Structural Requirements of Anticoagulant Protein S for Its Binding to the Complement Regulator C4b-binding Protein

Artigo Acesso aberto Revisado por pares

Structural Requirements of Anticoagulant Protein S for Its Binding to the Complement Regulator C4b-binding Protein

2002; Elsevier BV; Volume: 277; Issue: 17 Linguagem: Inglês

10.1074/jbc.m103036200

ISSN

1083-351X

Autores

Tusar Giri, Sara Linse, Pablo Garcı́a de Frutos, Tomio Yamazaki, Bruno O. Villoutreix, Björn Dahlbäck,

Tópico(s)

Coagulation, Bradykinin, Polyphosphates, and Angioedema

Resumo

The vitamin K-dependent anticoagulant protein S binds with high affinity to C4b-binding protein (C4BP), a regulator of complement. Despite the physiological importance of the complex, we have only a patchy view of the C4BP-binding site in protein S. Based on phage display experiments, protein S residues 447–460 were suggested to form part of the binding site. Several experimental approaches were now used to further elucidate the structural requirements for protein S binding to C4BP. Peptides comprising residues 447–460, 451–460, or 453–460 of protein S were found to inhibit the protein S-C4BP interaction, whereas deletion of residues 459–460 from the peptide caused complete loss of inhibition. In recombinant protein S, each of residues 447–460 was mutated to Ala, and the protein S variants were tested for binding to C4BP. The Y456A mutation reduced binding to C4BP ∼10-fold, and a peptide corresponding to residues 447–460 of this mutant was less inhibitory than the parent peptide. A further decrease in binding was observed using a recombinant variant in which a site for N-linked glycosylation was moved from position 458 to 456 (Y456N/N458T). A monoclonal antibody (HPSf) selective for free protein S reacted poorly with the Y456A variant but reacted efficiently with the other variants. A second antibody, HPS 34, which partially inhibited the protein S-C4BP interaction, reacted poorly with several of the Ala mutants, suggesting that its epitope was located in the 451–460 region. Phage display analysis of the HPS 34 antibody further identified this region as its epitope. Taken together, our results suggest that residues 453–460 of protein S form part of a more complex binding site for C4BP. A recently developed three-dimensional model of the sex hormone-binding globulin-like region of protein S was used to analyze available experimental data. The vitamin K-dependent anticoagulant protein S binds with high affinity to C4b-binding protein (C4BP), a regulator of complement. Despite the physiological importance of the complex, we have only a patchy view of the C4BP-binding site in protein S. Based on phage display experiments, protein S residues 447–460 were suggested to form part of the binding site. Several experimental approaches were now used to further elucidate the structural requirements for protein S binding to C4BP. Peptides comprising residues 447–460, 451–460, or 453–460 of protein S were found to inhibit the protein S-C4BP interaction, whereas deletion of residues 459–460 from the peptide caused complete loss of inhibition. In recombinant protein S, each of residues 447–460 was mutated to Ala, and the protein S variants were tested for binding to C4BP. The Y456A mutation reduced binding to C4BP ∼10-fold, and a peptide corresponding to residues 447–460 of this mutant was less inhibitory than the parent peptide. A further decrease in binding was observed using a recombinant variant in which a site for N-linked glycosylation was moved from position 458 to 456 (Y456N/N458T). A monoclonal antibody (HPSf) selective for free protein S reacted poorly with the Y456A variant but reacted efficiently with the other variants. A second antibody, HPS 34, which partially inhibited the protein S-C4BP interaction, reacted poorly with several of the Ala mutants, suggesting that its epitope was located in the 451–460 region. Phage display analysis of the HPS 34 antibody further identified this region as its epitope. Taken together, our results suggest that residues 453–460 of protein S form part of a more complex binding site for C4BP. A recently developed three-dimensional model of the sex hormone-binding globulin-like region of protein S was used to analyze available experimental data. The major isoform of the human complement regulator C4b-binding protein (C4BP) 1The abbreviations used are: C4BPC4b-binding proteinAPCactivated protein CGlaγ-carboxyglutamic acidSHBGsex hormone-binding globulinHRPhorseradish peroxidaseLGlaminin globularCCPcomplement control proteinELISAenzyme-linked immunosorbent assaywtwild-type circulates in a 1:1 high affinity noncovalent complex with the vitamin K-dependent anticoagulant protein S (KD, 0.1–0.6 nm), thus bringing the complement and coagulation systems into close interplay (1.Schwalbe R. Dahlbäck B. Hillarp A. Nelsestuen G. J. Biol. Chem. 1990; 265: 16074-16081Abstract Full Text PDF PubMed Google Scholar, 2.He X. Shen L. Malmborg A.C. Smith K.J. Dahlbäck B. Linse S. Biochemistry. 1997; 36: 3745-3754Crossref PubMed Scopus (41) Google Scholar, 3.Greengard J.S. Fernandez J.A. Radtke K.P. Griffin J.H. Biochem. J. 1995; 305: 397-403Crossref PubMed Scopus (32) Google Scholar, 4.Nelson R.M. Long G.L. Biochemistry. 1991; 30: 2384-2390Crossref PubMed Scopus (19) Google Scholar, 5.Dahlbäck B. Frohm B. Nelsestuen G. J. Biol. Chem. 1990; 265: 16082-16087Abstract Full Text PDF PubMed Google Scholar). C4BP attenuates the classical complement pathway by serving as a decay-accelerating factor for the C4b-C2a complex and as a cofactor to factor I in the proteolytic degradation of C4b (6.Law S.K.A. Reid K.B.M. Male D. Focus, Complement. IRL Press, Oxford, United Kingdom1988: 22-24Google Scholar). Protein S is an anticoagulant, acting as a cofactor to activated protein C (APC) in the proteolytic degradation of the activated forms of coagulation factors V (7.Walker F.J. J. Biol. Chem. 1981; 256: 11128-11131Abstract Full Text PDF PubMed Google Scholar, 8.Rosing J. Hoekema L. Nicolaes G.A. Thomassen M.C. Hemker H.C. Varadi K. Schwarz H.P. Tans G. J. Biol. Chem. 1995; 270: 27852-27858Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) and VIII (9.Walker F.J. Chavin S.I. Fay P.J. Arch. Biochem. Biophys. 1987; 252: 322-328Crossref PubMed Scopus (95) Google Scholar, 10.Koedam J.A. Meijers J.C. Sixma J.J. Bouma B.N. J. Clin. Invest. 1988; 82: 1236-1243Crossref PubMed Scopus (192) Google Scholar, 11.O'Brien L.M. Mastri M. Fay P.J. Blood. 2000; 95: 1714-1720Crossref PubMed Google Scholar). Protein S and APC form a complex on the surface of negatively charged phospholipid membranes, and protein S is involved in localizing and orienting the active site of APC toward its substrates (7.Walker F.J. J. Biol. Chem. 1981; 256: 11128-11131Abstract Full Text PDF PubMed Google Scholar, 12.Yegneswaran S. Wood G.M. Esmon C.T. Johnson A.E. J. Biol. Chem. 1997; 272: 25013-25021Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Protein S has also been reported to exert a direct anticoagulant activity independent of APC (13.Heeb M.J. Mesters R.M. Tans G. Rosing J. Griffin J.H. J. Biol. Chem. 1993; 268: 2872-2877Abstract Full Text PDF PubMed Google Scholar, 14.Hackeng T.M. van't Veer C. Meijers J.C. Bouma B.N. J. Biol. Chem. 1994; 269: 21051-21058Abstract Full Text PDF PubMed Google Scholar, 15.Koppelman S.J. Hackeng T.M. Sixma J.J. Bouma B.N. Blood. 1995; 86: 1062-1071Crossref PubMed Google Scholar, 16.van Wijnen M. van't Veer C. Meijers J.C. Bertina R.M. Bouma B.N. Thromb. Haemostasis. 1998; 80: 930-935Crossref PubMed Scopus (28) Google Scholar). The physiological importance of the anticoagulant function of protein S is supported by the association between heterozygous protein S deficiency and an increased risk of thrombosis (17.Aiach M. Borgel D. Gaussem P. Emmerich J. Alhenc Gelas M. Gandrille S. Semin. Hematol. 1997; 34: 205-216PubMed Google Scholar). Upon binding to C4BP, protein S loses its APC cofactor activity, whereas the functions of C4BP are not perturbed (18.Dahlbäck B. J. Biol. Chem. 1986; 261: 12022-12027Abstract Full Text PDF PubMed Google Scholar, 19.Dahlbäck B. Thromb. Haemostasis. 1991; 66: 49-61Crossref PubMed Scopus (328) Google Scholar, 20.Garcı́a de Frutos P. Dahlbäck B. J. Immunol. 1994; 152: 2430-2437PubMed Google Scholar). It has been suggested that protein S helps anchor C4BP to negatively charged phospholipid exposed on cell surfaces at sites of injury, thereby assisting regulation of inflammation (1.Schwalbe R. Dahlbäck B. Hillarp A. Nelsestuen G. J. Biol. Chem. 1990; 265: 16074-16081Abstract Full Text PDF PubMed Google Scholar). C4b-binding protein activated protein C γ-carboxyglutamic acid sex hormone-binding globulin horseradish peroxidase laminin globular complement control protein enzyme-linked immunosorbent assay wild-type C4BP and protein S are multidomain proteins. Protein S contains a γ-carboxyglutamic acid (Gla) domain, a thrombin-sensitive loop (thrombin-sensitive region), four epidermal growth factor-like domains, and a COOH-terminal region that is homologous to sex hormone-binding globulin (SHBG). The SHBG-like region comprises two laminin globular (LG) domains (LG1 and LG2), a fold present in the COOH-terminal part of the laminin α chain, and many other extracellular matrix proteins (21.Joseph D.R. Baker M.E. FASEB J. 1992; 6: 2477-2481Crossref PubMed Scopus (96) Google Scholar, 22.Hohenester E. Tisi D. Talts J.F. Timpl R. Mol. Cell. 1999; 4: 783-792Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 23.Grishkovskaya I. Avvakumov G.V. Sklenar G. Dales D. Hammond G.L. Muller Y.A. EMBO J. 2000; 19: 504-512Crossref PubMed Scopus (134) Google Scholar). LG2 contains three glycosylation sites, two of which are conserved in several species (3.Greengard J.S. Fernandez J.A. Radtke K.P. Griffin J.H. Biochem. J. 1995; 305: 397-403Crossref PubMed Scopus (32) Google Scholar,24.Dahlbäck B. Lundwall A. Stenflo J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4199-4203Crossref PubMed Scopus (106) Google Scholar, 25.Lundwall A. Dackowski W. Cohen E. Shaffer M. Mahr A. Dahlbäck B. Stenflo J. Wydro R. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6716-6720Crossref PubMed Scopus (150) Google Scholar, 26.He X. Dahlbäck B. Eur. J. Biochem. 1993; 217: 857-865Crossref PubMed Scopus (35) Google Scholar, 27.Chu M.D. Sun J. Bird P. Biochim. Biophys. Acta. 1994; 1217: 325-328Crossref PubMed Scopus (25) Google Scholar, 28.Yasuda F. Hayashi T. Tanitame K. Nishioka J. Suzuki K. J. Biochem. (Tokyo). 1995; 117: 374-383Crossref PubMed Scopus (29) Google Scholar). C4BP is an approximately 570-kDa glycoprotein composed of 6–8 polypeptide chains connected at their COOH-terminal ends by disulfide bridges, giving the oligomer a spider-like shape, as revealed by high resolution electron microscopy (29.Dahlbäck B. Smith C.A. Muller-Eberhard H.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3461-3465Crossref PubMed Scopus (178) Google Scholar). The major isoform of C4BP comprises seven α chains and a β chain, whereas the minor isoform has no β chain and does not interact with protein S (30.Hillarp A. Hessing M. Dahlbäck B. FEBS Lett. 1989; 259: 53-56Crossref PubMed Scopus (72) Google Scholar). Although C4BP is an acute phase protein, it is primarily the form lacking the β chain that increases during the acute phase inflammatory response so that levels of free protein S remain stable (31.Garcı́a de Frutos P. Alim R.I.M. Härdig Y. Zöller B. Dahlbäck B. Blood. 1994; 84: 815-822Crossref PubMed Google Scholar, 32.Criado Garcı́a O. Gonzalez Rubio C. López Trascasa M. Pascual Salcedo D. Munuera L. Rodrı́guez de Cordoba S. Haemostasis. 1997; 27: 25-34PubMed Google Scholar). The α and β chains are composed of repeating domains of about 60 amino acids denoted complement control protein (CCP) domains. The binding site for protein S is contained in CCP1-CCP2 of the β chain (33.Härdig Y. Dahlbäck B. J. Biol. Chem. 1996; 271: 20861-20867Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 34.van de Poel R.H. Meijers J.C. Bouma B.N. J. Biol. Chem. 1999; 274: 15144-15150Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 35.Webb J.H. Villoutreix B.O. Dahlbäck B. Blom A.M. J. Biol. Chem. 2001; 276: 4330-4337Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Using a molecular model of the β chain in combination with recombinant β chain expression and site-directed mutagenesis, it has been shown that a solvent-exposed hydrophobic patch in CCP1 lined by a positively charged area on an otherwise negatively charged surface forms the key binding site for protein S (35.Webb J.H. Villoutreix B.O. Dahlbäck B. Blom A.M. J. Biol. Chem. 2001; 276: 4330-4337Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Several studies have demonstrated that the C4BP-binding site in protein S is fully contained in the two LG domains (2.He X. Shen L. Malmborg A.C. Smith K.J. Dahlbäck B. Linse S. Biochemistry. 1997; 36: 3745-3754Crossref PubMed Scopus (41) Google Scholar, 36.Van Wijnen M. Stam J.G. Chang G.T. Meijers J.C. Reitsma P.H. Bertina R.M. Bouma B.N. Biochem. J. 1998; 330: 389-396Crossref PubMed Scopus (47) Google Scholar). Using recombinant chimeric proteins created between protein S and the structurally related protein Gas6, it was recently shown that both LG domains contribute independently to the interaction (37.Evenas P. Garcia De Frutos P. Linse S. Dahlbäck B. Eur. J. Biochem. 1999; 266: 935-942Crossref PubMed Scopus (32) Google Scholar). Synthetic peptides corresponding to protein S residues 413–434 (38.Fernandez J.A. Heeb M.J. Griffin J.H. J. Biol. Chem. 1993; 268: 16788-16794Abstract Full Text PDF PubMed Google Scholar), 447–460 (39.Linse S. Hardig Y. Schultz D.A. Dahlbäck B. J. Biol. Chem. 1997; 272: 14658-14665Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), and 605–614 (40.Walker F.J. J. Biol. Chem. 1989; 264: 17645-17648Abstract Full Text PDF PubMed Google Scholar, 41.Weinstein R.E. Walker F.J. J. Clin. Invest. 1990; 86: 1928-1935Crossref PubMed Scopus (15) Google Scholar) have been reported to compete with protein S for binding to C4BP. Recombinant truncated protein S variants lacking the COOH-terminal 28–58 residues demonstrated very low affinity for C4BP (4.Nelson R.M. Long G.L. Biochemistry. 1991; 30: 2384-2390Crossref PubMed Scopus (19) Google Scholar, 42.Chang G.T. Maas B.H. Ploos van Amstel H.K. Reitsma P.H. Bertina R.M. Bouma B.N. Thromb. Haemostasis. 1994; 71: 461-467Crossref PubMed Scopus (29) Google Scholar). In addition, specific substitutions of amino acids Lys423, Lys427, and Lys429 with polar amino acids resulted in a 5–10-fold reduction in the affinities (43.Fernandez J.A. Griffin J.H. Chang G.T. Stam J. Reitsma P.H. Bertina R.M. Bouma B.N. Blood Cells Mol. Dis. 1998; 24: 101-112Crossref PubMed Scopus (20) Google Scholar). In the present study, we have continued characterizing the binding site in protein S for C4BP, focusing on the region encompassing residues 447–460, which, based on phage display experimentation, was suggested to be involved in C4BP binding (39.Linse S. Hardig Y. Schultz D.A. Dahlbäck B. J. Biol. Chem. 1997; 272: 14658-14665Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Using Ala scanning mutagenesis, peptide inhibition assays, surface plasmon resonance, and monoclonal epitope mapping, the involvement of this region in C4BP binding was elucidated. To gain better insight into the characteristics of the C4BP-binding site, the now presented experimental data and those on record were evaluated on a recently created three-dimensional model for the SHBG-like region of protein S (44.Villoutreix B.O. Dahlbäck B. Borgel D. Gandrille S. Müller Y.A. Proteins Struct. Funct. Genet. 2001; 43: 203-216Crossref PubMed Scopus (28) Google Scholar). Rabbit polyclonal antibodies against human protein S (PK-anti-hPS) and mouse monoclonal antibodies against human protein S (HPS 54, HPS 34, HPS 67, HPS 21, and HPS 42) have been described previously (45.Dahlbäck B. Hildebrand B. Malm J. J. Biol. Chem. 1990; 265: 8127-8135Abstract Full Text PDF PubMed Google Scholar). HPS 54 was conjugated with HRP as described previously (46.Giri T.K. Hillarp A. Härdig Y. Zöller B. Dahlbäck B. Thromb. Haemostasis. 1998; 79: 767-772Crossref PubMed Scopus (46) Google Scholar). Protein S and C4BP were prepared from human plasma as reported previously (47.Dahlbäck B. Biochem. J. 1983; 209: 837-846Crossref PubMed Scopus (131) Google Scholar, 49.Hillarp A. Dahlbäck B. J. Biol. Chem. 1988; 263: 12759-12764Abstract Full Text PDF PubMed Google Scholar). A monoclonal antibody specific for Gla residues (M3B) (50.Brown M.A. Stenberg L.M. Persson U. Stenflo J. J. Biol. Chem. 2000; 275: 19795-19802Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) was a kind gift of Drs. Mark Brown and Johan Stenflo. HRP was obtained from Roche Molecular Biochemicals. 1,2-Phenylene diamine tablets and HRP-conjugated goat anti-mouse IgG were obtained from DAKO. N-Glycosidase F was from Roche Molecular Biochemicals. Five peptides (Table I) with acetylated NH2 termini and amidated COOH termini were synthesized on a MilliGen 9050 Plus synthesizer and purified by high pressure liquid chromatography, as described previously (39.Linse S. Hardig Y. Schultz D.A. Dahlbäck B. J. Biol. Chem. 1997; 272: 14658-14665Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar).Table ISynthetic peptidesPeptideProtein S residuesAmino acid sequenceSL8447–460Ac-SGIAQFHIDYNNNVS-NH2SL9451–460Ac-QFHIDYNNNVS-NH2SL10453–460Ac-HIDYNNNVS-NH2SL11447–458Ac-SGIAQFHIDYNNN-NH2SL15447–460 (Y456A)Ac-SGIAQFHIDANNNVS-NH2 Open table in a new tab The cDNA encoding human protein S (in vector pcDNA3; Invitrogen) was mutated using the QuikChange kit (Stratagene) and a series of oligonucleotides containing the desired mutation, as described previously (51.Giri T.K. Garcia de Frutos P. Yamazaki T. Villoutreix B.O. Dahlbäck B. Thromb. Haemostasis. 1999; 82: 1627-1633Crossref PubMed Google Scholar). A total of 13 cDNAs were produced, encoding variants designated S447A, G448A, I449A, Q451A, F452A, H453A, I454A, D455A, Y456A, N457A, N458A, V459A, and S460A. In a fourteenth variant, a new glycosylation consensus sequence was created around position 456 through the substitutions Y456N and N458T. The double mutant (Y456N/N458T) lacked the wild-type carbohydrate side chain at position 458. The mutations were confirmed by DNA sequence analysis using an ABIprism Taqpolymerase-based sequencing kit with fluorescent dye terminators (PerkinElmer Life Sciences). Vectors encoding wild-type protein S, the various Ala variants, and the Y456N/N458T double variant were used to transfect monkey kidney COS-1 cells by the DEAE-dextran method (51.Giri T.K. Garcia de Frutos P. Yamazaki T. Villoutreix B.O. Dahlbäck B. Thromb. Haemostasis. 1999; 82: 1627-1633Crossref PubMed Google Scholar). Expression levels were determined with an ELISA essentially as described previously (46.Giri T.K. Hillarp A. Härdig Y. Zöller B. Dahlbäck B. Thromb. Haemostasis. 1998; 79: 767-772Crossref PubMed Scopus (46) Google Scholar), except that the wells were coated with PK-anti-hPS, and samples were incubated overnight. The expression levels from confluent 10-cm Petri dishes with 10 ml of added Optimem were found to vary between 35 and 150 ng/ml/24 h. Most mutant expression levels were comparable to those seen for wild-type protein S. Exceptions were F452A (50% of wild type; p = 0.02), I454A (76% of wild type; p = 0.07), Y456A (35% of wild type; p = 0.009), and S460A (75% of wild type;p = 0.04), which demonstrated lower expression. The reported values are the mean of three independent experiments and were compared with the expression of wild-type protein S using a paired Student's t test. Conditioned media containing recombinant proteins were concentrated in Centricon concentrators (Amicon) and stored at −20 °C until further analysis. SDS-PAGE and Western blotting were performed following standard procedures. To deglycosylate the recombinant proteins, 5–10 μl of the concentrated culture medium containing ∼1 μg/ml protein S were treated withN-glycosidase F (0.5 unit/sample) under reducing and denaturing conditions and then analyzed by Western blotting using a polyclonal protein S antiserum The cDNAs encoding wt protein S and the D455A and Y456A protein S variants were used to transfect human embryonic kidney 293 cells using the Lipofectin method, and stable cell lines resistant to G418 were established, as described previously (51.Giri T.K. Garcia de Frutos P. Yamazaki T. Villoutreix B.O. Dahlbäck B. Thromb. Haemostasis. 1999; 82: 1627-1633Crossref PubMed Google Scholar). The recombinant proteins grown in the presence of vitamin K were collected in Optimem and purified using an immobilized calcium-dependent monoclonal antibody (HPS 21) directed against the Gla domain essentially as described previously (51.Giri T.K. Garcia de Frutos P. Yamazaki T. Villoutreix B.O. Dahlbäck B. Thromb. Haemostasis. 1999; 82: 1627-1633Crossref PubMed Google Scholar). The expression level was determined with the ELISA; wt protein S and the D455A mutant were present in ∼2–3 mg/liter, whereas the expression of the Y456A variant was 10–20-fold less. The purified proteins were analyzed by SDS-PAGE and Western blotting using polyclonal protein S antibodies or the M3B monoclonal antibody recognizing Gla residues (50.Brown M.A. Stenberg L.M. Persson U. Stenflo J. J. Biol. Chem. 2000; 275: 19795-19802Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The concentration of the proteins was determined by amino acid analysis after acid hydrolysis in 6 m HCl, and the Gla content was measured after base hydrolysis using methods outlined previously (26.He X. Dahlbäck B. Eur. J. Biochem. 1993; 217: 857-865Crossref PubMed Scopus (35) Google Scholar). Surface plasmon resonance experiments were carried out using a BIAcore 1000 system. Immobilization was performed using 10 mm Hepes, 0.15m NaCl, 3.4 mm EDTA, and 0.005% Tween 20, pH 7.4, as flow buffer and a flow rate of 5 μl/min. Equal volumes of 0.1m N-hydroxysulfosuccinimide and 0.4m 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were mixed, and 40 μl of this solution were injected to activate the carboxymethylated dextran. Then 40 μl of either 25 μg/ml monoclonal antibody against HPS 34 in 10 mm sodium acetate, pH 4.75, or 60 μg/ml C4BP in 10 mm sodium acetate, pH 4.5, were injected. Unreacted N-hydroxysulfosuccinimide-ester groups were deactivated by injecting a 20-μl pulse of 1 methanolamine hydrochloride, pH 8.5, and uncoupled protein was removed with 20 μl of 0.1 m HCl. Flow rates of 5–20 μl/min yielded identical results for the binding reactions. Surface plasmon resonance data were fitted as described previously (2.He X. Shen L. Malmborg A.C. Smith K.J. Dahlbäck B. Linse S. Biochemistry. 1997; 36: 3745-3754Crossref PubMed Scopus (41) Google Scholar, 39.Linse S. Hardig Y. Schultz D.A. Dahlbäck B. J. Biol. Chem. 1997; 272: 14658-14665Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Binding of protein S to HPS 34 was performed using 10 mmHepes, 0.15 m NaCl, 2 mm CaCl2, and 0.005% Tween 20, pH 7.4, as flow buffer. Wild-type recombinant protein S was injected at 12.5, 25, 50, 100, and 200 nmconcentrations. In addition, 50 nm of a 1:1 complex between protein S and C4BP was injected. The association phase was monitored for 12 min, and the dissociation into pure buffer was followed for 4 h. The remaining bound protein S was then removed by washing with 25 μl of 0.1 m HCl. Flow rates of 5–30 μl/min were tested and gave identical results. Binding experiments were also performed using the F452A, I454A, and Y456A mutants. The data were fitted as described previously (2.He X. Shen L. Malmborg A.C. Smith K.J. Dahlbäck B. Linse S. Biochemistry. 1997; 36: 3745-3754Crossref PubMed Scopus (41) Google Scholar). Binding of protein S to C4BP was performed in a flow buffer comprising 10 mm Hepes, 0.15 m NaCl, 2 mmCaCl2, and 0.005% Tween 20, pH 7.4. Wild-type protein S and variants were injected at concentrations of 1–20 nm. The association phase was monitored for 15 min, and the dissociation into pure buffer was followed for 300 min. Bound protein S was then removed by washing with 25 μl of 0.1 m HCl. Flow rates of 5–30 μl/min were tested and gave identical results. Peptide inhibition of protein S binding to C4BP was performed using 10 mm Hepes, 0.15 m NaCl, 3.4 mm EDTA, and 0.005% Tween 20, pH 7.4, as flow buffer. Each peptide was injected at concentrations ranging from 1 to 400 μm together with 60 nm protein S. The association phase was monitored for 15 min, and bound protein S or peptide was then removed by washing with 25 μl of 0.1 m HCl. Flow rates of 5–30 μl/min were tested and gave identical results. Conditioned medium containing each recombinant protein S variant was tested for direct binding to immobilized C4BP using the enzyme-linked ligandsorbent assay method, as described previously (46.Giri T.K. Hillarp A. Härdig Y. Zöller B. Dahlbäck B. Thromb. Haemostasis. 1998; 79: 767-772Crossref PubMed Scopus (46) Google Scholar), except that an overnight incubation step was used. The concentration of protein S in each sample was standardized before performing the assays. In brief, wells were coated with purified C4BP (10 μg/ml), blocked with bovine serum albumin, and washed with 50 mm Tris-HCl, 150 mm NaCl, 2 mm CaCl2, and 0.1% Tween 20, pH 7.5. Conditioned medium containing wild-type protein S or the variants (serially diluted with 50 mm Tris-HCl, 150 mm NaCl, 2 mm CaCl2, and 0.1% bovine serum albumin, pH 7.5) was added, and the plates were incubated overnight at 4 °C. The wells were washed, and HRP-labeled antibody HPS 54 directed against the epidermal growth factor 1-like module of protein S (45.Dahlbäck B. Hildebrand B. Malm J. J. Biol. Chem. 1990; 265: 8127-8135Abstract Full Text PDF PubMed Google Scholar) was used for detection of bound protein S. The apparent dissociation constant, KDapp, for the interaction was calculated by fitting the absorbance data to the formulaA n = A/(1 + ( KDapp/C)), whereA n is the observed absorbance, A is the maximum absorbance obtained with wild-type protein S, and Cis the concentration of protein S. It was assumed that the amount of protein S bound was negligible compared with the total concentration of protein S, such that C reflects free protein S. Conditioned medium containing wild-type protein S or the variant proteins was tested for binding to immobilized HPS 34 by ELISA. Bound protein S was detected with the antibody HPS 54 as described previously (46.Giri T.K. Hillarp A. Härdig Y. Zöller B. Dahlbäck B. Thromb. Haemostasis. 1998; 79: 767-772Crossref PubMed Scopus (46) Google Scholar). Apparent dissociation constants were calculated as described above. The ability of HPS 34 to inhibit the binding of human protein S to immobilized C4BP was tested using the enzyme-linked ligandsorbent assay method (46.Giri T.K. Hillarp A. Härdig Y. Zöller B. Dahlbäck B. Thromb. Haemostasis. 1998; 79: 767-772Crossref PubMed Scopus (46) Google Scholar). In brief, aliquots of 5 nm plasma-purified protein S were preincubated with various concentrations of HPS 34 (up to a 500-fold molar excess) for 30 min at room temperature, and then the samples were added to C4BP-coated wells and incubated for 1 h at room temperature. After washing, HRP-labeled HPS 54 was used as the detecting antibody. Phage display experiments using immobilized HPS 34 as a target and random linear 15-mer peptides displayed on the bacteriophage surface were performed as described previously (39.Linse S. Hardig Y. Schultz D.A. Dahlbäck B. J. Biol. Chem. 1997; 272: 14658-14665Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The selected peptides were aligned against the protein S sequence using the HOMOFILE and AVEHOM programs (39.Linse S. Hardig Y. Schultz D.A. Dahlbäck B. J. Biol. Chem. 1997; 272: 14658-14665Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Five peptides (Table I) were tested for their ability to compete with protein S for binding to C4BP using surface plasmon resonance analysis. Peptides corresponding to residues 447–460, 451–460, and 453–460 of protein S were found to inhibit protein S binding to C4BP (Fig. 1) at concentrations comparable to those observed previously for peptides 439–460 and 447–468 (39.Linse S. Hardig Y. Schultz D.A. Dahlbäck B. J. Biol. Chem. 1997; 272: 14658-14665Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The 447–460/Y456A peptide required an ∼3-fold higher concentration to give half-maximum inhibition, and the 447–458 peptide showed no inhibition at the concentrations tested. Thirteen recombinant protein S variants were generated by replacing each amino acid in the 447–460 region of protein S (Fig. 2 A) with Ala. The transiently expressed mutants were analyzed by SDS-PAGE and detected by Western blotting using a polyclonal antibody (PK-anti-hPS) (Fig. 2 B). All migrated as single bands. The N458A and S460A variants exhibited an increased mobility, consistent with loss of the carbohydrate moiety present at residue Asn458 in wild-type protein S (52.Lu D. Xie R. Rydzewski A. Long G. Thromb. Haemostasis. 1997; 77: 1156-1163Crossref PubMed Scopus (26) Google Scholar). After deglycosylation, the different recombinant proteins demonstrated migration rates similar to that of deglycosylated wild-type protein S, suggesting that all recombinant proteins were glycosylated to the expected degree. Furthermore, the Western blotting patterns of the deglycosylated recombinant proteins were identical to that of deglycosylated plasma-derived protein S (data not shown). Recombinant protein S was tested in Western blotting and ELISA techniques with a panel of carefully characterized monoclonal antibodies that reacted with conformation-dependent epitopes in different domains to investigate the structural integrity of the proteins and their correct folding. The antibodies tested were HPS 21 (reacting with a calcium-dependent epitope in the Gla domain), HPS 67 (reacting with a calcium-dependent epitope in thrombin-sensitive region), and HPS 54 (reacting with a calcium-dependent epitope in epidermal growth f

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Structural Requirements of Anticoagulant Protein S for Its Binding to the Complement Regulator C4b-binding Protein