Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.
J Thromb Haemost. 2011 Dec;9(12):2424-35. doi: 10.1111/j.1538-7836.2011.04524.x.
Serpins form a widely distributed protein superfamily, but no integral membrane serpins have been described.
To anchor three serpins -α(1) -proteinase inhibitor (α(1) PI) (M358R), antithrombin (AT), and heparin cofactor II (HCII) - in the plasma membranes of transfected mammalian cells and assess their ability to inhibit thrombin.
Serpin cDNAs were altered to include N-terminal, non-cleavable plasma membrane-targeting sequences from the human transferrin receptor (TR) (TR-serpin) or the human asialoglycoprotein receptor (AR) (AR-serpin), and used to transfect COS-1 or HEK 293 cells. Cells were analyzed for serpin expression by immunoblotting of subcellular fractions, by immunofluorescence microscopy, or by flow cytometry, with or without exposure to exogenous thrombin; AR-serpins and TR-serpins were also compared with their soluble recombinant counterparts.
Both TR-α(1) PI (M358R) and AR-α(1) PI (M358R) were enriched in the integral membrane fraction of transfected COS-1 or HEK 293 cells, and formed inhibitory complexes with thrombin, although less rapidly than soluble α(1) PI (M358R). Thrombin inhibition was abrogated by an additional T345R mutation in AR-α(1) PI (M358R). Surface-displayed AR-AT also formed serpin-enzyme complexes with thrombin, but to a lesser extent than AR-α(1) PI (M358R); AR-HCII inhibitory function was not detected. Immunofluorescence detection and flow cytometric quantification of bound thrombin also supported the status of AR-α(1) PI (M358R) and AR-AT as thrombin inhibitors.
Two of three thrombin-inhibitory serpins retained functionality when expressed as integral membrane proteins. Our findings could be applied to create and screen hypervariable serpin libraries expressed in mammalian cells, or to confer protease resistance on engineered cells in vivo.
丝氨酸蛋白酶抑制剂（serpins）构成了广泛分布的蛋白质超家族，但尚未发现整合膜丝氨酸蛋白酶抑制剂。
将三种丝氨酸蛋白酶抑制剂——α1-蛋白酶抑制剂（α1PI）（M358R）、抗凝血酶（AT）和肝素辅因子 II（HCII）——锚定在转染的哺乳动物细胞的质膜上，并评估它们抑制凝血酶的能力。
对丝氨酸蛋白酶抑制剂 cDNA 进行修饰，加入来自人转铁蛋白受体（TR）（TR-丝氨酸蛋白酶抑制剂）或人去唾液酸糖蛋白受体（AR）（AR-丝氨酸蛋白酶抑制剂）的非切割性质膜靶向序列的 N 端，用于转染 COS-1 或 HEK 293 细胞。通过亚细胞级分的免疫印迹、免疫荧光显微镜或流式细胞术分析转染细胞中丝氨酸蛋白酶抑制剂的表达情况，或在有无外源性凝血酶存在的情况下进行分析；还比较了 AR-丝氨酸蛋白酶抑制剂和 TR-丝氨酸蛋白酶抑制剂与其可溶性重组对应物的差异。
TR-α1PI（M358R）和 AR-α1PI（M358R）在转染的 COS-1 或 HEK 293 细胞的完整质膜级分中均得到富集，并与凝血酶形成抑制性复合物，尽管其形成速度不及可溶性α1PI（M358R）。在 AR-α1PI（M358R）中加入 T345R 突变可使凝血酶抑制作用丧失。表面展示的 AR-AT 也与凝血酶形成丝氨酸蛋白酶抑制剂-酶复合物，但程度低于 AR-α1PI（M358R）；未检测到 AR-HCII 的抑制功能。用结合的凝血酶的免疫荧光检测和流式细胞术定量也支持 AR-α1PI（M358R）和 AR-AT 作为凝血酶抑制剂的状态。
当作为整合膜蛋白表达时，三种凝血酶抑制性丝氨酸蛋白酶抑制剂中的两种保留了功能。我们的发现可用于在哺乳动物细胞中构建和筛选高变异性丝氨酸蛋白酶抑制剂文库，或赋予体内工程细胞对蛋白酶的抗性。
