Retention of thrombin inhibitory activity by recombinant serpins expressed as integral membrane proteins tethered to the surface of mammalian cells
| Autor: | Patricia C. Liaw, William P. Sheffield, Lisa J. Toltl, Richard F. Gierczak, Varsha Bhakta, J. S. Sutherland |
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| Rok vydání: | 2011 |
| Předmět: |
animal structures
medicine.medical_treatment Serpin Biology Thrombomodulin Polymerase Chain Reaction Antithrombins Cell Line Thrombin medicine Animals Humans Integral membrane protein Serpins DNA Primers Protease Base Sequence Membrane Proteins Hematology Transfection Flow Cytometry Molecular biology Recombinant Proteins carbohydrates (lipids) Microscopy Fluorescence embryonic structures Asialoglycoprotein receptor Electrophoresis Polyacrylamide Gel Discovery and development of direct thrombin inhibitors medicine.drug |
| Zdroj: | Journal of thrombosis and haemostasis : JTH. 9(12) |
| ISSN: | 1538-7836 |
| Popis: | Summary. Background: Serpins form a widely distributed protein superfamily, but no integral membrane serpins have been described. Objectives: To anchor three serpins –α1-proteinase inhibitor (α1PI) (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-α1PI (M358R) and AR-α1PI (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 α1PI (M358R). Thrombin inhibition was abrogated by an additional T345R mutation in AR-α1PI (M358R). Surface-displayed AR-AT also formed serpin–enzyme complexes with thrombin, but to a lesser extent than AR-α1PI (M358R); AR-HCII inhibitory function was not detected. Immunofluorescence detection and flow cytometric quantification of bound thrombin also supported the status of AR-α1PI (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. |
| Databáze: | OpenAIRE |
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