Study of a new C-type lectin with a long Carbohydrate recognition Domain (CRD) from Artemia parthenogenetica

摘要

C-type lectin is a family of Ca2+ dependent carbohydrate-recognition proteins whichplay crucial roles in the innate immunity of invertebrates by mediating therecognition of host cells to pathogens and clearing micro-invaders as a patternrecognition 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 was 1077 bp, consisting of a 5'-terminal untranslated region(UTR) of 24 bp and an 3' UTR of 219 bp with a polyadenylation signal sequencesAATAAA and a poly(A) tail. The ApLC cDNA encoded a polypeptide of 284 aminoacids without a putative signal peptide. Analysis of the protein domain featuresindicated a typical long-form carbohydrate-recognition domain (CRD) of 222residues in the ApLC deduced amino acid sequence. The expression pattem of ApLCtranscripts in healthy and bacterial challenged brine shrimp was studied by farsouthem blotting. mRNA transcripts ofApLC could be mainly detected in the thoraxofthe brine shrimp during the ovoviviparous reproductive mode and in the thorax andthe abdomen during the oviparous reproductive mode of the brine shrimp. Whereasthe expression ofApLC transcripts was increased in thorax after bacteria challenge.The temporal expression of ApLC mRNA after challenge with Gram-negativebacteria Vibrio alginaliticus, Vibrio parahaemoliticus, Vibrio fluvialis, Aeromonashydrophyla, Gram-positive bacteria Staphylococcus aureus was up-regulated andreached the maximum level between 3 and 12 h post stimulation, and then droppedback to the original level. In order to investigate its immune functions, ApLC wasrecombined and expressed in Escherichia coli BL21(DE3) as a fusion protein with aHis-tag. The recombinant ApLC agglutinated Gram-negative bacteria Vibrioalginaliticus, Vibrio parahaemoliticus, Vibrio fluvialis, Aeromonas hydrophyla,Gram-positive bacteria Staphylococcus aureus and erythrocytes rabbit in vitro, andthe agglutination was Ca2+ dependent which could be inhibited by EDTA.Meanwhile, the recombinant ApLC could inhibit the growth of Gram-negativebacteria Vibrio alginaliticus, Vibrio parahaemoliticus, Vibrio fluvialis, Aeromonashydrophyla, but no inhibition activity against Gram-positive bacteria Staphylococcusaureus. These result indicated that ApLC was a constitutive and inducible PRP whichwas involved in the reorganization and clearance of invaders in Artemiaparthenogenetica.