Retention of thrombin inhibitory activity by recombinant serpins expressed as integral membrane proteins tethered to the surface of mammalian cells
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BACKGROUND: Serpins form a widely distributed protein superfamily, but no integral membrane serpins have been described. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
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