Expression, Purification And Characterization Of BmGalectin-4in Silkworm, Bombyx Mori

Galectins, which can bind β-galactoside specifically, are a group of lectins. Because oftheir wide distribution and highly specificity, galectins have been studied as importantimmune factors since they were first characterized in the1970s. So far,15galectins havebeen found in mammals, and they were generally considered to bind endogenouscarbohydrate moieties, function as modulators of cell adhesion, signal transduction, apoptosis,immune homeostasis and so on. Recently, people found galectins can also bind exogenouscarbohydrate moieties, function as pattern recognition receptors (PRRs). In insects, somestudies indicated that galectin can influence the interaction between parasites and their hostinsects, but seldom related to other immune functions. Here, we chose a galectin namedBmGalectin-4, to explore its immune function in silkworm, Bombyx mori. Our works asfollows:First, sequence analysis showed BmGalectin-4is a tandem repeat galectin.BmGalectin-4and tandem repeat type galectins in other organisms are highly conservedaccording to sequence alignment. The carbohydrate recognition domain of BmGalectin-4comprised all conserved binding sites interact with β-galactoside. Phylogenetic analysisrevealed that the tandem repeat galectins from insects, nematodes and mammals formed threehighly distinctive clusters. We used Galectin-8crystal structure as a template to predictBmGalectin-4’s structure. The results showed that both of the CRDs’ structure inBmGalectin-4have the typical characteristics of the CRD in galectin, with the highlysimilarity between.Semi-quantitative RT-PCR was used to detect BmGalectin-4expression at different timepoints after bacterial and fungal challenge in vivo. We found BmGalectin-4are expressed inthe fat body, midgut, and hemocytes simultaneously, and the expression level changedsignificantly at some time points.We cloned and sequenced the full-length of BmGalectin-4sequence, obtained a1089bpnucleic acid sequence, which encoding363amino acids. Prokaryotic expression system wasused to express the recombinant protein. We got a56kD purified recombinant protein byNi-NAT affinity chromatography. The recombinant protein was then digested with protease, followed a second Ni-NAT affinity chromatography purification. We finally obtained a43kDpurified BmGalectin-4.ELISA technique was used to detect the binding property of recombinant protein withdifferent concentrations to Gram-negative bacteria Pseudomonas aeruginosa, Gram-positivebacteria Staphylococcus aureus and the fungus Candidiasis albicans. We found that purifiedrecombinant BmGalectin-4exhibited significant bacterial binding ability with all threepathogens in a dose-dependent mannerIn order to detect the binding ability of BmGalectin-4to insoluble polysaccharides, weincubated the recombinant protein with peptidoglycans in Gram-positive bacteria orGram-negative bacteria, or curdlan in fungus, respectively. Protein was then eluted and thesamples were detected by Western blot to test the binding ability of BmGalectin-4with thesepolysaccharides. The results showed that BmGalectin-4can bind all the peptidoglycan inbacterias, and curdlan in fungus, except the peptidoglycan in Micrococcus lutes, indicatingthe bindinding specificity of BmGalectin-4to saccharides.In addition, BmGalectin-4can not activate prophenoloxidase cascade pathway alone.But in the group added both protein and elicitor, prophenoloxidase activation pathway wasinhibited in some cases. We found BmGalectin-4protein did not promote phagocytosis topathogens.All the results showed that BmGalectin-4is an immune related protein. This protein canrecognize pathogens of different kinds, and this recognition maybe based on the identificationof polysaccharides.