The factor VIII (FVIII) is a cofactor of the coagulation cascade. The FVIII C2 domain is a critical domain that participates in the interactions with the von Willebrand factor and the phospholipidic surfaces. To assess the importance of each residue of this domain in the maintenance of the structure and the function of FVIII, a number (n=139) of mutants were generated by substituting the original residues, from Ser2173 to Gly2325, by an alanine. Mutants were built within a complete B domain- deleted FVIII and expressed in COS-1 cells. Mutant antigen levels and procoagulant activities were measured. Two in silico analyses, a sliding average procedure and an analysis of the mutation energy cost were conducted in parallel on the FVIII structure. Both results were in agreement with the functional data, and illustrated the benefit of using such strategies prior to targeting specific residues in the aim of generating active recombinant molecules. The functional assays identify the residues that are important to maintaining the structure of the C2 domain, mainly those forming β-sheet, and those that can afford substitution, establishing a detailed functional relation with the available crystallographic data. This study provided a comprehensive functional mapping of the FVIII C2 domain and discussed the implication of specific residues in respect to the maintenance in the activity and structure stability, the efficiency in secretion, the binding to phospholipids and the formation of epitope.
Keywords
Coagulation factors -
factor VIII -
protein structure / folding
* Present address: Global Bioenergies, Evry, France
1
Fay PJ.
et al. von Willebrand factor mediates protection of factor VIII from activated protein C-catalyzed inactivation. J Biol Chem 1991; 266: 2172-2177.
6
Ngo JC.
et al. Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex. Structure 2008; 16: 597-606.
9
Foster PA.
et al. Synthetic factor VIII peptides with amino acid sequences contained within the C2 domain of factor VIII inhibit factor VIII binding to phos- phatidylserine. Blood 1990; 75: 1999-2004.
10
Saenko EL,
Scandella D.
A mechanism for inhibition of factor VIII binding to phospholipid by von Willebrand factor. J Biol Chem 1995; 270: 13826-13833.
11
Kuwabara I.
et al. Mapping of the minimal domain encoding a conformational epitope by lambda phage surface display: factor VIII inhibitor antibodies from haemophilia A patients. J Immunol Methods 1999; 224: 89-99.
20
Pellequer JL.
et al. Homology models of the C domains of blood coagulation factors V and VIII: a proposed membrane binding mode for FV and FVIII C2 domains. Blood Cells Mol Dis 1998; 24: 448-461.
21
Meeks SL.
et al. Antihuman factor VIII C2 domain antibodies in hemophilia A mice recognize a functionally complex continuous spectrum of epitopes dominated by inhibitors of factor VIII activation. Blood 2007; 110: 4234-4242.
22
Nagashima M.
et al. Alanine-scanning mutagenesis of the epidermal growth factor-like domains of human thrombomodulin identifies critical residues for its cofactor activity. J Biol Chem 1993; 268: 2888-2892.
23
Dimitrov JD.
et al. Induction of heme oxygenase-1 in factor VIII-deficient mice reduces the immune response to therapeutic factor VIII. Blood 2010; 115: 2682-2685.
24
Spiegel PC.
et al. Surface-exposed hemophilic mutations across the factor VIII C2 domain have variable effects on stability and binding activities. J Biol Chem 2004; 279: 53691-53698.
25
Lewis DA.
et al. Contributions of Asn2198, Met2199, and Phe2200 in the factor VIII C2 domain to cofactor activity, phospholipid-binding, and von Willebrand factor-binding. Thromb Haemost 2003; 89: 795-802.
27
Schatz SM.
et al. Mutation of the surface-exposed amino acid Trp to Ala in the FVIII C2 domain results in defective secretion of the otherwise functional protein. Br J Haematol 2004; 125: 629-637.
28
Gilbert GE.
et al. Four hydrophobic amino acids of the factor VIII C2 domain are constituents of both the membrane-binding and von Willebrand factor-binding motifs. J Biol Chem 2002; 277: 6374-6381.
29
Chen L.
et al. The enhancing effects of the light chain on heavy chain secretion in split delivery of factor VIII gene. Mol Ther 2007; 15: 1856-1862.
30
Webb E.
et al. Enhanced secretory ability for the human factor VIII light chain produced in baculovirus-infected insect cells. Cytotechnology 1996; 21: 165-170.
32
Healey JF.
et al. Residues Glu2181-Val2243 contain a major determinant of the inhibitory epitope in the C2 domain of human factor VIII. Blood 1998; 92: 3701-3709.
33
Scandella D.
et al. Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site. Blood 1995; 86: 1811-1819.
34
Shima M.
et al. A factor VIII neutralizing monoclonal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 domain inhibit factor VIII binding to von Willebrand factor and to phosphatidylserine. Thromb Haemost 1993; 69: 240-246.
35
Nogami K.
et al. Relationship between the binding sites for von Willebrand factor, phospholipid, and human factor VIII C2 inhibitor alloantibodies within the factor VIII C2 domain. Int J Hematol 2007; 85: 317-322.
36
Lewis DA.
et al. Binding of factor VIII inhibitors to discrete regions of the factor VIII C2 domain disrupt phospholipid binding. Blood Coagul Fibrinolysis 2003; 14: 361-368.
37
d’Oiron R.
et al. Deletion of alanine 2201 in the FVIII C2 domain results in mild hemophilia A by impairing FVIII binding to VWF and phospholipids and destroys a major FVIII antigenic determinant involved in inhibitor development. Blood 2004; 103: 155-157.
38
Liu Z.
et al. Trp2313-His2315 of factor VIII C2 domain is involved in membrane binding: structure of a complex between the C2 domain and an inhibitor of membrane binding. J Biol Chem 2010; 285: 8824-8829.
39
Gale AJ.
et al. Structural basis for hemophilia A caused by mutations in the C domains of blood coagulation factor VIII. Thromb Haemost 2000; 83: 78-85.
40
Lewis DA.
et al. Phospholipid vesicles interfere with the binding of antibody fragments to the light chain of factor VIII. Thromb Haemost 2005; 93: 833-841.
41
Nogami K.
et al. Identification of a factor VIII peptide, residues 2315-2330, which neutralizes human factor VIII C2 inhibitor alloantibodies: requirement of Cys2326 and Glu2327 for maximum effect. Br J Haematol 1999; 107: 196-203.
42
Spiegel PC.
Jr.
et al. Structure of a factor VIII C2 domain-immunoglobulin G4kappa Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII. Blood 2001; 98: 13-19.
43
Parker ET.
et al. Reduction of the inhibitory antibody response to human factor VIII in hemophilia A mice by mutagenesis of the A2 domain B-cell epitope. Blood 2004; 104: 704-710.
46
Chen S-wW.
et al. Structure-activity relationships in peptide-antibody complexes: implications for epitope prediction and development of synthetic peptide vaccines. Curr Med Chem 2009; 16: 953-964.
