An immune responsive multidomain galectin from bay scallop Argopectens irradians

Galectins are a family of β-galactoside-binding lectins which play crucial roles in innate immunity of vertebrates and invertebrates. In the present study, the cDNA of a galectin with multiple carbohydrate-recognition domains (CRDs) was cloned from bay scallop Argopectens irradians (designated AiGal1) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of AiGal1 was of 2235 nucleotides, encoding a polypeptide of 549 amino acids. SMART program analysis revealed that AiGal1 contained four galectin CRDs, and all the CRDs contained the two consensus motifs essential for ligand-binding. Quantitative real-time PCR was employed to investigate the tissue distribution of AiGal1 mRNA and temporal expression in haemocytes of scallops challenged with Vibrio anguillarum, Micrococcus luteus and Pichia pastoris. The AiGal1 mRNA could be detected in all tested tissues with the highest expression level in hepatopancreas. After challenged by V. anguillarum and M. luteus, the expression level of AiGal1 mRNA was both up-regulated and reached the maximum level at 9 h (1.52 fold, P < 0.05) and 18 h (2.89 fold, P < 0.01) post challenge, respectively. However, there was no significant difference in the mRNA expression of AiGal1 in haemocytes after P. pastoris challenge (P > 0.05). These results collectively indicated that AiGal1 was a new member of the galectin family and involved in the immune responses against bacterial infection.     

Himasthla elongata: Effect of infection on expression of the LUSTR-like receptor mRNA in common periwinkle haemocytes

The first mollusc mRNA coding G-protein-coupled transmembrane receptor (GPСR), homologous to human receptors LUSTR 1 (GPR107) and LUSTR 2 (GPR108), was isolated from haemocytes of common periwinkle Littorina littorea. The analyses showed that the full-length cDNA is 1935 bp long and is predicted to encode a 614 amino acid protein (named Lit-LUSTR) with a calculated molecular mass of 69.6 kDa and theoretical isoelectric point 7.59. Pair-wise comparisons between Lit-LUSTR and LUSTR proteins from human or mouse have approximately 38% identity and 56% similarity. Lit-LUSTR clusters with LUSTR-A sub-family proteins and is a first characterization of proteins containing Lung7TM-R domain in Mollusca. Significant differences were found between the Lit-LUSTR mRNA levels in haemocytes of healthy periwinkles and those naturally infected with the echinostome trematode Himasthla elongata. Down regulated expression of the LUSTR-like receptor caused by infection illustrates modification of the haemocyte receptor system and may be attributed to the previously demonstrated greater numbers of “immature” haemocytes in the circulation of infected snails.     

AiCTL-6, a novel C-type lectin from bay scallop Argopecten irradians with a long C-type lectin-like domain

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, a novel C-type lectin gene from scallop Argopecten irradians (designated as AiCTL-6) was cloned by rapid amplification of cDNA ends (RACE) approach based on expression sequence tag (EST) analysis. The full-length cDNA of AiCTL-6 was 1080 bp. The open reading frame encoded a polypeptide of 307 amino acids, including a signal sequence and a C-type lectin-like domain (CTLD) of 150 amino acid residues longer than any usual CTLD. It contained six conserved cysteine residues involved in the formation of three internal disulfide bridges and an EPD (Glu₂₆₉-Pro₂₇₀-Asp₂₇₁) motif at the Ca²⁺-binding site 2. The deduced amino acid sequence of AiCTL-6 showed high similarity to members of C-type lectin superfamily. By fluorescent quantitative real-time PCR, AiCTL-6 mRNA was found mainly in hepatopancreas and gill, and marginally expressed in other tissues. After the scallops were challenged by Listonella anguillarum for 6 h, the mRNA expression of AiCTL-6 was up-regulated significantly to 7.2-fold compared to the blank group. While at 9 h post Micrococcus luteus challenge, its expression level was 60.1 times higher than that of the blank group. The functional activity of AiCTL-6 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta gami (DE3). The recombinant AiCTL-6 could agglutinate Gram-negative bacteria E. coli TOP10F′, Gram-positive bacteria M. luteus and Staphylococcus aureus. These results collectively suggested that AiCTL-6, as a novel member of C-type lectin family, contributed to the host defense mechanisms against invading microorganism in A. irradians.