Lipovitellin In Rosy Barb (Puntius Conchonius) Is A Pattern Recognition Receptor With An Opsonic Activity

Lipovitellin (Lv), a glycolipoprotein, is the major component of egg yolk, which is used to be regarded only as energy reserves for nutrients essential for growth and development. In this study, Lv was purified from the ovulated eggs of the rosy barb Puntius conchonius by double-step chromatography (ion exchange chromatography on a DEAE-23 cellulose column and gel filtration on a Superdex 200 column).The protein purified from ovulated eggs of P. conchonius had an apparent molecular weight of approximately 370 kDa under native PAGE,and was stained positively for carbohydrate and lipid but stained rather weakly for phosphorus. Under reducing SDS-PAGE, the purified protein was resolved into three bands of approximately 125,110 and 76 kDa with 110 kDa being the major band, resembling medaka and frog Lvs. Amino acid composition of the purified protein was closely similar to rainbow trout, Sakhalin taimen and medaka Lvs. Peptide mass fingerprinting revealed that the peptides from the bands of 125 kDa and 110 kDa were identical to the heavy chain Lv of carp(Cyprinus carpio) vitellogenin B1,and the peptide from the band of 76 kDa was identical to the light chain Lv of C. carpio vitellogenin B1.Both amino acid composition analysis and peptide mass fingerprinting showed the protein purified double-step chromatography was Lv. Polyclonal antisera against the purified Lv from rosy barb were raised in rabbit firstly, Native PAGE and western blotting demonstrated that specificity of antisera were good and suggest that Vg in the plasm of female rosy barb can be detected by anti-Lv serum. In addition, tissue-specificity of Lv was also demonstrated using double immunodiffusion.Lv is used to be regarded only as energy reserves for nutrients essential for growth and development, but other roles of Lv remain lacking. Eggs of most fish are released and fertilized externally, and the resulting embryos and larvae are therefore exposed to an aquatic environment full of potential pathogens capable of causing various types of diseases. During the early stages of development, fish embryos and larvae have little or only limited ability to synthesize immune-relevant molecules endogenously and their lymphoid organs are not yet fully matured.In this study, it was demonstrated for the first time that the native form of P. conchonius Lv purified acted as a pattern recognition molecule with an opsonic activity.FITC-labeled Lv was incubated with E. coli and S. aureus. It was found that Lv bound to both E. coli and S. aureus. We then incubated the microbes E. coli and S. aureus with egg extracts and Lv solution, respectively, the proteins bound to the microbes were eluted and subjected to Western blotting. Lv was monitored in the eluates derived from the microbes incubated with egg extracts and Lv solution, All these demonstrated that the native form of Lv was able to specifically bind to both E. coli and S. aureus. To better understand the mechanisms of the bacterial binding activity of native Lv, an enzyme-linked immunosorbent assay (ELISA) was carried out to investigate which molecules on the microbial surfaces were recognized by Lv. It was observed that Lv strongly bound to pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide, lipoteichoic acid and peptidoglycan rather than self components in a dose-dependent manner.It was also been tested through phagocytosis test that if the native form of P. conchonius was capable of enhancing macrophage phagocytosis. The number of macrophages with phagocytosed microbes and the number of microbes in each macrophage were recorded. The phagocytic ability (PA) was defined as the percentage of macrophages with one or more engulfed microbes within the total cell population, and the phagocytic index (PI) as the number of engulfed microbes per cell.It is clear that the percent of macrophages phagocytosing Lv-treated microbes was significantly higher than that of cells phagocytosing BSA-treated and untreated microbes (p<0.05): Furthermore, the number of Lv-treated microbes engulfed by each macrophage was also significantly higher than that of BSA-treated and untreated microbes engulfed by each macrophage (p<0.05).These together suggest that in P. conchonius developing embryos/larvae, the native form of Lv may be physiologically involved in the sensing of invading pathogens via interaction with PAMPs as well as the recruitment of the primitive macrophages appeared in the early embryos to phagocytose and digest the pathogens, protecting them from pathogenic attacks.