Lead Discovery Wuppertal, Bayer AG, Pharma Research and Development Center, Aprather Weg 18a, D-42096 Wuppertal, Germany.
Broad Institute of MIT and Harvard, 415 Main Street, Cambridge, MA, USA.
Biochem Pharmacol. 2019 Feb;160:62-70. doi: 10.1016/j.bcp.2018.12.009. Epub 2018 Dec 13.
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 Ca-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.
心钠肽转换酶（atrial natriuretic peptide-converting enzyme，EC 3.4.21）是一种在心肌细胞中表达的跨膜丝氨酸蛋白酶。心钠肽通过蛋白水解切割和激活前心钠肽（pro-atrial natriuretic peptide，pro-ANP）为心钠肽（ANP）发挥其心脏保护作用。我们最近描述了一种稳定表达 ANP 受体的 ANP 报告细胞系，该受体是一种 cGMP 依赖性阳离子通道，用作实时 cGMP 生物传感器，以及钙敏感的光蛋白水母发光蛋白（aequorin）。在这里，我们描述了一种新的报告细胞系的产生，该细胞系表达钙生物传感器 GCaMP6 而不是水母发光蛋白。与基于发光的测定法不同，ANP 刺激我们的新型 GCaMP6 报告细胞导致稳定、持久的荧光信号。使用这种新型报告系统，我们能够检测到纯化的可溶性野生型心钠肽转换酶（solCorin）而不是活性位点突变体 solCorin（S985A）对前心钠肽的转化，从而导致浓度-反应曲线左移。此外，在瞬时表达野生型心钠肽转换酶后，可在 HEK 293 细胞上检测到细胞内前心钠肽酶活性。相比之下，在用无活性的心钠肽转换酶（S985A）变体转染后，未检测到心钠肽转换酶活性。在心肌细胞衍生的 HL-1 细胞的上清液中也可以检测到前心钠肽到心钠肽的转化，而在 HL-1 心钠肽敲除细胞中则没有观察到转化。这些发现强调了心钠肽转换酶在心钠肽转换中的作用。我们新的基于荧光的 ANP 报告细胞系非常适合敏感检测心钠肽转换酶的活性，并且可用于鉴定和表征新型心钠肽转换酶调节剂。
