Big defensin 3

Tachylectin-5, a 41-kDa protein with a common fold of the C-terminal globular domain of the γ-chain of fibrinogen, is purified from horseshoe crab hemolymph plasma by affinity column chromatography, using acetyl-group-immobilized resin. Two types of isolectins, tachylectin-5A and tachylectin-5B, are obtained by stepwise elution with GlcNAc at 25 and 250 mM, respectively. Tachylectins-5A and -5B exhibit extraordinarily strong hemagglutinating activity against all types of human erythrocytes (the minimum agglutinating concentration of 0.004-0.008 μg/mL for tachylectin-5A and 0.077-0.27 μg/mL for tachylectin-5B). Their hemagglutinating activities are inhibited by acetyl group-containing sugars and noncarbohydrates such as sodium acetate, acetylcholine, and acetyl CoA (the minimum inhibitory concentrations of 1.3-1.6 mM), indicating that the acetyl group is required and sufficient for recognition by tachylectins-5A and -5B. EDTA inhibits their hemagglutinating activity, whereas the inhibition is overcome by adding an excess amount of Ca2+. Tachylectins-5A and -5B also exhibit bacterial agglutinating activity against both Gram-negative bacteria (the minimum agglutinating concentrations of 0.04-0.08 μg/mL for tachylectin-5A and 0.05-0.11 μg/mL for tachylectin-5B) and Gram-positive bacteria (the minimum agglutinating concentrations of 0.3-2.4 μg/mL for tachylectin-5A and 15.1-26.8 μg/mL for tachylectin-5B). Interestingly, tachylectins-5A and -5B enhance the antimicrobial activity of a hemocyte-derived peptide, big defensin.

Antimicrobial peptides (AMPs) are important mediators of the primary defense mechanism against microbial invasion. In the present study, a big defensin was identified from Venerupis philippinarum haemocytes (denoted as VpBD) by RACE and EST approaches. The VpBD cDNA contained an open reading frame (ORF) of 285 bp encoding a polypeptide of 94 amino acids. The deduce amino acid sequence of VpBD shared the common features of big defensin including disulfide array organization and helix structure, indicating that VpBD should be a new member of the big defensin family. The mRNA transcript of VpBD was up-regulated significantly during the first 24 hr after Vibrio anguillarum challenge, which was 7.4-fold increase compared to that of the control group. Then the expression decreased gradually from 24 hr to 96 hr, and the lowest expression level was detected at 96 hr post-infection, which was still 3.9-fold higher than that of control. The mature peptide of VpBD was recombined in Escherichia coli and purified for minimum inhibitory concentration (MIC) determination. The rVpBD displayed broad-spectrum inhibitory activity towards all tested bacteria with the highest activity against Staphyloccocus aureus and Pseudomonas putida. These results indicated that VpBD was involved in the host immune response against bacterial infection and might contribute to the clearance of invading bacteria.

The noble scallop Chlamys nobilis has been an important marine cultured bivalve in the Southern Sea of China for decades. However, large-scale mortality events often occurred during the scallop' cultivation. As one of AMPs (antimicrobial peptides), big defensin is an important component of the innate immunity against pathogenic microorganisms in invertebrates. In order to investigate whether the big defensin can play a role in the immune defense against pathogenic microorganisms in noble scallop, a big defensin gene from the hemocytes of Chlamys nobilis (CnBD) was cloned, and the mRNA level was measured after an acute Vibrio parahaemolyticus challenge of 36 h. The CnBD cDNA contains an open reading frame (ORF) of 381 bp encoding a peptide of 126 amino acids residues. The deduce amino acid sequence of CnBD shows a high similarity with that from Argopecten irradians and displays common features of big defensin, indicating that CnBD is a new member of the big defensin family. Compared with the control group, the relative mRNA level of CnBD was significantly up-regulated at 3, 24 and 36 h. The present result indicated that CnBD played an immune role against bacterial infection in noble scallop.