Study Of A New C-type Lectin With A Long Carbohydrate Recognition Domain(CRD)from Artemia Parthenogenetica

C-type lectin is a family of Ca2+dependent carbohydrate-recognition proteins which play crucial roles in the innate immunity of invertebrates by mediating the recognition of host cells to pathogens and clearing micro-invaders as a pattern recognition protein (PRP). The cDNA of Artemia parthenogenetica C-type lectin (designated ApLC) was cloned by expressed sequence tag (EST) and PCR. The full-length cDNA of ApLC was1077bp, consisting of a5'-terminal untranslated region (UTR) of24bp and an3'UTR of219bp with a polyadenylation signal sequences AATAAA and a poly (A) tail. The ApLC cDNA encoded a polypeptide of284amino acids without a putative signal peptide. Analysis of the protein domain features indicated a typical long-form carbohydrate-recognition domain (CRD) of222residues in the ApLC deduced amino acid sequence. The expression pattern of ApLC transcripts in healthy and bacterial challenged brine shrimp was studied by far southern blotting. mRNA transcripts of ApLC could be mainly detected in the thorax of the brine shrimp during the ovoviviparous reproductive mode and in the thorax and the abdomen during the oviparous reproductive mode of the brine shrimp. Whereas the expression of ApLC transcripts was increased in thorax after bacteria challenge. The temporal expression of ApLC mRNA after challenge with Gram-negative bacteria Vibrio alginaliticus, Vibrio parahaemoliticus, Vibrio fluvialis, Aeromonas hydrophyla, Gram-positive bacteria Staphylococcus aureus was up-regulated and reached the maximum level between3and12h post stimulation, and then dropped back to the original level. In order to investigate its immune functions, ApLC was recombined and expressed in Escherichia coli BL21(DE3) as a fusion protein with a His-tag. The recombinant ApLC agglutinated Gram-negative bacteria Vibrio alginaliticus, Vibrio parahaemoliticus, Vibrio fluvialis, Aeromonas hydrophyla, Gram-positive bacteria Staphylococcus aureus and erythrocytes rabbit in vitro, and the agglutination was Ca2+dependent which could be inhibited by EDTA. Meanwhile, the recombinant ApLC could inhibit the growth of Gram-negative bacteria Vibrio alginaliticus, Vibrio parahaemoliticus, Vibrio fluvialis, Aeromonas hydrophyla, but no inhibition activity against Gram-positive bacteria Staphylococcus aureus. These result indicated that ApLC was a constitutive and inducible PRP which was involved in the reorganization and clearance of invaders in Artemia parthenogenetica.