Gene Cloning Characterization of Lectin Containing Low-Density Lipoprotein Receptor Domain from Hemocytes of Fenneropenaeus merguiensis

Abstract

As the pattern recognition molecules, lectins are related to many biological processes such as protein trafficking, cell signaling, pathogen recognition etc. These proteins are generally found in almost all living organisms. Almost lectins have a carbohydrate recognition domain (CRD). They can interact via CRD domain to the carbohydrate molecules on different types of cells including bacterial cells and viral particles. Since a lectin molecule has at least two binding sites that are specific to surface of cells, the protein causes cell agglutination when it attaches to specific carbohydrates on the cell surfaces. C-type lectin (CTL) is a group of lectin that requires Capt to complete its functions. There are several reports of CTLs in marine shrimp. The interaction of CTLs with shrimp pathogens activates various immune processes such as phagocytosis, encapsulation and prophenoloxidase activating system. These processes are important mechanisms to combat with shrimp pathogens. According to the elements in their molecules, shrimp CTLs are divided into 3 groups; CTLs with only one CRD domain, CTLs with dual CRD domains and CTLs with one CRD domain plus additional another domain. Although most common shrimp CTLS are classified in the first two groups, recent study reported that CTL containing CRD and low-density lipoprotein receptor class A domain (LDLR) of Litopenaeus vannamei and Marsupenaeus japonicus contributed in shrimp immune system against yellow head virus (YHV) and White spot syndrome virus (WSSV), respectively. In this study, we clarified the function of a CTL containing CRD and LDLR of F. merguiensis designated as FmLdlr and also illustrated the role of two domains (CRD and LDLR) of FmLdlr in the shrimp immune system. In addition, the previous studies concerning vitellogenin (Vg) which is a precursor protein in oogenesis of female shrimp found that transporting Vg from hepatopancreas (synthesized organ) to the ovary (target organ) via hemolymph relies on the receptor. Type of this Vg receptor is the LDLR family protein. Therefore, it is interesting to study a function of LDLR domain within FmLdlr molecule whether it is related to the vitellogenin transportation or not. The full-length cDNA of FmLdlr gene was cloned from hemocytes of F. merguiensis by rapid amplification of cDNA ends (RACE) method and RT-PCR. It contained 1425 bp including a 100 bp of 5' UTR, a 410 bp of 3' UTR and a 915 bp of open reading frame (ORF) sequence. The deduced amino acid sequence of FmLdlr was composed of 305 amino acid residues. The mature protein had a calculated molecular mass of 31.78 kDa and pl of 5. It contained a LDLR domain at the Nterminus and a CRD with a QAP motif at the C-terminus. The amino acid sequence alignment of FmLdlr showed the highest identity (89%) with LdlrLecl, the LDLRcontaining CTL from M. japonicus. RT-PCR result in different tissues showed that the expression of FmLdlr was detected only in hemocytes. After shrimp were inoculated with pathogens, the transcriptional expression levels of FmLdlr were detected by quantitative real time-PCR ( RT-PCR). Vibrio parahaemolyticus and WSSV challenge showed the similar results that the mRNA expression of FmLdir responded to the pathogens by being up-regulated significantly. The RNA interference-mediated knockdown of FmLdlr resulted in severe down-regulation of FmLdlr gene. The knockdown with pathogenic co-injection caused reducing in the median lethal time and increasing the cumulative mortality of shrimp. Recombinant proteins of FmLdlr (rFmLdlr) and its domains (rCRD and LDLR) were successfully produced in bacterial system and applied in the work to characterize function of this lectin. The results of bacterial agglutination test showed that rFmLdlr and its domains could agglutinate all kinds of tested bacteria including two gram-positive bacteria (Staphylococcus aureus, Bacillus cereus) and four Gram-negative bacteria (Vibrio harveyi, V. parahaemolyticus, Escherichia coli, Salmonella typhi) at different concentrations. All agglutination was displayed in a Ca?t-dependent manner. Among recombinant proteins, rFmLdlr exhibited the maximal activity towards all tested microorganisms while rCRD had more activity than rLDLR. Moreover, all lectin recombinant proteins had the maximal activity against V. parahaemolyticus. The rFmLdlr and CRD could enhance the in vitro phagocytic activity and in vitro encapsulation of F. merguiensis hemocytes whereas LDLR could not. The rFmLdir could promote the in vivo bacterial clearance activity via the main functional CRD domain. FmLdlr and its domains had inhibitory effect on the growth of V. parahaemolyticus in a dose-dependent manner. ELISA assay demonstrated that rFmLdlr and CRD possessed the binding activity to all tested WSSV recombinant proteins (rVP15, rVP28 and rVP39A) at different affinities. ELISA was also used to detect the binding between recombinant proteins of lectin (rFmLdlr, rLDLR and TCRD) and purified native Vg. Recombinant proteins of the lectin could bind to Vg with different affinities while rLDLR showed the highest specific binding to Vg. Altogether, we concluded that the unique CTL named FmLdlr existed in F. merguiensis might contributed in the shrimp immune response against invading pathogens and its LDLR domain exhibited the potent binding to Vg that might function as a Vg receptor to transport this protein in the hemolymph.