Retention of thrombin inhibitory activity by recombinant serpins expressed as integral membrane proteins tethered to the surface of mammalian cells

Abstract

To cite this article: Gierczak RF, Sutherland JS, Bhakta V, Toltl LJ, Liaw PC, Sheffield WP. Retention of thrombin inhibitory activity by recombinant serpins expressed as integral membrane proteins tethered to the surface of mammalian cells. J Thromb Haemost 2011; 9: 2424-35.Summary. Background: Serpins form a widely distributed protein superfamily, but no integral membrane serpins have been described. Objectives: To anchor three serpins -a 1 -proteinase inhibitor (a 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 Nterminal, 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-a 1 PI (M358R) and ARa 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 a 1 PI (M358R). Thrombin inhibition was abrogated by an additional T345R mutation in AR-a 1 PI (M358R). Surfacedisplayed AR-AT also formed serpin-enzyme complexes with thrombin, but to a lesser extent than AR-a 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-a 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.

Comparison of mammalian and bacterial expression library screening to detect recombinant alpha-1 proteinase inhibitor variants with enhanced thrombin inhibitory capacity☆

“…The T345R mutation eliminates the ability of API M358R to form denaturation-resistant complexes with thrombin (Gierczak et al, 2011). Sonicates (Fig.…”

“…We previously described the expression of antithrombin and API M358R as membrane proteins tethered to the surface of mammalian cells in culture, and the retention of thrombin inhibitory activity by the tethered serpins (Gierczak et al, 2011). This finding suggested a strategy for selection of novel API variants by cell sorting of transfected cells acquiring the ability to bind thrombin (Gierczak et al, 2011).…”

“…Plasmid DNA from plasmids pC3-AR-API (M358R) and pC3-AR-API (T345R/M358R) (Gierczak et al, 2011) was restricted with HindIII and XhoI, and the 1492 bp minor fragment was inserted between these sites in pCEP4 (Invitrogen/Life Technologies, Carlsbad, CA), forming pCEP4-AR-API M358R and pCEP4-AR-API T345R/M358R, respectively (both 11,661 bp).…”

“…Following transfection, cells were resuspended in serum-free culture medium and reacted with thrombin, affinity-purified sheep anti-human prothrombin IgG, and Alexa Fluor488-conjugated donkey anti-sheep IgG as previously described (Gierczak et al, 2011). They were then characterized by flow cytometry and 0.5 to 1.0 × 10 6 transfected cells were sorted using a BD LSR II bench-top flow cytometer operated by the McMaster Flow Cytometry Facility powered by FACSDiva 6.0 software (BD Biosciences, San Diego, CA).…”

“…This finding suggested a strategy for selection of novel API variants by cell sorting of transfected cells acquiring the ability to bind thrombin (Gierczak et al, 2011). We also described a thrombin capture assay and its use to select functional serpin variants from pools of candidates expressed in libraries expressed in Escherichia coli (Bhakta et al, 2013).…”