Immune Roles Of Fish Vitellogenin

Vitellogenin (Vg) is the egg yolk protein precursor in non-mammalian vertebrates and invertebrates. It is a high molecular mass phospholipoglycoprotein response to estrogen stimulation. Vg is known as its nutritional role to provide developing embryos and larvae nutrients. Many reports proved that Vg has evolved pleiotropic functions in many physiological processes. Vg has recently been demonstrated to possess both hemagglutinating and antibacterial activities in the protochordate amphioxus (Branchiostoma belcheri) as well as the bony fish rosy barb (Puntius conchonius). However, the mode of action by which Vg is involved in anti-infectious response remains unknown. The main objective of this study is to demonstrate the underlying mechanisms by which Vg is involved in anti-infectious response.First, fish Hexagrammos otakii (H. otakii) Vg was purified and characterized. A 17β-estradiol induced protein was purified by DEAE-23 anion exchange chromatography and Sephadex G-200 gel filtration chromatography from 17β-estradiol-injected intraperitoneally fish plasma. The purified protein was identified as Vg by MALDI TOF/TOF MS analysis. H. otakii Vg appeared to exist as a homogenous trimer of approximately 450 kDa in native-PAGE. Vg was proved as a phospholipoglycoprotein by staining using Sudan black B, periodic acid/Schiff reagent, and methyl green.Second, to test if Vg can bind to microbes, FITC-labeled Vg was incubated with Gram-negative bacterium Escherichia coli (E. coli), Gram-positive bacterium Staphylococcus aureus (S. aureus) and fungus Pichia pastoris (P. pastoris). It is found that Vg is able to bind both E. coli and S. aureus as well as P. pastoris. To better understand the mechanisms of binding activity, an enzyme-linked immunosorbent assay (ELISA) was carried out to investigate what molecules on the microbial surfaces are recognized by Vg, and it is found that Vg had a significantly stronger affinity to the immobilized ligands including LPS from Gram-negative bacteria, LTA from Gram-positive bacteria, PGN from both Gram-positive and Gram-negative bacteria,β-1, 3-glucan from fungi and laminarin from brown algae concentration-dependently than to BSA, the serum proteins (SPs) and total muscle proteins (TMPs) extracted from H. otakii itself. These results indicated that Vg was a multivalent pattern recognition receptor. In addition, we addressed the effects of Vg on the phagocytosis of H. otakii macrophages. It is demonstrated that Vg can promote macrophage phagocytosis. The head kidney of teleostean fish is considered to be the functional and structural homologue of mammalian bone marrow. H. otakii head kidney macrophages were isolated by discontinuous gradient centrigugation. Then phagocytosis assays were performed and two parameters phagocytic ability (PA) and phagocytic index (PI) were used to test the effects of Vg on phagocytosis. Compared with the microbes pre-incubated with BSA and PBS alone, those pre-incubated with Vg were readily phagocytosed by the macrophages. The PA and PI values of the macrophages phagocytosing Vg-treated E. coli, S. aureus and P. pastoris were significantly higher than those of cells phagocytosing BSA-treated and untreated microbes (p<0.05). These denoted that Vg was able to promote the macrophage phagocytosis. Furthermore, it is found that FITC-labeled Vg is able to bind to the surface of macrophages specifically. These results indicate that Vg maybe act as an opsonin.Finally, the way by which Vg plays antibacterial activities and its active components which are essential for the execution of immune activities were studied in this part. It is demonstrated that fish Vg is capable of killing E. coli and S. aureus whole cells via interaction with LPS and LTA existing in the bacterial cell walls, rather than targeting plasma membranes,and the integrity of the polypeptide chain and the carbohydrate residues of Vg are indispensible for its antibacterial activity. Antimicrobial proteins and peptides exert their bactericidal effects by several different pathways. Most proteins exert their antibacterial effects by interacting with and destabilizing the microbial cell membrane, but some proteins were demonstrated to kill the bacteria by damaging cell wall directly via interacting with components of cell walls. It is demonstrated by SEM and bacterial cell and protoplast lysis assays that Vg is able to cause lysis of both E. coli and S. aureus whole cells rather than their cell wall-lacking protoplasts. To test the effects of LPS, LTA and PGN on the antibacterial activities of Vg, these chemicals were preincubated with the protein, then antagonism assays were performed on a petri dish. It is found that the antibacterial activity of Vg against E.coli is able to be inhibited by LPS from E.coli, agreeing with the binding of LPS to Vg, and the antibacterial activity against S. aureus is able to be inhibited by LTA from S. aureus, according with the binding of LTA to Vg. It is believable that Vg acts as a direct microbial killing protein via interaction with LPS and LTA.The components of Vg active in bacteriostasis were indentified in this part. Post-translational processing and modifications of proteins are often important for their biological function. To identify the components of Vg active in bacteriostasis, the protein was degraded by trypsin, oxidated by NaIO4, delipidated by either and dephosphorylated by AP, and antibacterial assays were performed on a petri dish. It is found that neither delipidation nor dephosphorylation of Vg had any influence on its antibacterial activities, but degradation of polypeptide chain and oxidation of carbohydrate residues significantly reduced its antibacterial activities. These show that the glycoprotein component of Vg plays a crucial role in inhibiting the bacterial growth. This is further supported by the fact that the antibacial activities of Vg are inhibited by the sugars like D-mannose, GluNAc and D-fucose, suggesting that Vg functions like lectins.In summary, the present study demonstrates for the first time that Vg from fish H. otakii can act as a multivalent pattern recognition receptor binding to several PAMPs, and functions as an opsonin that can enhance macrophage phagocytosis; It is also demonstrated for the first time that Vg is capable of killing E. coli and S. aureus whole cells via interaction with LPS and LTA existing in the bacterial cell walls, and it highlights the correlation between the polypeptide integrity and glycosylation of Vg and its bactericidal activities.