Hunter Alison R, Turner Anthony J
Proteolysis Research Group, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom.
Exp Biol Med (Maywood). 2006 Jun;231(6):718-22.
Endothelin-converting enzyme (ECE)-1 is a membrane-bound metalloprotease responsible for production of vasoactive endothelin (ET)-1 from inactive big ET-1. ECE-1 exists as four separate isoforms, ECE-1a, b, c, and d, which differ only in their amino-terminal regions. We investigated the expression and localization of the ECE-1 isoforms in primary human umbilical vein endothelial cells (HUVECs) and EAhy926 cells. Reverse transcriptase polymerase chain reaction showed expression of all four isoforms in both cell lines, with ECE-1d seeming, at least qualitatively, to be the predominant isoenzyme. Isoform-specific polyclonal antibodies were used to investigate isoform protein expression. ECE-1a, b, and c protein was detected in EAhy926 cells by immunoblotting; only ECE-1a and ECE-1c were detected in HUVECs. Using immunofluorescence microscopy analysis, both HUVEC and EAhy926 cells showed nuclear immunoreactivity with a monoclonal antibody recognizing all ECE-1 isoforms. The ECE-1a antibody also showed nuclear immunoreactivity in both cell lines; this seemed to colocalize with nucleolin. The ECE-1b antibody showed nuclear immunoreactivity in EAhy926 cells, but no overlap with nucleolin was seen. Intracellular immunoreactivity was seen in both cell lines using the ECE-1c antibody; this showed some colocalization with concanavalin A (an endoplasmic reticulum marker). von Willebrand Factor was used as a marker for Weibel-Palade bodies in HUVECs, but no colocalization with ECE-1 was seen during this study. The data presented here sheds new light on the localization of ECE-1a, b, and c in cultured human endothelial cells, which may further understanding of the ET system and aid design of therapeutic ECE inhibitors.
内皮素转化酶（ECE）-1是一种膜结合金属蛋白酶，负责将无活性的大内皮素（big ET）-1转化为具有血管活性的内皮素（ET）-1。ECE-1以四种不同的同工型存在，即ECE-1a、b、c和d，它们仅在氨基末端区域有所不同。我们研究了ECE-1同工型在原代人脐静脉内皮细胞（HUVECs）和EAhy926细胞中的表达和定位。逆转录聚合酶链反应显示两种细胞系中均表达所有四种同工型，至少从定性角度来看，ECE-1d似乎是主要的同工酶。使用同工型特异性多克隆抗体研究同工型蛋白表达。通过免疫印迹在EAhy926细胞中检测到ECE-1a、b和c蛋白；在HUVECs中仅检测到ECE-1a和ECE-1c。使用免疫荧光显微镜分析，HUVEC和EAhy926细胞均显示与识别所有ECE-1同工型的单克隆抗体发生核免疫反应。ECE-1a抗体在两种细胞系中也显示出核免疫反应；这似乎与核仁素共定位。ECE-1b抗体在EAhy926细胞中显示出核免疫反应，但未观察到与核仁素重叠。使用ECE-1c抗体在两种细胞系中均观察到细胞内免疫反应；这显示与伴刀豆球蛋白A（一种内质网标记物）有一定共定位。在HUVECs中使用血管性血友病因子作为魏尔-帕拉德小体的标记物，但在本研究中未观察到与ECE-1共定位。本文提供的数据为ECE-1a、b和c在培养的人内皮细胞中的定位提供了新的线索，这可能有助于进一步了解ET系统并辅助治疗性ECE抑制剂的设计。
