studies on chicken riboflavin carrier protein : Recombinant expression and receptor characterization

Abstract

Investigations detailed in this thesis constitute part of an ongoing research programme in our laboratory on riboflavin carrier protein (RCP), with particular reference to recombinant expression of chicken RCP in E. coli and characterization of specific receptors for RCP in the chicken oocyte. Chapter I (General Introduction) summarizes available information on the biochemical and physicochemical aspects of chicken RCP. This chapter also outlines relevant information on vitellogenesis-i.e., deposition of yolk during chicken oocyte growth-which is directly related to Chapter III of this thesis, dealing with the mechanism of RCP uptake by the chicken oocyte. RCP ensures adequate vitamin deposition in the developing chicken oocyte. The protein is highly conserved across evolution, from fish through birds to mammals including primates, in both physicochemical and immunological properties. In rodents and subhuman primates, immunization with chicken eggwhite RCP leads to functional neutralization of endogenous maternal RCP, resulting in curtailment of early pregnancy. Thus, RCP plays a crucial role in the maintenance of pregnancy and has been identified as a potential candidate vaccine for immunocontraception.

Chapter II: Production of Chicken RCP in E. coli The goal of these studies was to determine whether RCP produced in E. coli could serve as an antigen capable of generating antibodies that would neutralize endogenous RCP during early pregnancy in mammals. The RCP cDNA encoding a protein of predicted Mr 27,000 was cloned into a T7polymerasedriven expression vector. Highlevel expression was achieved upon IPTG induction in E. coli. At 37°C, the recombinant protein localized mainly to inclusion bodies. When the induction temperature was reduced to 22°C, RCP was present predominantly in the cytosol, and Nterminal sequencing showed removal of the pelB leader peptide from the vector. However, RCP was not detected in the periplasm. At 37°C, two major protein species were observed; sequence and molecularweight analysis showed that these represented RCP with and without the pelB leader peptide. This differential production at different temperatures may be due to alternative secondary structures adopted by the RCP mRNA in E. coli. Extraction of inclusion bodies with detergents preferentially solubilized the protein lacking the pelB signal sequence. This recombinant RCP was recognized by polyclonal antibodies to native RCP, though radioimmunoassay revealed differences in epitope affinity. Sequencespecific monoclonal antibodies crossreacted effectively, while conformationdependent antibodies showed reduced recognition, indicating that only partial conformational epitopes were present. Polyclonal antibodies raised against recombinant RCP recognized native chicken RCP and, when administered to pregnant mice, caused embryonic resorption and early termination of pregnancy. These findings indicate that recombinant RCP is suitable for studying pregnancy termination mechanisms and for analysing folding, ligand binding, and antigenic determinants of RCP.

Chapter III: Identification and Characterization of RCP Receptors in Chicken Oocytes Membranes prepared from whole chicken oocytes were used to investigate RCP uptake. RCP binding to membranes was inhibited by serum, eggwhite and yolkderived RCPs, as well as by vitellogenin. Binding required calcium absolutely, suggesting that RCP interacts with members of the lowdensity lipoprotein receptorrelated protein (LRP) family. The binding affinity (Kd 10 M) was high enough to be physiologically relevant. Reduced and carboxyamidated RCP inhibited the binding of ¹²Ilabelled RCP as effectively as native RCP, suggesting that native tertiary conformation is not essential for receptor interaction. Ligand blotting of solubilized oocyte membrane proteins revealed three RCPbinding proteins of Mr 380, 260, and 110 kDa. These were distinct from the known LRP receptor ligands such as RAP (receptorassociated protein) but resembled receptors identified using radiolabelled vitellogenin. Dephosphorylated RCP failed to inhibit receptor binding, indicating that the phosphaterich region of RCP is critical for interaction. A purified phosphopeptide derived from tryptic digests of RCP inhibited receptor binding with affinity comparable to native RCP, indicating that this phosphopeptide acts as a novel ligand for LRPfamily receptors in chickens.