a protein of 237 amino acids with a putative selenocysteine residue encoded by an unusual stop codon. MmeGPx shares a higher level of similarity with human GPx 3 than with other human GPx isozymes. The level of MmeGPx mRNA roughly paralleled GPx enzyme activity in different tissues except in gills, with the highest mRNA expression and enzyme activity occurring in hepatopancreas. MmeGPx mRNA expressions were detected in different larval stages and the results showed that MmeGPx mRNA increased significantly in pediveliger stage, which may be a response to oxidative stress. After challenge of clam with a Vibrio parahaemolyticus-related bacterium (MM21), the expression of MmeGPx was significantly up-regulated at 6 h and 12 h in hepatopancreas, which suggested that MmeGPx may be involved in the immune response to MM21 infection. To better understand its role in the immunity of clam, the expression of MmeGPx in hepatopancreas was compared between a selected Vibrio-resistant population and a control population after immersion challenge with MM21. Early up-regulation of MmeGPx was observed in the resistant population. These results suggested that MmeGPx might be involved in maintaining the redox state of immune system, and the early immune response to pathogen infection may help the clam against pathogen infection.     

Molecular properties and immune defense of two ferritin subunits from freshwater pearl mussel,Hyriopsis schlegelii

Ferritin is a conserved iron-binding protein involved in cellular iron metabolism and host defense. In the present study, two distinct cDNAs for ferritins in the freshwater pearl mussel Hyriopsis schlegelii were identified (designated as HsFer-1 and HsFer-2) by SMART RACE approach and expressed sequence tag (EST) analysis. The full-length cDNAs of HsFer-1 and HsFer-2 were of 760 and 877 bp, respectively. Both of the two cDNAs contained an open reading frame (ORF) of 522 bp encoding for 174 amino acid residues. Sequence characterization and homology alignment indicated that HsFer-1 and HsFer-2 had higher similarity to H-type subunit of vertebrate ferritins than L-type subunit. Analysis of the HsFer-1 and HsFer-2 untranslated regions (UTR) showed that both of them had an iron response element (IRE) in the 5′-UTR, which was considered to be the binding site for iron regulatory protein (IRP). Quantitative real-time PCR (qPCR) assays were employed to examine the mRNA expression profiles. Under normal physiological conditions, the expression level of both HsFer-1 and HsFer-2 mRNA were the highest in hepatopancreas, moderate in gonad, axe foot, intestine, kidney, heart, gill, adductor muscle and mantle, the lowest in hemocytes. After stimulation with bacteria Aeromonas hydrophila, HsFer-1 mRNA experienced a different degree of increase in the tissues of hepatopancreas, gonad and hemocytes, the peak level was 2.47-fold, 9.59-fold and 1.37-fold, respectively. Comparatively, HsFer-2 showed up-regulation in gonad but down-regulation in hepatopancreas and hemocytes. Varying expression patterns indicate that two types of ferritins in H. schlegelii might play different roles in response to bacterial challenge. Further bacteriostatic analysis showed that both the purified recombinant ferritins inhibited the growth of A. hydrophila to a certain degree. Collectively, our results suggest that HsFer-1 and HsFer-2 are likely to be functional proteins involved in immune defense against bacterial infection.