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

Abstract

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.

Elabela/Toddler and apelin bind differently to the apelin receptor

“…C11P is the only Elabela/Toddler isoform detected under endogenous conditions by mass spectrometry on embryos containing APELA mRNA 10 . The presence of dibasic sites strongly suggests that the precursor would be processed by PCSK3 in a cell type‐dependent manner, as recently shown for proapelin 26 . In this context, we can speculate that the processing of Elabela/Toddler may, like that of apelin, differ between cell types, resulting in different isoform‐mediated biological effects.…”

“…10 The presence of dibasic sites strongly suggests that the precursor would be processed by PCSK3 in a cell type-dependent manner, as recently shown for proapelin. 26 In this context, we can speculate that the processing of Elabela/Toddler may, like that of apelin, differ between cell types, resulting in different isoform-mediated biological effects. Longer Elabela/Toddler isoforms appear to be very potent apelin receptor agonists, whereas C11P seems to be a poor agonist of the apelin receptor.…”

The emerging role of the apelinergic system in kidney physiology and disease

“…The distribution, function and processing of these specific isoforms have yet to be fully elucidated. APLN is expressed as a 77-amino acid precursor, preproapelin, which is further processed by tissue-specific proteases [7]. The longest secretable form is apelin-55 [8,9].…”

In vitro metabolism of synthetic Elabela/Toddler (ELA-32) peptide in human plasma and kidney homogenates analyzed with mass spectrometry and validation of endogenous peptide quantification in tissues by ELISA

“…ELA-21, which is one amino acid less than the expected furin cleavage site, has never been reported in degradation studies. Interestingly, apelin-55 also contain these di-arginine residues, and it was shown that proprotein convertase subtilisin/kexin 3 (PCSK3) cleaves immediately after these dibasic residues in the N-terminal to generate apelin-17 and apelin-13 [ 27 , 28 ]. Here, ELA-11 and ELA-22 were identified as fragments of ELA-32 generated in vitro in human plasma, but there was no evidence for the presence of ELA-21, either in the plasma or tissue homogenates.…”