Functional Consequences of Mutations in Amino Acid Residues that Stabilize Calcium Binding to the First Epidermal Growth Factor Homology Domain of Human Protein C

Summary

Charge-to-alanine mutations of three amino acid residues, viz., D⁴⁶, D⁴⁸, and D/Hya⁷¹, which are known to be important in stabilizing Ca²⁺ binding to epidermal growth factor (EGF) domains of vitamin K-de-pendent blood coagulation proteins, have been engineered into recombinant human protein C (r-PC), The resulting variants were then employed to assess the importance of this Ca²⁺ binding site in the activation properties of r-PC and in the activity of activated protein C (APC). Another mutation, of D⁴⁸ to E, was constructed in order that a more conservative mutation at the Ca²⁺ binding site could be similarly examined.

The mutant proteins were fully processed with regard to proper signal peptide cleavage, γ-carboxylation, and β-hydroxylation, except, of course, for the D⁷¹A mutant in this latter case. The D⁴⁸E variant possessed an additional residue of γ-carboxyglutamic acid (Gla), showing that E⁴⁸ was γ-carboxylated. All of the mutants were reactive against a monoclonal antibody (MAb) specific for a Ca²⁺-dependent epitope within the amino-terminus of the Gla domain of r-PC, demonstrating that a proper Ca²⁺-dependent conformation was adopted in this region of the protein. None of the mutants, except for [D⁴⁸γ]r-PC, were reactive against another Ca²⁺-dependent MAb which possessed specificity for Ca²⁺ binding to the EGF1 region of PC-this being the area of the protein that contained the mutated residues. These data strongly suggest that the alanine mutations present at D⁴⁶, D⁴⁸, and D⁷¹ diminished Ca²⁺ binding to the EGF1 domain of r-PC.

Steady state kinetic analysis demonstrated that determinants for the Ca²⁺-dependent inhibition of the thrombin (flla)-catalyzed activation of r-PC, and for the kinetic recognition of the flla/thrombomodulin complex, were not dependent on the integrity of the Ca²⁺ sites present in EGF1. The lone exception was [D⁴⁸γ]r-PC, which did not undergo inhibition by Ca²⁺, an effect likely due to the potential for altered coordination of Ca²⁺ due to the Gla insertion, rather than to a dependency on D⁴⁸. Plasma-based anticoagulant assays, as well as individual factor Va and factor Villa inactivation assays, showed that only [D71A]r-APC possessed a significantly reduced activity compared to wild-type r-APC. These observations suggest that D/Hya⁷¹ is likely an important determinant for activity of APC toward its physiological substrates, factor Va and factor Villa.

• References

• 1 Kisiel W, Canfield WM, Ericsson LH, Davie EW. Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry 1977; 16: 5824-5831
• 2 Vehar GA, Davie EW. Preparation and properties of bovine factor VIII (antihemophilic factor). Biochemistry 1980; 19: 401-410
• 3 Esmon NL, Owen WG, Esmon CT. Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. J Biol Chem 1982; 257: 859-864
• 4 Amphlett GW, Kisiel W, Castellino FJ. Interaction of calcium with bovine plasma protein C. Biochemistry 1981; 20: 2156-2161
• 5 Clarke JH, Light DR, Blasko E, Parkinson JF, Nagashima M, McLean K, Vilander L, Andrews JH, Morser J, Glaser CB. The short loop between epidermal growth factor-like domains 4 and 5 is critical for human thrombomodulin function. J Biol Chem 1993; 268: 6309-6315
• 6 Colpitts TL, Castellino FJ. Calcium and phospholipid binding properties of synthetic gamma-carboxyglutamic acid-containing peptides with sequence counterparts in human protein C. Biochemistry 1994; 33: 3501-3508
• 7 Nelsestuen GL. Role of γ-carboxyglutamic acid. An unusual protein transition required for the calcium-dependent binding of prothrombin to phospholipid. J Biol Chem 1976; 251: 5648-5656
• 8 Öhlin A-K, Linse S, Stenflo J. Calcium binding to the epidermal growth factor homology region of bovine protein C. J Biol Chem 1988; 263: 7411-7417
• 9 Rezaie AR, Esmon NL. Esmon CT The high affinity calcium-binding site involved in protein C activation is outside of the first epidermal growth factor homology domain. J Biol Chem 1992; 267: 11701-11704
• 10 Rezaie AR, Esmon CT. The function of calcium in protein C activation by thrombin and the thrombin-thrombomodulin complex can be distinguished by mutational analysis of protein C derivatives. J Biol Chem 1992; 267: 26104-26109
• 11 Zhang L, Castellino FJ. Role of the hexapeptide disulfide loop present in the γ-carboxyglutamic acid domain of protein C in its activation properties and in the in vitro anticoagulant activity of activated protein C. Biochemistry 1991; 30: 6696-6704
• 12 Rezaie AR, Mather T, Sussman F, Esmon CT. Mutation of glu-80->lys results in a protein C mutant that no longer requires Ca²⁺ for rapid activation by the thrombin-thrombomodulin complex. J Biol Chem 1994; 269: 3151-3154
• 13 Rees DJG, Nones IM, Handford PA, Walter SJ, Esnouf MO, Smith KJ, Brownlee GG. The role of β-hydroxyaspartic acid and adjacent carboxylate residues in the first EGF domain of human factor IX. EMBO J 1988; 7: 2053-2061
• 14 Mayhew M, Handford P, Baron M, Tse AGD, Campbell ID, Brownlee GG. Ligand requirements for Ca²⁺ binding to EGF-like domains. Protein Eng 1992; 5: 489-494
• 15 Selander-Sunnerhagen M, Ullner M, Persson E, Teleman O, Stenflo J, Drakenberg T. How an epidermal growth factor (EGF)-like domain binds calcium. High resolution nmr structure of the calcium form of the NH2-terminal EGF-like domain in coagulation factor X. J Biol Chem 1992; 267: 19642-19649
• 16 Handford PA, Mayhew M, Baron M, Winship PR, Campbell ID, Brownlee GG. Key residues involved in calcium-binding motifs in EGF-like proteins. Nature 1991; 351: 164-167
• 17 Rao Z, Handford P, Mayhew M, Knott V, Brownlee GG, Stuart D. The structure of a Ca²⁺-binding epidermal growth factor-like domain: Its role in protein-protein interactions. Cell 1995; 82: 131-421
• 18 Zhang L, Castellino FJ. A γ-carboxyglutamic acid variant γ^6D, γ^7D) of human activated protein C displays greatly reduced activity as an anticoagulant. Biochemistry 1990; 29: 10828-10834
• 19 Meyback B, Heim J, Rink H, Zimmermann W, Maerki W. Desulfato-hirudin, a specific thrombin inhibitor: Expression and secretion in yeast. Thromb Res 1987; 7 (Suppl.) 33
• 20 Heeb MJ, Schwartz P, White T, Lammle B, Berrettini M, Griffin J. Immunoblotting studies of the molecular forms of protein C in plasma. Thromb Res 1988; 52: 33-43
• 21 Wakabayashi K, Sakata Y, Aoki N. Conformation-specific monoclonal antibodies to the calcium-induced structure of protein C. J Biol Chem 1986; 261: 11097-11105
• 22 Suzuki K, Kusumoto H, Deyashiki Y, Nishioka J, Maruyami I, Zushi M, Kawahara S, Honda G, Yamamoto S, Horiguchi S. Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO J 1987; 6: 1891-1897
• 23 Glaser CB, Morser J, Clarke JH, Blasko E, McLean K, Kuhn I, Chang RJ, Lin JH, Vilander L, Andrews WH, Light DR. Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity. A potential rapid mechanism for modulation of coagulation. J Clin Invest 1992; 90: 2565-2573
• 24 Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods in Enzymol 1987; 154: 367-382
• 25 Yu S, Zhang L, Jhingan A, Christiansen WT, Castellino FJ. Construction, expression, and properties of a recombinant human protein C with replacement of its growth factor-like domains by those of human coagulation factor IX. Biochemistry 1994; 33: 823-831
• 26 Zhang L, Jhingan A, Castellino FJ. Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity. Blood 1992; 80: 942-952
• 27 Jhingan A, Zhang L, Christiansen WT, Castellino FJ. The activities of recombinant gamma-carboxyglutamic acid-deficient mutants of activated human protein C toward human coagulation factor VIII and factor Va in purified systems and in plasma. Biochemistry 1994; 33: 1869-1875
• 28 Menhart N, Sehl LC, Kelley RF, Castellino FJ. Construction, expression and purification of recombinant kringle 1 of human plasminogen and analysis of its interaction with w-amino acids. Biochemistry 1991; 30: 1948-1957
• 29 De Serrano VS, Menhart N, Castellino FJ. The expression, purification and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator. Arch Biochem Biophys 1992; 294: 282-290
• 30 De Serrano VS, Castellino FJ. The role of tryptophan-74 of the recombinant kringle 2 domain of tissue-type plasminogen activator in its co-amino acid binding properties. Biochemistry 1992; 31: 3326-3335
• 31 Grinnell BW, Walls JD, Gerlitz B. Glycosylation of human protein C affects its secretion, processing, functional activities, and activation by thrombin. J Biol Chem 1991; 266: 9778-9785
• 32 Handford PA, Baron M, Mayhew M, Willis A, Beesley T, Brownlee GG, Campbell ID. The first EGF-like domain of human factor IX contains a high-affinity calcium binding site. EMBO J 1990; 9: 475-480
• 33 Rezaie AR, Neuenschwander PF, Morrissey JH. Esmon CT Analysis of the functions of the first epidermal growth factor-like domain of factor X. J Biol Chem 1993; 268: 8176-8180
• 34 Selander-Sunnerhagen MS, Persson E, Dahlqvist I, Drakenberg T, Stenflo J, Mayhew M, Robin M, Handford P, Tilley JW, Campbell ID, Brownlee GG. The effect of aspartate hydroxylation on calcium binding to epidermal growth factor-like modules in coagulation factors IX and X. J Biol Chem 1993; 268: 23339-23344
• 35 Valcarce C, Selander-Sunnerhagen M, Tamlitz AM, Drakenberg T, Bjork I, Stenflo J. Calcium affinity of the NH₂-terminal epidermal growth factorlike module of factor X. Effect of the gamma-carboxyglutamic acid-containing module. J Biol Chem 1993; 268: 26673-26678
• 36 Öhlin A-K, Landes G, Bourdon P, Oppenheimer C, Wydro R, Stenflo J. β-Hydroxyaspartic acid in the first epidermal growth factor-like domain of protein C. Its role in Ca2+ binding and biological activity J Biol Chem 1988; 263: 19240-19248
• 37 Davis LM, McGraw RA, Ware JL, Roberts H, Stafford DW. Factor IXAlabama-A point mutation in a clotting protein results in hemophilia B. Blood 1987; 69: 140-143
• 38 McCord DM, Monroe DM, Smith KJ, Roberts HR. Characterization of the functional defect in factor IX Alabama. Evidence for a conformational change due to high affinity calcium binding in the first epidermal growth factor domain. J Biol Chem 1990; 265: 10250-10254
• 39 Winship PR, Dragon AC. Identification of haemophilia B patients with mutations in the two Ca²⁺ binding domains of factor IX: Importance of a β-OH 64 Asn change. Br J Haematol 1991; 77: 102-109
• 40 Green PM, Bentley DR, Mibashan RS, Nilsson IM, Gianelli F. Molecular pathology of haemophilia B. EMBO J 1989; 8: 1067-1072
• 41 Bode W, Huber R, Rydel TJ, Tulinsky A. in Thrombin. 3-61
• 42 Colpitts TL, Castellino FJ. Binding of calcium to synthetic peptides containing gamma-carboxyglutamic acid. Int J Peptide Protein Res 1993; 41: 567-575
• 43 Bode W, Schwager P. Single calcium-binding site of crystalline bovine β-trypsin. FEBS Lett 1975; 56: 139-143
• 44 Sabharwal AK, Birktoft JJ, Gorka J, Wildgoose P, Petersen LC, Bajaj SP. High affinity Ca²⁺-binding site in the serine protease domain of human factor Vila and its role in tissue factor binding and development of catalytic activity. J Biol Chem 1995; 270: 15523-15530
• 45 Bajaj SP, Sabharwal AK, Gorka J, Birktoft JJ. Antibody-probed conformational transitions in the protease domain of factor IX upon calcium binding and zymogen activation: Putative high-affinity Ca²⁺-binding site in the protease domain. Proc Natl Acad Sci USA 1992; 89: 152-156
• 46 Esmon NL, DeBault LE, Esmon CT. Proteolytic formation and properties of γ-carboxyglutamic acid-domainless protein C. J Biol Chem 1983; 258: 5548-5553