Báo cáo khoa học: Selective modulation of protein C affinity for EPCR and phospholipids by Gla domain mutation

Selective Modulation of Protein C Affinity for EPCR and Phospholipids by Gla Domain Mutation

Tác giả: Roger J. S. Preston, Ana Villegas-Mendez, Yong-Hui Sun, José Hermida, Paolo Simioni, Helen Philippou, Björn Dahlbäck, and David A. Lane

Lĩnh vực: Huyết học, Hóa sinh, Y học phân tử

Nội dung tài liệu: Nghiên cứu này khám phá mối liên hệ giữa miền Gla của protein C, khả năng tương tác với thụ thể tế bào nội mô protein C (EPCR) và phospholipid, cũng như ảnh hưởng của các đột biến trên miền này đến hoạt động kháng đông. Các nhà khoa học đã phân tích các biến thể protein C tự nhiên và tái tổ hợp để đánh giá khả năng liên kết với sEPCR, sự hoạt hóa trên bề mặt tế bào nội mô và khả năng bất hoạt yếu tố Va (FVa) phụ thuộc phospholipid. Kết quả cho thấy sự gấp nếp chính xác của miền Gla là cần thiết cho các chức năng này, nhưng chúng có thể được điều chỉnh một cách chọn lọc thông qua đột biến. Nghiên cứu nhấn mạnh tầm quan trọng của các đột biến tự nhiên trên miền Gla của protein C trong việc làm suy giảm tương tác với EPCR và hoạt hóa trên bề mặt tế bào nội mô, có thể góp phần làm tăng nguy cơ huyết khối.

Mục lục chi tiết:

• Keywords
• Correspondence
• Present address
• Received 9 July 2004, revised 6 September 2004, accepted 9 September 2004
• doi:10.1111/j.1432-1033.2004.04401.x
• Uniquely amongst vitamin K-dependent coagulation proteins, protein C interacts via its Gla domain both with a receptor, the endothelial cell protein C receptor (EPCR), and with phospholipids.
• We have studied naturally occurring and recombinant protein C Gla domain variants for soluble (s)EPCR binding, cell surface activation to activated protein C (APC) by the thrombin-thrombomodulin complex, and phospholipid dependent factor Va (FVa) inactivation by APC, to establish if these functions are concordant.
• Wild-type protein C binding to SEPCR was characterized with surface plasmon resonance to have an association rate constant of 5.23 × 10⁵ M^-1.s^-1, a dissociation rate constant of 7.61 × 10^-2 s^-1 and equilibrium binding constant (K_D) of 147 nm.
• It was activated by thrombin over endothelial cells with a K_m of 213 nm and once activated to APC, rapidly inactivated FVa.
• Each of these interactions was dramatically reduced for variants causing gross Gla domain misfolding (R-1L, R-1C, E16D and E26K).
• Recombinant variants Q32A, V34A and D35A had essentially normal functions.
• However, R9H and H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y (QGNSEDY) variants had slightly reduced (< twofold) binding to sEPCR, arising from an increased rate of dissociation, and increased K_m (358 nm for QGNSEDY) for endothelial cell surface activation by thrombin.
• Interestingly, these variants had greatly reduced (R9H) or greatly enhanced (QGNSEDY) ability to inactivate FVa.
• Therefore, protein C binding to sEPCR and phospholipids is broadly dependent on correct Gla domain folding, but can be selectively influenced by judicious mutation.
• The protein C anticoagulant pathway is essential for normal haemostasis, downregulating thrombin generation after the coagulation cascade has been activated [1,2].
• When thrombin binds to the endothelial cell transmembrane protein thrombomodulin, its potent procoagulant functions are reversed, and its substrate specificity is redirected towards protein C, which it activates.
• This key step is enhanced by a second endothelial cell transmembrane protein, the endothelial cell protein C receptor (EPCR) [3–5], which concentrates protein C on the endothelial cell surface, reducing the K_m for protein C activation by the thrombin-thrombomodulin complex [6].
• Activated protein C (APC) exerts its anticoagulant activity by inactivating factors Va (FVa) and VIIIa by limited proteolysis, thereby attenuating thrombin generation [7–9].
• APC-mediated inactivation of FVa involves the cleavage of peptide bonds at positions Arg306, Arg506 and Arg679 of FVa [10,11].
• Cleavage at Arg506 occurs approximately 20-fold faster than cleavage at Arg306 [12], and this step is greatly accelerated by the presence of anionic phospholipids [13].
• The cleavage at Arg679 (the slowest of the three cleavage steps) is of uncertain functional significance, but may contribute to the inactivation of two naturally occurring FV variants, FV Cambridge and FV Hong Kong [14].
• APC also activates protease-activated receptor 1 (PAR1) [15], and has been shown to protect brain endothelial cells from p53-mediated apoptosis in an EPCR-dependent manner [16].
• Cell surface full-length EPCR (residues 1-221, mature protein numbering) and truncated EPCR (soluble or sEPCR, residues 1-193) bind protein C and APC with equal affinity [17-19].
• The interaction of protein C with EPCR/sEPCR is dependent upon Ca²⁺ ions [3,18].
• The Gla domain of protein C, through which the interaction with EPCR takes place, is the source of this Ca²⁺ dependence [20].
• The Gla domain contains post-translationally γ-carboxylated Glu residues and undergoes a large structural transition in the presence of physiological concentrations of Ca²⁺ ions [21-23].
• A critical step in the structural transition is the formation of the ω-loop (approximately residues 1-11) [24,25].
• This endows protein C, and other vitamin K-dependent proteins, with the ability to bind anionic phospholipid surfaces [16,26,27] and is therefore crucial for its activity.
• The crystal structures of recombinant sEPCR, and sEPCR in complex with the Gla domain of protein C, have recently been solved [25].
• As predicted [18,28-30], the overall fold of sEPCR is similar to that of the CD1/MHC class I family of proteins, consisting of a β-pleated sheet platform supporting two α-helices.
• A tightly bound phospholipid moiety was found to reside in the groove between the two α-helices of the EPCR.
• Although it does not seem to interact with protein C directly, the phospholipid moiety appears to be required for protein C binding to EPCR [25].
• A small clustered patch of residues on the EPCR, which include residues on both α-helices, was found to interact with protein C [31].
• These residues were positioned to interact with the Gla domain of protein C, specifically the ω-loop.
• Information on the functional consequences of residue substitution in the protein C Gla domain has come from two sources.
• Firstly, protein C deficiency is a known risk factor for venous thrombosis and the mutational analysis of this deficiency has identified causative amino acid substitutions [32].
• Type II (functional) deficiency is associated with normal protein C antigen levels but reduced activity.
• Type II clotting deficiency is diagnosed when the amidolytic activity with respect to synthetic substrates is normal, but the anticoagulant activity is reduced.
• In the latest published update of the protein C deficiency database, of the 335 mutations (161 unique events) that have been identified in patients in association with protein C deficiency, at least 30 are associated with type II clotting deficiency and 14 of these are located in the Gla domain [32].
• Secondly, more detailed structure-function relationships have been identified by in vitro mutagenesis and expression of recombinant protein C variants.
• Using this approach, loss- and gain-of-function variants have been characterized [33-39].
• Most investigations of the functional properties of protein C Gla domain variants have focused upon APC interaction with anionic phospholipids and the subsequent effect on FVa inactivation; that is the anticoagulant function of the enzyme.
• Before this anticoagulant function can be expressed, however, protein C must first be activated on the endothelial cell surface.
• Activation involves protein C interaction with EPCR and presentation of EPCR-bound protein C to the thrombin-thrombomodulin complex for proteolysis of its activation peptide.
• In this report, we examine how binding to EPCR is influenced by protein C Gla domain mutation, with particular reference to naturally occurring protein C Gla domain variants associated with type II clotting deficiency.
• We also provide the first evidence that the EPCR and membrane binding properties of protein C can be selectively influenced by specific mutation.
• Results
• Expression and characterization of recombinant EPCR
• Recombinant wild-type sEPCR was prepared using the yeast Pichia pastoris expression system.
• In addition to binding protein C with expected affinity (see below), the wild-type sEPCR was also able to inhibit the anticoagulant activity of APC in a modified clotting assay, as described previously [40].
• Using SDS/PAGE and Western blot analysis with the rat monoclonal RCR-2, sEPCR was found to be heterogeneous, probably due to N-linked glycosylation.
• Indeed, treatment of sEPCR with PNGase F resulted in increased mobility and the smeared bands resolved into a single defined band (data not shown).
• Variant forms of sEPCR were generated by site-directed mutagenesis (N30Q, L37A and E86A) and were expressed, concentrated and buffer-exchanged using gel filtration.
• These mutants migrated with similar mobility to that of wild-type sEPCR on SDS/PAGE, except for variant N30Q that had slightly increased mobility attributed to the removal of a predicted carbohydrate side chain.
• Expression and characterization of protein C Gla domain variants
• Two natural protein C variants, R-1L and R-1C, were selected for study and the variant component isolated from plasma.
• Two other naturally occurring protein C variants, R9H and E26K, were expressed using HEK293 cells.
• Protein C variants with point mutations (E16D, Q32A, V34A and D35A) were also generated.
• Finally, a variant with multiple residue substitutions, H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y (QGNSEDY), reported previously to exhibit enhanced anionic phospholipid affinity and increased anticoagulant activity [37], was also studied.
• Recombinant variants were expressed at concentrations ranging between 0.9 and 7 µg mL^-1 and migrated as closely spaced 62 kDa doublets under nonreducing conditions (data not shown), in accordance with previous reports [36,37,41,42].
• Expressed protein C was subsequently concentrated and partially/fully purified from conditioned medium by ion-exchange chromatography.
• To ensure that the catalytic site of each variant was functional, they were activated by the human protein C carrier, Protac, and their amidolytic properties evaluated.
• The wild-type and variant preparations could all be fully activated by Protac and efficiently cleaved the chromogenic substrate S-2366, with K_m and k_cat parameters comparable to those for wild-type APC described in the literature [43] (data not shown).
• Activation of protein C Gla domain variants on the surface of endothelial cells
• To investigate the activation of protein C variants by the thrombin-thrombomodulin complex in the presence of EPCR, each variant was activated by thrombin over the surface of an endothelial cell line, EA.hy926.
• The activation of recombinant wild-type protein C was characterized by a K_m of 213 ± 42 nm (Fig. 1 and Table 1), similar to the K_m for plasma protein C activation (155 nm; data not shown) and similar to previously reported values [4].
• The activation of variants E16D and E26K by thrombin on EA.hy926 cells at concentrations up to 1 µm was barely detectable (Fig. 1A and Table 1).
• However, small amounts of protein C could be activated on the surface of endothelial cells at concentrations higher than 1 µm, suggesting some activation by the thrombin-thrombomodulin complex had taken place (data not shown).
• The K_m values for activation of variants R9H, Q32A, V34A, D35A and QGNSEDY on the cell surface were normal or slightly increased (Fig. 1 and Table 1).
• Activation of QGNSEDY and R9H variants was also characterized by slight increases in the V_max for this reaction, with the increase for QGNSEDY being approximately twofold (Fig. 1A, Table 1).
• To further investigate this, the activation of QGNSEDY by thrombin on the surface of cells expressing thrombomodulin alone was determined.
• Interestingly, over the surface of HEK293-TM cells, the K_m for activation of wild-type protein C and QGNSEDY by thrombin were closely comparable (K_m = 666 ± 182 nm and 603 ± 112 nm, n = 3), respectively, with no difference in V_max.
• This indicated that the increased V_max was dependent upon cell-surface EPCR.
• Affinity of protein C Gla domain variants for sEPCR
• Surface plasmon resonance (SPR) was used to analyse the binding kinetics of each protein C variant for sEPCR.
• sEPCR has been previously reported to lose activity when bound directly to artificial surfaces [4].
• Therefore, the anti-EPCR monoclonal antibody, RCR-2, was first immobilized onto the surface of a CM5 sensor chip, and sEPCR captured onto one of the two flow cells of the sensor chip.
• To investigate the nature of RCR-2 binding to sEPCR, recombinant wild-type sEPCR and several sEPCR variants were expressed and their concentrations and binding to RCR-2 determined by SPR.
• Wild-type sEPCR bound to RCR-2 with an association rate, k_a, of 5.39 ± 0.97 × 10⁴ M^-1.s^-1 and dissociated with a k_d of 3.28 ± 0.58 × 10^-4 s^-1.
• The equilibrium constant (K_D) was 6.1 ± 0.1 nm (Fig. 2A and Table 2).
• Similar results were obtained with variant sEPCR with the substitution E86A.
• Glu86 is an important residue of the protein C binding site on EPCR [25,31].
• In contrast, sEPCR with substitutions N30Q and L37A had an impaired interaction, with the k_a for N30Q being particularly increased (≈ fourfold) to 1.15 ± 0.04 × 10^-3 s^-1 (Fig. 2B and Table 2).
• The K_D for this variant was also increased to 16.3 ± 1.4 nm.
• These results demonstrate a slow off rate (k_d) for the interaction between wild-type sEPCR and RCR-2 and furthermore suggest that the epitope for RCR-2 on sEPCR is on the face of sEPCR opposite to its known binding site for protein C.
• The stability of immobilized RCR-2 as a capture ligand for sEPCR and protein C is illustrated in Fig. 2C.
• This shows initial binding of sEPCR to RCR-2 (Fig. 2C; panel 1), the addition of increasing concentrations of protein C showing association, dissociation and regeneration experiments – (Fig. 2C; panel 2) and final regeneration of the RCR-2 immobilized chip by injection of 10 mm glycine/HCl (Fig. 2C; panel 3).
• The binding of protein C to sEPCR was initially characterized using wild-type and variant forms of sEPCR.
• Human plasma protein C associated with RCR-2 immobilized wild-type sEPCR in a concentration-dependent manner (Fig. 3A).
• The association and dissociation rate constants were 12.1 ± 1.29 × 10⁵ M^-1.s^-1 and 8.96 ± 1.09 × 10^-2 s^-1, yielding a calculated K_D of 74.8 ± 9.2 nm.
• Binding of plasma protein C was completely abolished when sEPCR with the substitution E86A was used (Fig. 3B).
• Recombinant wild-type protein C bound to sEPCR with a K_D