Gladysheva Inna P, Robinson Brian R, Houng Aiilyan K, Kováts Tímea, King Sarah M
Cardiovascular Research Center, Division of Cardiology, Medical College of Georgia, Augusta, GA 30912, USA.
J Mol Cell Cardiol. 2008 Jan;44(1):131-42. doi: 10.1016/j.yjmcc.2007.10.002. Epub 2007 Oct 11.
The multi-domain transmembrane serine protease corin cleaves pro-atrial natriuretic peptide (pro-ANP) in vitro to generate an active hormone, ANP. Corin may also contribute to the regulation of the natriuretic peptide system in vivo, and might be an attractive target for treatment of cardiovascular diseases. In order for corin to cleave its substrate pro-ANP, it should be catalytically active and located proximally. However, because knowledge of native corin is limited, we examined the expression, cardiac localization and molecular forms of the native corin protein. Immunofluorescence studies using a series of anti-corin antibodies directed against the stem and protease domains reveal that corin is present on the cell-surface of rat neonatal cardiomyocytes and murine HL-1 cardiomyocyte-like cells. Furthermore, we immunolocalized native corin in pro-ANP expressing cardiomyocytes. Immunoprecipitation of the membrane fraction of mouse heart extract showed that native corin had a relative mass of 205-210 kDa. Under reducing conditions native corin migrates as several different molecular weight forms corresponding to zymogen (uncleaved) and active (cleaved) forms. Studies using a FITC-tagged chloromethyl ketone that mimics the corin cleavage sequence in pro-ANP, suggest that an enzymatically active form of corin is localized to the cell surface of myocardial cells in vivo. Additionally, we showed that the 205-210 kDa form of corin is a glycosylated protein. Treatment of HL-1 cells with tunicamycin reduced the relative mass of expressed corin. We conclude that native corin is a glycosylated protease that is localized on the cell surface of pro-ANP-expressing cardiomyocytes in both zymogen and catalytically active forms.
多结构域跨膜丝氨酸蛋白酶corin在体外可切割前心钠素（pro-ANP）以生成活性激素心钠素（ANP）。Corin在体内可能也有助于利钠肽系统的调节，并且可能是治疗心血管疾病的一个有吸引力的靶点。为使corin能够切割其底物pro-ANP，它应具有催化活性并位于近端。然而，由于对天然corin的了解有限，我们研究了天然corin蛋白的表达、心脏定位及分子形式。使用一系列针对茎部和蛋白酶结构域的抗corin抗体进行的免疫荧光研究表明，corin存在于大鼠新生心肌细胞和小鼠HL-1心肌样细胞的细胞表面。此外，我们对表达pro-ANP的心肌细胞中的天然corin进行了免疫定位。对小鼠心脏提取物膜部分进行免疫沉淀显示，天然corin的相对分子质量为205 - 210 kDa。在还原条件下，天然corin以几种不同分子量形式迁移，分别对应酶原（未切割）形式和活性（已切割）形式。使用模拟pro-ANP中corin切割序列的异硫氰酸荧光素标记的氯甲基酮进行的研究表明，corin的一种酶活性形式在体内定位于心肌细胞的细胞表面。此外，我们表明205 - 210 kDa形式的corin是一种糖基化蛋白。用衣霉素处理HL-1细胞可降低所表达corin的相对分子质量。我们得出结论，天然corin是一种糖基化蛋白酶，以酶原形式和催化活性形式定位于表达pro-ANP的心肌细胞的细胞表面。
