Development of a novel, sensitive cell-based corin assay

Abstract

Corin (atrial natriuretic peptide-converting enzyme, EC 3.4.21) is a transmembrane serine protease expressed in cardiomyocytes. Corin exerts its cardioprotective effects via the proteolytic cleavage and activation of pro-atrial natriuretic peptide (pro-ANP) to ANP. We recently described an ANP reporter cell line stably expressing the ANP receptor, a cGMP-dependent cation channel used as a real-time cGMP biosensor, and the Ca²⁺-sensitive photoprotein aequorin. Here, we describe the generation of a novel reporter cell line expressing the calcium biosensor GCaMP6 instead of aequorin. In contrast to the luminescence-based assay, ANP stimulation of our novel GCaMP6 reporter cell resulted in stable, long-lasting fluorescence signals. Using this novel reporter system, we were able to detect pro-ANP to ANP conversion by purified, soluble wildtype corin (solCorin), but not the active site mutant solCorin(S985A), resulting in left-shifted concentration-response curves. Furthermore, cellular pro-ANPase activity could be detected on HEK 293 cells after transient expression of wildtype corin. In contrast, corin activity was not detected after transfection with the inactive corin(S985A) variant. In supernatants from cardiomyocyte-derived HL-1 cells pro-ANP to ANP conversion could also be detected, while in HL-1 corin knockout cells no conversion was observed. These findings underline the role of corin as the pro-ANP convertase. Our novel fluorescence-based ANP reporter cell line is well-suited for the sensitive detection of corin activity, and may be used for the identification and characterization of novel corin modulators.

Keywords: ANP receptor; Atrial natriuretic peptide; Corin; Reporter assay; Serine protease; cGMP.