Expanded insight into processing and isoform dependent properties of apelin

Abstract

Apelin is one of two peptide ligands for a class A G-protein-coupled receptor (the apelin receptor; AR/APJ). Apelin-AR signalling regulates many body systems, including the cardiovascular system, central nervous system, and adipoinsular axis. Notably, apelin can exist as various isoforms and demonstrates isoform-dependent variation in its potency and efficacy, with potency inversely correlated to isoform size. Thus, apelin processing may have an important regulatory role. The existing processing theory suggested that upon N-terminal signal peptide removal, the resulting 55-residue peptide would be an inactive proprotein (apelin-55), requiring an N-terminal truncation to a 36-residue isoform (apelin-36) for activation. Apelin-36 could then be further processed into 17- or 13-residue isoforms to increase potency. However, apelin-55 detection extracellularly did not fit this theory. Thus, I focused on better elucidating our understanding of apelin processing and the potential involvement of biological membranes in regulating the apelinergic system. I begin by demonstrating, through development of an in vitro assay using high performance liquid chromatography and mass spectrometry, that proprotein convertase subtilisin kexin subtype 3 (PCSK3) processes apelin-55 into apelin-13 specifically and preferentially, while PCSK1 and 7 could not process it. This showed that apelin-55 need not be initially processed to apelin-36. Secondly, I demonstrate that apelin processing can occur extracellularly. Specifically, the introduction of apelin-55 into the culture media of various cell lines resulted in observable changes in the level of apelin-55 with distinct processing patterns observable for different cell lines. Next, I examine apelin-55 biophysics. Through solution-state NMR spectroscopy, I provided clear demonstration that apelin-55 shows behaviour fully consistent with it being the longest bioactive isoform, rather than an inactive proprotein, correlating directly to our functional assays. I also examined interactions of both apelin-55 and -36 with zwitterionic and anionic micelles. Strikingly, both apelin isoforms preferentially interacted with anionic micelles and adopted a similar micelle-bound conformation independent of micelle headgroup. In combination, my studies have expanded the current understanding of the apelin processing pathway, increased the number of bioactive apelin isoforms, and demonstrated highly similar behaviour for all apelin isoforms. These factors have likely relevance to the apelinergic system function and regulation.