The Binding And Dissociation Mechanism Exploration Of Ligands To Vitellogenin Receptor In Silkworm, Bombyx Mori

Insect Vitellogenin receptor (VgR) belongs to the low-density lipoprotein receptor (LDLR) super-family, it is an important protein to ovary and embryonic development in oviparous animals, which mediates vitellogenin (Vg) into the ovary to supply nutrition with oocytes through endocytosis. So far, there are21species of insects have been reported in the VgR cDNA and genomic sequences, all of them have a typical LDLR conserved functional domains. BmVgR was reported which locates on chromosome20in silkworm, the full length cDNA sequences is5746bp in our laboratory. BmVgR was expressed during the entire life span of silkworm as ovary-specific protein. The oogenesis mutant’scanty vitelliri’(vit) was caused by the mutation of BmVgR, which had lacked150bp. The RNAi experiments of the female Bm VgR showed that the laid eggs had many small eggs with white color, almost less half of the normal and could not hatch. So BmVgR plays an important role in the ovary development. We have reported the full-length cDNA of BmVgR. In this study, we explored the function of BmVgR by gene clone, prokaryotic expression, cell expression, immunohistochemistry, Co-Immunoprecipitation, Far western blotting technique and so on. The results are as following:1. The structural analysis and clone of BmVgR in the silkwormBased on the reported21species of insects VgR, the bioinformatic analysis revealed the structural figures of VgRs were almost unanimous, they had two Ligand-binding domains (LBD1and LBD2) and EGF-precursor homology domains (EGFP1and EGFP2). The structure of BmVgR was the same with ApVgR and AsVgR (silkworm moths). Phylogenetic analysis indicated VgRs in the Lepidoptera insects evolved similarly; the homology of BmVgR^ApVgR and As VgR was the highest, which was the same as traditional evolutionary relationships. We cloned LBD1and LBD2of BmVgR, the amino acid sequence analysis indicated:LBD1consists of four ligand binding repeats (LBRs) with six cysteine residues each; LBD2has five different splicing forms, that is LBD2-(1-5), LBD2-2, LBD2-3and LBD2-4were inserted serval amino acid sequences, but LBD2-5lacked some amino acid sequences;Post-translational modifications of proteins showed LBD1and LBD2-(1-5) had many phosphorylation sites, and only LBD2-(1-5) had a little of N-glycosylation sites.2. The antibodies preparation of BmVg and BmVgR in the silkwormBmVg-ND and BmVgR-YWTD peptides were cloned and prokaryotic expressed, and they were expressed in inclusion body. After the purification by Ni-NTA affinity chromatography, the two purified proteins were sended to the company for making polyclonal rabbit antibodies. The titers of BmVg and BmVgR antibodies were more than1:512000, and the concentrations were0.4mg/mL and0.26mg/mL, respectively. So the antibodies of BmVg and BmVgR could be used for later experiments.3. Purification of BmVg from silkworm pre-pupal hemolymphAfter ammonium sulfate precipitation, SDS-PAGE showed BmVg was existed40%-60%mainly, and then we obtained purer BmVg (O.lmg/mL) after anion exchange chromatography, dialysis, concentrated. Western blotting showed the molecular weight of this protein was the same as predicted with BmVg polyclonal antibody. So the purified protein (?)Vg, and it could be used for binding experiment.4. Binding and dissociation between BmVgR and BmVgFor expression of normal and mutational BmVgR-LBD1+EGF1(p50:C11D, d50:C11V) with a Myc-tag and the signal peptide, Sf9cell line was used, the results showed expressed protein was secreted into the culture medium mainly. Immunohistochemistry in C11D and C11V showed there were no clear differences in expression between dazao and vit mutant. We determined the pH of different eggs for confirming the pH of BmVgR and BmVg dissociating in vitro, the result indicated the pH of egg was acidic, that was5.1-6.3. Co-IP assay showed the C11D and C11V could bind BmVg at neutral pH; BmVg could be dissociated from the C11D, but not from the C11V at acidic pH, These results showed the BmVgR of the vit mutant, which is mutated in the3rd Class B region of the EGF1domain, can bind ligands, however, ligands cannot be dissociated from mutational BmVgR in the acidic environment of the silkworm egg, so BmVg is lacked in the vit mutant.5. Interactions between BmVgR and different LigandsFor expression of BmVgR-LBDl and BmVgR-LBD2with a Myc-tag, Sf9cell line was used to express, the results showed expressed proteins exist in the cytoplasm. After cell expressing proteins by lysis and simple purification, BmVgR-LBD2(LBD2-1.. LBD2-4and LBD2-5) were incubated with purified BmVg. Far western blotting showed the three forms could bind BmVg, but their binding ability needs to be confirmed in further experiment. Binding of the three forms of proteins and four30K proteins had not got a clear result, further experiments will be needed.