Factor VII Deficiency Caused by a Structural Variant N57D of the First Epidermal Growth Factor Domain

Abstract

We have previously described a kindred with factor VII (FVII) deficiency whose members exhibited reduced procoagulant activity relative to FVII antigen concentration. In this report, the molecular genetic basis of the FVII defect has been determined to be a heterozygous substitution of Asp for Asn at position 57 in the first epidermal growth factor (EGF) domain. Recombinant FVII (N57D) cDNA was created by site-directed mutagenesis and transiently expressed in human 293 cells. The transfected cells synthesized an immunoprecipitable protein with an apparent molecular weight of 50 kD. Quantitation of expression by FVII enzyme-linked immunosorbent assay indicated that mutant protein yields were consistently low, typically 10% to 30% of wild-type FVII. FVII (N57D) protein did not accumulate intracellularly, and Northern blot analysis indicated equivalent FVII mRNA levels in 293 cells expressing either wild-type FVII or FVII (N57D). Secreted FVII (N57D) protein did not bind tissue factor, exhibited no procoagulant activity, and failed to bind a conformation-dependent monoclonal antibody specific for the first EGF domain of FVII. Molecular modeling of the first EGF domain of FVII predicted that the N57D amino acid substitution would disrupt tertiary bonding structure. We conclude that the N57D mutation affects folding of the first EGF domain of FVII resulting in decreased cellular secretion of a mutant FVII molecule, which is unable to bind tissue factor and is therefore biologically inactive.

Interspecies exchange mutagenesis of the first epidermal growth factor‐like domain of human factor VII

“…We previously described two naturally occurring mutations of the FVII EGF1 domain which affect binding to TF. Both mutants rFVII(N57D) [15] and rFVII(R79Q) [14,31] exhibited a five‐ to ten‐fold decrease in TF binding but the mechanisms of the two defects differed. Amino acid residue R79 of FVII was shown to form both hydrophobic and hydrogen bonds with TF [20], whereas the mutation FVII(N57D) did not directly alter FVII contact with TF but caused a misfolding of the FVII EGF1 domain [15].…”

“…Both mutants rFVII(N57D) [15] and rFVII(R79Q) [14,31] exhibited a five‐ to ten‐fold decrease in TF binding but the mechanisms of the two defects differed. Amino acid residue R79 of FVII was shown to form both hydrophobic and hydrogen bonds with TF [20], whereas the mutation FVII(N57D) did not directly alter FVII contact with TF but caused a misfolding of the FVII EGF1 domain [15]. rFVII(R79Q) mutant protein bound normally to the EGF1 conformation‐sensitive monoclonal antibody 231‐7 but rFVII(N57D) mutant protein did not [15].…”

“…Human FVII cDNA was subcloned in the Eco RI– Hin dIII site of the expression vector pCMV5 as described [15]. Cloning vector pUC19 DNA was obtained from New England Biolabs (Beverly, MA, USA).…”

“…Oligonucleotide site‐directed mutagenesis (Clontech, Palo Alto, CA, USA) was performed on the FVII EGF1 domain in the vector pUC19 as previously described [15]. The mutagenic primers employed were S53(5′‐AGT‐3′)→N (5′‐GTGTGCCTCAA AC CCATGCCAGAATG‐3′), K62(5′‐AAG‐3′)→E (5′‐GGGCTCCTGC G AGGACCAGCTC‐3′), P74(5′‐CCT‐3′)→A (5′‐GCTTCTGCCTC G CTGCCTTCGAG‐3′), A75(5′‐GCC‐3′)→D (5′‐CTGCCTCCCTG A CTTCGAGGGC‐3′), and T83(5′‐ACG‐3′)→K (5′‐GCCGGAAGTGTGAGA AA CACAAGGATGACC‐3′).…”

“…A different approach resulted in the description of inhibitory peptides to exosites in the heavy chain of human FVII [12,13]. To date, studies of both natural [14–16] and site‐directed mutants of FVII [17] have generally described FVII mutant proteins with decreased affinity for TF with one exception being the M156Q mutation [18]. The impetus for this work was an early observation of Janson et al [19] who described a fourfold increased clotting activity of rabbit‐plasma FVII vs. human‐plasma FVII, on incubation with human TF.…”