Proapelin is processed extracellularly in a cell line-dependent manner with clear modulation by proprotein convertases

Apelin is a peptide hormone that binds to a class A GPCR (the apelin receptor/APJ) to regulate various bodily systems. Upon signal peptide removal, the resulting 55-residue isoform, proapelin/apelin-55, can be further processed to 36-, 17-, or 13-residue isoforms with length-dependent pharmacological properties. Processing was initially proposed to occur intracellularly. However, detection of apelin-55 in extracellular fluids indicates that extracellular processing may also occur. To test for this, apelin-55 was applied exogenously to HEK293A cells overexpressing proprotein convertase subtilisin kexin 3 (PCSK3), the only apelin processing enzyme identified thus far, and to differentiated 3T3-L1 adipocytes, which endogenously express apelin, PCSK3 and other proprotein convertases. Analysis of culture media constituents from each cell type by high performance liquid chromatography–mass spectrometry and western blot demonstrated a time-dependent decrease in apelin-55 levels. This decrease was partially, but not fully, attenuated by PCSK inhibitor treatment in both cell lines. Comparison of the resulting apelin-55-derived peptide profile between the two cell lines demonstrated distinct processing patterns, with apelin-36 production apparent in 3T3-L1 adipocytes vs. detection of the prodomain of a shorter isoform (likely the apelin-13 prodomain, observed after additional proteolytic processing) in PCSK3-transfected HEK293A cells. Extracellular processing of apelin, with distinct cell type dependence, provides an alternative mechanism to regulate isoform-mediated physiological effects of apelin.