Exploring Serum Proteins to Stabilize the Conformation of the Precursor Protein of ANP

Abstract

The atrial natriuretic peptide (ANP) is expressed in the form of a precursor, pre-pro-ANP, and is then processed into pro-ANP, which consists of the pro-peptide and the mature peptide. The biological role of ANP has been extensively investigated, but the role of the pro-peptide region continues to remain unclear. Therefore, to study the role of the pro-peptide region of pro-ANP, recombinant pro-ANP molecules were prepared using an E. coli expression system and the resulting proteins were examined in our laboratory. Secondary structure analyses of the pro-ANP by CD in our previous study indicated that pro-ANP is an intrinsically disordered protein and the formation of an α-helical structure rapidly occurs by the addition of organic solvents, such as ethanol and trifluoroethanol. The results suggest that some currently unknown factors contribute to stabilizing the conformation of pro-ANP in vivo. Therefore, to explore these in vivo factors that stabilize the conformation of pro-ANP, the interactions of human serum albumin (HSA) and lipocalin-type prostaglandin D synthase (L-PGDS), major serum proteins, with pro-ANP were examined. The interactions between pro-ANP and candidate interactor proteins were estimated by pull-down assays, DLS, and CD measurements. The binding experiments were carried out in the presence of various concentrations of HSA. The concentrations of HSA bound to pro-ANP in pull-down assays were determined by densitometric analyses of the SDS-PAGE data. The results indicated that HSA specifically binds to pro-ANP in a concentration-dependent manner, suggesting the existence of specific in vivo interactions between the two proteins. In addition, the results of DLS measurements also indicated that the pro-ANP molecule forms a complex with HSA molecule(s). In addition, a pro-ANP and L-PGDS complexes signal was also detected by DLS measurements.