Functional identification of C-type lectin in the diamondback moth,Plutella xylostella(L.)innate immunity

C-type lectins(CTLs)are a superfamily of Ca^(2+)-dependent carbohydrate-recognition proteins,and an important pattern recognition receptor(PRR)in insect innate immunity which can mediate humoral and cellular immunity in insects.In this study,we report a novel dual carbohydrate-recognition domain(CRD)CTL from Plutella xylostella which we designate PxIML.PxIML is a protein with a 969 bp open reading frame(ORF)encoding 322 amino acids,containing a signal peptide and a dual-CRD with EPN(Glu_(124)-Pro_(125)-Asn_(126))and QPD(Gln_(274)-Pro_(275)-Asp_(276))motifs.The expression of PxIML mRNA in the fat body was significantly higher than in hemocytes and midgut.The relative expression levels of PxIML in the whole insect and the fat body were significantly inhibited after infection with Bacillus thuringiensis 8010(Bt8010)at 18 h,while they were significantly upregulated after infection with Serratia marcescens IAE6 or Pichia pastoris.The recombinant PxIML(rPxIML)protein could bind to the tested pathogen-associated molecular patterns(PAMPs),and the bacteria of Enterobacter sp.IAE5,S.marcescens IAE6,Staphylococcus aureus,Escherichia coli BL21,and Bt8010 in a Ca^(2+)-dependent manner,however,it showed limited binding to the fungus,P.pastoris.The rPxIML exhibited strong activity in the presence of Ca^(2+) to agglutinate Bt8010,Enterobacter sp.IAE5 and S.aureus,but it only weakly agglutinated with E.coli BL21,and could not agglutinate with S.marcescens IAE6 or P.pastoris.Furthermore,the rPxIML could bind to hemocytes,promote the adsorption of hemocytes to beads,and enhance the phenoloxidase(PO)activity and melanization of P.xylostella.Our results suggest that PxIML plays an important role in pathogen recognition and in mediating subsequent humoral and cellular immunity of P.xylostella.

Molecular characterization and expression analysis of a novel dual-CRD C-type lectin in kuruma shrimp(Marsupenaeus japonicus)

C-type lectins are among the most significant pattern recognition receptors（PRRs） found in invertebrate. They are a class of carbohydrate-binding proteins that can recognize specific sugar moieties on the surface of pathogens. In the present study, a novel C-type lecitn（termed Mj Lectin） from kuruma shrimp Marsupenaeus japonicus was identified. The full-length c DNA of Mj Lectin was 1 245 bp with a 1 011 bp open reading frame（ORF） that encoded a polypeptide of 336 amino acid residues. Mj Lectin consisted of two tandemly arrayed carbohydrate-recognition domains（CRDs）, unlike other reported M. japonicus C-type lectins with only one CRD. It showed a high similarity to other shrimp dual-CRD lectins. Among the Ca2＋-binding Site 2, the tripeptide motif dictating the carbohydrate binding specificity was exhibited as a rare mutant LPN（Leu134-Pro135-Asn136） in CRD1 and a traditional EPN（Glu299-Pro300-Asn301） in CRD2, respectively. Mj Lectin showed a specific expression pattern in both tissue and cellular levels, for its m RNA transcript was mainly expressed in the F-cells of the hepatopancreas. After white spot syndrome virus（WSSV） challenge（3.6×108 virions/μL）, the expression of Mj Lectin in the hepatopancreas was up-regulated significantly at 48 h（P〈0.01） compared with the control group. These results suggested that Mj Lectin might be involved in the innate immune defense against WSSV infection.

Comparative analysis of C-type lectin domain proteins in the ghost moth, Thitarodes xiaojinensis (Lepidoptera: Hepialidae)

Insects have a large family of C-type lectins involved in cell adhesion, pathogen recognition and activation of immune responses. In this study, 32 transcripts encoding C-type lectin domain proteins (CTLDPs) were identified from the Thitarodes xiaojinensis transcriptome. According to their domain structures, six CTLDPs with one carbohydraterecognition domain (CRD) were classified into the CTL-S subfamily. The other 23 CTLDPs with two CRDs were grouped into the immulectin (IML) subfamily. The remaining three with extra regulatory domains were sorted into the CTL-X subfamily. Phylogenetic analysis showed that CTL-S and CTL-X members from different insects could form orthologous groups. In contrast, no T. xiaojinensis IML orthologues were found in other insects. Remarkable lineage-specific expansion in this subfamily was observed reflecting that these CTLDPs, as important receptors, have evolved diversified members in response to a variety of microbes. Prediction of binding ligands revealed that T. xiaojinensis, a coldadapted species, conserved the ability of CRDs to combine with Ca^2+ to keep its receptors from freezing. Comparative analysis of induction of CTLDP genes after different immune challenges indicated that IMLs might play critical roles in immune defenses. This study examined T. xiaojinensis CTLDPs and provides a basis for further studies of their characteristics.