Oct 25, 2017 - ic' 1989 by The American Soclety for Biochemistry and Molecular Biology, Inc. ... From the American Red Cross Blood Services and the.
THE,JOURNAL
Communication
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Characterization of a Synthetic Peptide That Inhibits the Interaction between Protein S and C4b-bindingProtein* (Received for publication, April 10, 1989)
Frederick J. Walker From the American Red Cross Blood Services and the Departments of Laboratory Medicine and Medicine, University of Connecticut School of Medicine, Farmington, Connecticut 06032
Protein S is unique among the vitamin K-dependent proteins found in blood plasma because it is a cofactor rather than a zymogen of a serine protease. Instead of a trypsin-like doma.in,protein S contains a domain that has sequence homology with steroid binding proteins. In order to understand the function of this structural domain, peptides have been synthesized with amino acid sequences that are homologous between human protein S and rat androgen binding protein. Two peptides, c0rrespondin.g to amino acids 400-407 (PINPRLDG) and 605-6114 (GVQLDLDEAI) of the protein S sequence have been tested for their effects on protein S function. Neither peptide altered the clotting of bovine or human plasma. The peptide GVQLDLDEAI enhanced the antilcoagulant activity of human-activated protein C in human plasma while the peptide PINPRLDG had no effect. The peptide GVQLDLDEAI was observed to inhibit the binding of protein S to C4bbinding protein in plasma, resulting in increased concentrations of free protein s. GVQLDLDEAI was also observed to enhance the disassociation of the protein S*C4b-bindingprotein complex when purified complex was used. Finally, C4b-binding protein was observed to bind to GVQLDLDEAI. These results suggest that the carboxyl-terminal region of protein S, which contains the sequence GVQLDLDEAI, is involved in the interaction between protein S and C4b-binding protein.
OF BIOLOGICAL CHEMISTRY
Vol. 264. No. 30,Issue of October 25, pp. 17645-17648, 1989 1989 by The American Soclety for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
presence of a cofactor, protein S (Walker, 1980). Protein S is a vitamin K-dependent protein that has been isolated from human (DiScipio and Davie, 1979; Dahlback, 1983a) and bovine plasma (Stenflo and Jonsson, 1979). Protein S and activated protein C have been observed to form a complex on the surface of membranes, and this complex appears to be important in the expression of the anticoagulant activity of activated protein C (Walker, 1981). Protein S has been observed to stimulate the proteolysis of factor V (Walker, 1980), factor VI11 (Walker et al., 1987), and plasminogen activator inhibitor (D’Angelo et al., 1987) by activated protein C. Its action as a cofactor makes protein S a unique member of the vitaminK-dependentprotein family found in blood since other members of this family are zymogens of serine proteases. The determination of the amino acidsequencefrom both human (Lundwall et al., 1986) and bovine plasma (Dahlback et al., 1986) confirms that protein S is unique since it does not contain thehomologies with trypsin that are found in the other vitamin K-dependent proteins. In theregion where one would expect to finda serine protease, one finds a region that is homologous to steroid binding proteins (Baker et al., 1987). In addition to its unusual structure, protein S also appears to be regulated in a unique fashion. In human plasma, protein S is foundpredominantlyboundto C4b-binding protein (Dahlback and Stenflo, 1981). When in complex with this protein, it is not able to acta as cofactor foractivated protein C (Dahlback, 1986).Severallines of evidencesuggest that protein S may also be bound to other plasma proteins (Walker, 1986). In this paper, synthetic peptides that contain sequences homologous with regions in protein S and androgen binding protein have beentested for their effects on proteinS function to determine if these regions may be involved in interactions of protein S with activated proteinC or C4b-binding proteins. EXPERIMENTAL PROCEDURES
Preparation of Proteins-The peptides PINPRLDG and GVQLDLDEAI were synthesized by Pennisula Laboratories, Inc. The peptides were greaterthan 90% homogeneous asdetermined by HPLC’ and contained the correct ratio of amino acids as determined by amino acid analysis. The peptides GVQLGLDEAI, DEGEQLADLIV, LDLDEAISK, MEVNINGVQ, and KHNDIRAHS were synthesized by Multiple Peptide Systems and were greater than 85% pure as determined by HPLC. Bovine activated protein C was prepared by the method previously described (Walker et al., 1979). Human actiProtein C is a vitamin K-dependent protein found inblood vated protein C was prepared as described by Comp and co-workers plasma. The active !form, activatedprotein C, is a serine (Comp etal., 1984). Human C4b-binding proteinswas purified using proteasethatexhibitsanticoagulant (Seegers et al., 1972; a previously described method (Dahlback, 1983b). The C4b-binding protein contained tightly bound protein S which was removed by gel Zolton and Seegers, 1978; Kisiel et al., 1977) and profibrinol- filtration in 3 M sodium thiocyanate buffered with 0.02 M Tris-HC1, ytic properties (Zolton and Seegers, 1978; Comp and Esmon, pH 7.5 Thiocyanate was removed fromthe C4b-binding protein 1981). The anticoagulant activityof this protease is due to itssample by gel filtration. Bovine protein S was prepared by a previously recognition of factor!< V(Kisiel et al., 1977; Walker et al., described method (Walker, 1980). Protein S was radioiodinated by 1979; Marlar et al., 1982) and VI11 (Marlar et al., 1982; Walker the chloramine-T method (McConahey and Dixon, 1980). Radiolaet al., 1987; Vehar and Davie, 1980), cofactors in the coagu- beled protein S had a specific activity of 2000 cpm/ng. Biotinylated protein S was prepared by treating proteinS dialyzed in 1% carbonate lation cascade, as substrates which it inactivates by proteol- with sulfosuccinimidyl 6-(biotinamido) hexanoate (1 mg/mg protein) ysis. Maximum activity of activated protein C requires the for 2 h a t room temperature. Excess biotinwas removed by dialysis. Electrophoresis-Purity of proteins was assured by electrophoresis * This work was supported by Grant HL 38388 from the National in sodium dodecyl sulfate-polyacrylamide gels (Laemmli, 1970). Gels Heart, Lung, and Blood Institute and by funds from the American were stained either with Coomassie Blue or with silver nitrate (MorRed Cross. The costs of publication of this article were defrayed in risey, 1981). part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section The abbreviationused is: HPLC, high pressure liquid chromatog1734 solely to indicate this fact. raphy.
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Characterization of Synthetic Peptide That Inhibits Interaction
Assay for the Anticoagulant Actiuity of Activated Protein C-The anticoagulant activityof activated protein C was determined in a one-stage clotting assay which contained 0.1 ml of either human or bovine plasma, 0.1 ml of rabbit thromboplastin, 0.1 ml of 0.025 M CaC12,and activated protein C (0.1 ml of 1 pg/ml in 0.1 M NaCl, 0.02 M Tris HCI, pH 7.5, and 1 mg/ml bovine serum albumin). Clotting was initiated by the addition of calcium. Determination of Free Protein S - T protein S (10 ng/ml final concentration) was added toplasmathathad been dialyzed into 0.38% sodium citrate, 0.02 M Tris-HC1, pH 7.5, 0.1 M NaCl. To determine free protein S samples were added to 25% polyethylene glycol 8000 to a final polyethylene glycol concentration of 3.1% (w/ v). Samples were mixed, and then the precipitate was removed by centrifugation in a Beckman Microfuge at full speed. A sample was I , , I removed from the supernatant and counted ina y counter. All points 0 20 40 60 were taken in triplicate and the data averaged. In the experiments [Peptide] ug/ml reported inthispaperthevariationinthetriplicatepoints was approximately 5%. FIG. 1. The effect ofPINPRLDG and GVQLDLDEAI on the Determination of the Interaction between ProteinS and C4b-bindanticoagulant activityofactivated protein C in human ingProtein-The interaction between protein S and C4b-binding plasma. Clotting was carried out as described under “Experimental protein was determinedin a two-stage assay. Inthefirststage Procedures” with the indicated quantities of eitherPINPRLDG hiotinylated protein S (10 pg) was incubated with C4b-bindingprotein (squares) or GVQLDLDEAI (circles). The valuesshown arethe (50 pg) in a total volume of 0.200 ml. A sample of this mixture was average of two determinations. This experiment is representativeof added to microtiter plates that hasbeen coated with rabbit anti-C4bthree separate experiments. binding protein (0.1 ml of 1/100 antiserum from Calhiochem) for 2 h. The wells were washed and blocked with 3% albumin. The plates TABLE I were then washed with 0.5 M NaC1, 0.02 M Tris-HC1, pH 7.5, and avidin-peroxidase was added and incubated for 1 h.After 1 h the Effect of various peptidesfrom the proteinS sequence on the plates were again washed and developed using the peroxidase subanticoagulant actiuity of activated protein C strate o-phenylenediamine. All experiments were runinduplicate Relative Peptide Protein S sequence and the resultsaveraged. activitv“ -~ Binding of Peptides to C4b-binding Protein-Binding of C4b-bindGVQLDLDEAI 605-614 100 ing protein to synthetic peptides was carried out on the surface of GVQLGLDEAI 605-614 (ASP”’ to Gly) 11 microtiter plates. Peptides were incubated in the wells of microtiter DGEQLADLIV Scrambled 605-614 8 plates for 18 h. The wells were then washed and blocked by incubating PINPRLDG 400-407 5 them with 3% bovine serum albuminfor 30 min. C4b-binding protein MEVNINGVQ 599-607 18 was then added to the wells and incubated for 1 b. Wells were again LDLDEAISK 608-616 47 washed and blocked with 3% bovine serum albumin. The amount of KHNDIRAHS 616-624 45 boundC4b-binding protein was determined by enzyme-linked immunosorbent assay using rabbit antiX4b-binding protein as the first a Relative activity of the peptides was determined using a modifiantibody. Concentrations of antibodies used were determined to be cation of the factor Xa-initiated clotting assay. Clotting times were in a range such that the rate of o-phenylanediamine hydrolysis was measured inthe presence of various concentrations of activated proportional to the amount of C4b-binding protein bound to the protein C, and a standard curvewas constructed. Peptideswere added plate. Thedataarereported as therate of o-phenylenediamine to plasma at a final concentration of 50 pg/ml, and then the ant,icohydrolysis. agulant effect of activated protein C (0.75 pg/ml) was determined. In this experiment GVQLDLDEAI (50 pg/ml) with activated protein C (0.75 pg/ml) had the same effect on clotting as 2.3 pg/ml activated RESULTSANDDISCUSSION protein C alone. GVQLDLDEAI was assigned a relative activity of The addition of either PINPRLDG or GVQLDLDEAI to 100. Relative activity is defined as, relative activity = (APC activity bovine or human plasma had no effect on the clotting initiated with peptide - APC activity without peptide) X 100/(APC activity with factor Xa. In addition, neither peptide altered the anti- with GVQLDLDEAI - APC activity without peptide). ~~
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