Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 142-148
Factor VII Deficiency Caused by a Structural Variant N57D of the
First Epidermal Growth Factor Domain
By
Blair J.N. Leonard,
Qi Chen,
Morris A. Blajchman,
Frederick A. Ofosu,
Sampath Sridhara,
Daniel Yang, and
Bryan J. Clarke
From the Departments of Pathology, Biochemistry, and Biomedical
Sciences, McMaster University; and the Canadian Red Cross Society,
Hamilton, Ontario, Canada.
 |
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.
| |
INTRODUCTION |
HUMAN FACTOR VII (FVII) is a 406-amino
acid, 50-kD plasma protein that is essential for the initiation of the
extrinsic pathway of blood coagulation. The FVII gene consists of nine
exons spanning 12.8 kb of DNA in the q34 region of chromosome
13.2,3 Synthesized in hepatocytes, FVII circulates in
normal human plasma at levels of 4 to 12 nmol/L, approximately 98% of
which is in the zymogen form.4 Blood clotting is initiated
when FVII binds in a calcium-dependent reaction to its cofactor, the
transmembrane protein tissue factor (TF). This interaction occurs on
exposure of plasma FVII to either subendothelial cells, which
constitutively express TF,5-7 or to monocytes and
endothelial cells, which transiently express TF in response to various
stimuli, eg, bacterial endotoxin.8
Several epitopes of FVII important to the interaction between FVII and
TF have been previously identified by biochemical
analysis.9-13 Crystallization of the activated FVII
(FVIIa)/TF complex has extended these observations and directly
identified three main contact points between FVIIa and
TF.14 One contact point exists within the
-carboxyglutamic acid (GLA) domain of FVIIa, a second is in the
first epidermal growth factor domain (EGF-1) module of FVIIa, and the
last spans both the second epidermal growth factor domain (EGF-2)
module and the catalytic domain of FVIIa. Initially, both EGF modules
of FVII were identified as being equally important for TF
binding.15 Subsequent studies using a monoclonal antibody
(MoAb) specific for the FVII EGF-1 domain16 and analysis of
the EGF-1 mutant R79Q11,17-19 have provided strong evidence
for a critical role of the FVII EGF-1 module in TF binding,
observations which have been corroborated by the FVIIa/TF
crystallization data.14
In this report we have established the existence of a naturally
occurring novel mutation (N57D) of the FVII EGF-1
domain1,20 and analyzed the structure and function of
recombinant FVII (rFVII; N57D) protein using a variety of biological
assays in combination with molecular modeling techniques.
| |
MATERIALS AND METHODS |
Materials.
FVII cDNA in the vector pCMV5 was the generous gift of Dr Katherine
High (Children's Hospital, Philadelphia, PA). COS-1 (ATCC CRL 1650)
and 293 cells (ATCC CRL 1573) were obtained from the American Type
Culture Collection, Rockville, MD. Rabbit anti-human FVII polyclonal
antibody was purchased from Diagnostica Stago (Wellmark Diagnostics,
Guelph, Ontario, Canada). Human FVII EGF-1 specific murine MoAb 231-7
and FVII-depleted plasma were prepared as previously
described.16 The FVII-specific MoAb E.A.8.1 was the
generous gift of Dr George Broze (Jewish Hospital of St Louis, St
Louis, MO). 
-Actin cDNA probe for Northern hybridizations was the
kind gift of Dr Carl Richards (McMaster University). Recombinant TF
apoprotein was the kind gift of Dr Robert Kelley (Genentech, San
Francisco, CA) and was relipidated with
phosphatidylcholine-phosphatidylserine vesicles as previously
described.17,21 Immulon II 96-well polystyrene microtitre
plates were purchased from Dynatech Laboratories Inc (Chantilly, VA).
Purified human FVII was obtained from Enzyme Research Laboratories Inc
(Southbend, IN). Factor Xa chromogenic substrate S-2222 was obtained
from Helena Laboratories (Mississauga, Ontario). Goat anti-rabbit IgG
conjugated to alkaline phosphatase was obtained from Jackson
Laboratories (West Grove, PA). Tween 20, bovine serum albumin fraction
V, and disodium-p-nitrophenyl phosphate were purchased from Sigma
Chemical Company (St Louis, MO). All other chemical reagents were of
the highest quality available.
DNA analysis.
After purification of genomic DNA from peripheral blood mononuclear
cells, each of the nine exons of the FVII gene was isolated using
exon-specific EcoRI-containing oligonucleotide primers and the
polymerase chain reaction (PCR) technique essentially as previously
described.11 Products of the PCR reactions were purified
after agarose gel electrophoresis and subcloned into the EcoRI
site of the vector pGEM3Z (Promega, Madison, WI). DNA sequence analysis
of multiple cloned samples from each exon was performed on both strands
using a commercial version of the Sanger dideoxy technique
(Pharmacia-Biotec, Uppsala, Sweden).
Site-directed mutagenesis.
Oligonucleotide site-directed mutagenesis (Clontech, Palo Alto, CA) was
performed on FVII cDNA in the vector pGEM7Z (Promega) to create rFVII
(N57D) cDNA as per the method of Deng and Nickoloff.22 The
selection primer used was the 26 mer
5
-GAATTGGGCCCGTCGACGCATGCTCC-3 converting an
AatII site in the polylinker region to an
Sal I site. The mutagenic primer was the 27 mer
5-CAAGTCCATGCCAGGATGGGGGCTCCT-3 converting the A at the
FVII cDNA nucleotide position 330 to G. The cDNA was then subcloned
into the EcoRI-HindIII site of the mammalian expression
vector pCMV5.23 rFVII (R79Q) cDNA was prepared as
previously described.11
Cell culture and transfection.
Wild-type rFVII (WT), rFVII (R79Q), and rFVII (N57D) cDNA in the vector
pCMV5 were transfected into either 293 or COS-1 cells by
liposome-mediated transfer using Lipofectin reagent (GIBCO-BRL,
Gaithersberg, MD) as previously described.24 Both 293 and
COS-1 cells were routinely maintained in an equal mixture of
Dulbecco's Modified Eagles-Ham's F12 tissue culture media containing
10% fetal calf serum and 100 ng/mL vitamin K. Cell culture conditioned
media were collected for analysis 72 hours posttransfection.
FVII antigen determination.
Total rFVII antigen levels were determined in conditioned media by
solid-phase immunoassay using a commercially available enzyme-linked
immunosorbent assay (ELISA) from Diagnostica Stago (Wellmark
Diagnostics). Briefly, the ELISA incorporated rabbit anti-human FVII
Fab fragments as the trapping antibody and rabbit anti-human FVII
conjugated to horseradish peroxidase as the detecting antibody. Serial
dilutions of purified human FVII in tissue culture media were used to
generate a standard curve for relative FVII antigen determination in
the recombinant samples.
FVII-TF binding assay.
rFVII proteins secreted by transfected 293 and COS-1 cells were assayed
for binding to TF essentially as described by Sridhara et
al.17 Briefly, relipidated, recombinant TF was coated onto
96-well microtiter plates, nonspecific binding blocked, and the wells
overlaid with conditioned media containing FVII protein. Rabbit
anti-human FVII IgG conjugated to biotin was used as the primary
antibody, followed by streptavidin-alkaline phosphatase as the
secondary detecting component. Serial dilutions of purified human FVII
in tissue culture media were used to generate a standard curve.
A405nm was directly proportional to FVII bound to
immobilized TF in the range 1 to 20 ng FVII/mL.17 All rFVII
test samples were diluted in pCMV5 mock-transfected conditioned media
to maintain total protein levels and uniformity among samples.
FVII-MoAb binding assay.
rFVII proteins secreted by transfected 293 cells were assayed for
binding to purified IgG of FVII-specific murine MoAb according to the
method of Ofosu et al.25
Amidolytic/prothrombin time assay.
Both the amidolytic26 and procoagulant activity of rFVII in
conditioned tissue culture media from transfected 293 and COS-1
cells were assayed as previously described.18
Northern blot analysis.
Total RNA was isolated from 293 cells transfected with FVII cDNA in
pCMV5 using a commercial RNA isolation kit (Qiagen, Chatsworth, CA).
Total RNA was electrophoresed in formaldehyde-agarose gels and
transferred to a nylon membrane (Zetaprobe 30; Biorad, Mississauga,
Ontario, Canada) as described by the manufacturer. Radioactive
32P-
-dATP-labeled probes used for the quantitation of
FVII mRNA were synthesized by the random primer27 method
(GIBCO-BRL) using either gel-purified FVII cDNA insert or human
-actin cDNA insert as templates for DNA synthesis.
Molecular modeling analysis.
Molecular modeling of the EGF-1 domain of FVII was accomplished using
Insight II (version 2.3) and Homology (version 2.3)
software (Biosym/MSI, San Diego, CA) on an SGI INDIGO 2 workstation
(SiliconGraphics, Mississanga, Ontario, Canada). The x-ray
crystallographic coordinates of the FIX EGF-1 domain28 and
the FVIIa-sTF complex14 used to generate the FVII models
were graciously provided by Dr David Stuart and Dr David Banner,
respectively.
Other methods.
rFVII was immunoprecipitated from the cell culture conditioned media
using rabbit anti-human FVII polyclonal antibody and protein
A-sepharose according to the method of Harlow and Lane.29
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
was performed as previously described.30
| |
RESULTS |
FVII antigen levels and procoagulant activity of (N57D) kindred.
To confirm the status of the extrinsic pathway of coagulation in the
affected kindred,1,20 both the FVII antigen concentration
and procoagulant activity assays were conducted by two separate
laboratories using fresh, unfrozen plasma samples from each family
member (Table 1). Normal pooled plasma
control FVII antigen levels are defined as 100% (450 ng/mL), with a
procoagulant value of 1 U/mL. The plasma of the propositus
(III3, Table 1), the most severely affected clinically, had
a mean FVII antigen level of 62% and a mean procoagulant activity of
0.35 U/mL. Individual II2, a nonaffected family member,
exhibited a mean FVII antigen level of 110% and procoagulant activity
of 1.56 U/mL. Individual III5, an affected family member
(Fig 1), was unavailable for repeat FVII
antigen and activity determinations. Mean FVII antigen levels in the
four affected family members investigated were 84% and 85% as
determined by each lab independently, values that are within the normal
range of 60% to 140%.31 Similarly, the mean FVII
procoagulant activity in plasmas from the four affected family members
were determined to be 0.58 and 0.59 U/mL by each laboratory,
respectively. Antigen to procoagulant activity ratio (Ag/C), a measure
of procoagulant activity normalized to 100% FVII antigen
concentration, was 0.56 for the propositus and 0.68 for the affected
family members collectively. Thus, we initially concluded that this
kindred was affected by a type 2 FVII deficiency.20
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| Fig 1.
Electrophoresis of Fok I-digested PCR-amplified
exon 4 DNA samples from the FVII (N57D) kindred. Lanes 1 to 7 are
radiolabeled, PCR-amplified DNA samples from a control and individuals
II2, II1, III5, III2,
III3, and IV3, respectively (see Table 1). Lane
M contained radiolabeled DNA molecular weight markers of 220 bp, 154
bp, and 75 bp (top to bottom) derived from a HinfI digest of
pBR322.
|
|
Genetic basis of FVII deficiency.
DNA sequencing of multiple samples from each exon of the propositus
identified a single base pair heterozygous substitution of adenosine
for guanine at nucleotide 5989 in exon 4 of the FVII gene. This
mutation predicted the amino acid change N57D in the EGF-1 domain of
the FVII protein and the creation of a second Fok I restriction
endonuclease site in the 264 bp PCR product of the mutant allele of
exon 4. For determination of the existence of the second Fok I
restriction endonuclease site, we amplified exon 4 of each family
member using a standard PCR reaction mix11 with added 10
µCi 32P--dATP. After digestion with
Fok I, the radiolabeled PCR products were separated on a DNA
sequencing gel containing 6% polyacrylamide/8mol/L urea
and the dried gel was analyzed by autoradiography. As shown in Fig 1
(lanes 1 and 2) exon 4 DNA from a normal control and the unaffected
family member II2 had one Fok I restriction site
yielding the predicted bands at 186 bp and 78 bp. Lanes 3 through 7 in
Fig 1 confirmed that the five affected family members, including the
propositus (III3), were heterozygous for the A to G
mutation and therefore exhibited bands of 186 bp and 78 bp from the
normal allele plus two additional bands at 95 bp and 91 bp, a result of
the new Fok I restriction site in the 186-bp DNA fragment of
the mutant allele.
Expression of rFVII (N57D) in mammalian cells.
In view of the technical difficulties expected in the purification of
FVII (N57D) from the plasma of family members, we chose to express and
analyze rFVII (N57D). The recombinant cDNA encoding FVII (N57D) was
created by site-directed mutagenesis. After confirmation of the
fidelity of the entire FVII (N57D) cDNA by DNA sequence analysis, it
was subcloned into the mammalian expression vector pCMV5 and
transfected into both COS and 293 cells. SDS-PAGE and autoradiographic
analysis of 35S-methionine-labeled proteins
immunoprecipitated from 293 cell conditioned media with a FVII-specific
polyclonal antibody showed a specific 50-kD band in both the rFVII
(N57D) and rFVII (WT) samples (Fig 2).Three days after transfection into both COS and 293 cells, total FVII
antigen in the conditioned media was quantitated by an ELISA using a
polyclonal FVII-specific antibody (Fig 3).In cultures containing equal numbers (5 × 105) of 293
cells, rFVII (WT) antigen levels routinely (n = 5) exceeded 30 ng/mL of
conditioned media, whereas rFVII (N57D) antigen levels varied from 1.5
to 10 ng/mL of FVII protein. This latter result is not a general
characteristic of mutants of the FVII EGF-1 domain as the previously
described variant rFVII (R79Q)11,18 was secreted at levels
equivalent to that of rFVII (WT). Levels of rFVII (N57D) secreted by
transfected COS cells (n = 2) were similar to those obtained from 293
cells, with rFVII (N57D) protein concentration being approximately 20%
of rFVII (WT). To eliminate the possibility of vector bias, rFVII (WT)
cDNA and rFVII (N57D) cDNA were excised from their respective pCMV5
vectors by EcoRI-HindIII restriction digestion, and the
gel purified cDNA inserts were resubcloned into the opposite pCMV5 host
vector and transfected into 293 cells with no change in secretion
levels of either rFVII (WT) or rFVII (N57D) protein. After lysis of the
293 cells by sonication the intracellular concentration of rFVII (N57D)
protein (4.1 ± 0.1 ng per 35-mm plate) was found to be comparable
to the concentration of rFVII (WT) protein (6.5 ± 0.1 ng per 35-mm
plate) in an equivalent cell number, indicating that sequestration of
rFVII (N57D) protein was not occurring within the secretory pathway.
Lastly, to eliminate the possibility that low rFVII (N57D) antigen
expression was caused by poor efficiency of either transfection or
transcription of the rFVII (N57D) cDNA, Northern blot analysis of total
RNA from 293 cells transfected with either rFVII (N57D) cDNA or rFVII
(WT) cDNA was performed. Repeated experiments (n = 3, data not shown)
indicated that FVII mRNA expression was equivalent in 293 cells
transfected with either rFVII (WT) or rFVII (N57D) cDNA when FVII mRNA
levels in the two cell populations were normalized to the concentration
of the constitutively expressed -actin gene.
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| Fig 2.
PAGE analysis of radiolabeled, immunoprecipitated rFVII
(N57D) protein. Lanes 1 to 3 contain immunoprecipitated protein from
293 cells expressing FVII (WT), mock transfected control, and FVII
(N57D), respectively. Protein molecular weight markers ranging from 106
kD to 18.5 kD are indicated on the right.
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| Fig 3.
Quantitation of rFVII proteins after transient expression
in mammalian cells. ( ) FVII antigen concentration in conditioned
media from transfected 293 cells; ( ) FVII antigen expressed by COS
cells. FVII (R79Q) was expressed in 293 cells only. Error bars
represent the standard error of the mean.
|
|
TF binding of rFVII (N57D).
Figure 4 shows the TF binding of equal
quantities of rFVII (WT), FVII (N57D), and FVII (R79Q) proteins derived
from 293 cell conditioned media. As previously
reported11,18 rFVII (R79Q) exhibited reduced binding to TF
as compared with rFVII (WT) protein. rFVII (N57D) protein also showed
reduced ability to bind TF compared with that seen with rFVII (WT). The
ability of rFVII (N57D) to bind TF was also significantly reduced as
compared with rFVII (WT) after expression in COS cells (data not
shown). Mixed samples of rFVII (WT) and rFVII (N57D) in equimolar
concentrations in 293 cell conditioned media showed an approximate 50%
decrease in TF binding (data not shown), indicating that there was no
apparent interaction between the two FVII molecular populations.
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| Fig 4.
TF binding of rFVII proteins. TF binding of FVII (WT),
FVII (N57D), and FVII (R79Q) at a FVII protein concentration of 3 ng/mL
in conditioned media from transfected 293 cells. Error bars represent
the standard error of the mean.
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|
Prothrombin time/amidolytic activity of rFVII (N57D).
Figure 5 illustrates the procoagulant and
amidolytic activities of rFVII (WT), rFVII (N57D), and rFVII (R79Q)
proteins derived from 293 cell conditioned media. The rFVII (WT)
protein synthesized by transfected 293 cells exhibited a procoagulant
activity similar to plasma-derived FVII. The procoagulant activity of
rFVII (R79Q) was approximately 60% of rFVII (WT), whereas rFVII (N57D)
protein had no procoagulant activity. Similarly, the activity of 60
pmol/L rFVII (WT) and rFVII (R79Q) proteins in the coupled,
TF-dependent S-2222 amidolytic assay for FVII26 was
determined to be 0.027 and 0.006 A405nm U/min,
respectively. The rFVII (N57D) protein exhibited no amidolytic
activity.
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| Fig 5.
Procoagulant and amidolytic activity of rFVII proteins.
Prothrombin time and amidolytic activity of the rFVII proteins were
measured at a rFVII concentration of 3 ng/mL in tissue culture media.
The dashed line indicates the specific activity of plasma-derived FVII
(2,222 U/mg). Error bars represent the standard error of the mean.
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Antibody recognition of rFVII (N57D).
Figure 6 illustrates the binding of equal
concentrations of rFVII(WT), rFVII (N57D), and rFVII (R79Q) to two
human FVII-specific antibodies in ELISA assay. All three rFVII
molecular populations were bound equally by a FVII-specific polyclonal
antibody previously shown to react with both native and denatured FVII
(Fig 6A). In contrast, the FVII EGF-1 conformation-specific MoAb
231-716 did not bind rFVII (N57D) but exhibited normal
binding to both rFVII (WT) and another mutant of the EGF-1 domain rFVII
(R79Q) (Fig 6B). The binding of MoAb 231-7 is dependent on the
integrity of the disulphide bonds and overall conformation of residues
51-88 of the first EGF module of FVII (unpublished
data).16,25 As an additional test of the conformation of
rFVII(N57D), we determined that the FVII light chain-specific MoAb
E.A.8.1 isolated by Broze et al32 did not bind to
rFVII(N57D), whereas rFVII(WT) was bound normally (data not shown).
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| Fig 6.
Binding of rFVII proteins to polyclonal and monoclonal
FVII-specific antibodies. Antibody binding of FVII (WT), FVII (N57D),
and FVII (R79Q) in 293 cell conditioned media to a rabbit anti-human
FVII-specific polyclonal antibody (A) and the human FVII-specific MoAb
231-7 (B). Error bars represent the standard error of the mean.
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| |
DISCUSSION |
Reexamination of the plasma FVII antigen and procoagulant activities in
a kindred with combined FVII and FXI coagulation defects confirmed that
FVII antigen concentration in affected family members was low but
remained in the normal range, whereas FVII coagulant activity was
abnormally low (Table 1).1 Detailed molecular genetic
analyses of FVII DNA from the propositus (individual III3)
showed that she did not encode the R353Q polymorphism known to affect
FVII antigen levels in plasma33 but revealed that she, as
well as four other affected family members, were heterozygous for a G
A substitution at nucleotide 5989 in exon 4 of the FVII gene
(Fig 1). This mutation predicted the amino acid change N57D in the
EGF-1 domain of the FVII protein, but the mechanism whereby the N57D
substitution might affect FVII coagulant activity was not readily
apparent because initial studies of TF binding by kindred plasma FVII
appeared to be normal.1,20 The combined clinical and
molecular genetic data suggested that plasma FVII protein in affected
individuals was a mixture of wild-type and mutant FVII molecules.
Because functional analyses of the FVII in kindred plasma samples would
be difficult to interpret, homogeneous FVII (N57D) protein was
generated by recombinant means.
Transient expression of rFVII (N57D) in both COS and 293 cells showed
reduced synthesis of rFVII (N57D) protein versus rFVII (WT) protein
(Figs 2 and 3). Northern blot analysis indicated that the rFVII (N57D)
cDNA was transfected and transcribed at levels equivalent to rFVII (WT)
cDNA. In addition, the intracellular levels of rFVII (N57D) protein
were low and similar to rFVII (WT) protein, suggesting that the mutant
protein was not being sequestered in the protein secretory pathway as
has been reported for the FVII (T359M) variant.34 Inclusion
of a protease inhibitor in the culture medium failed to increase rFVII
(N57D) protein levels in the media, providing evidence that the mutant
protein was not being proteolytically digested by trypsin-like enzymes
outside the cell. Collectively, the evidence suggested that the
majority of the rFVII (N57D) protein synthesized was being degraded
before secretion into the extracellular space, a result also observed
for the FVII heavy chain variant FVII Mie.35 Secreted rFVII
(N57D) was dysfunctional in both TF binding and enzymatic activity
(Figs 4 and 5), a result in accord with the approximately 10%
coagulant activity of the analogous mutant factor IX N58K36
(mutation UK373 in the hemophilia B database,
http://www.ebi.ac.uk/pub/database/ haemb/). Thus, both rFVII (N57D) and
another EGF-1 domain mutant (R79Q) exhibit sharply decreased binding to
TF, a result consistent with previous evidence that the FVII EGF-1
module is essential for TF binding.11,16-18
Both rFVII (WT) and rFVII (R79Q) bound the conformation-dependent,
EGF-1 domain-specific MoAb 231-7 in a dose-dependent manner (Fig 6B).
In contrast rFVII (N57D) did not bind to either of the EGF-1-specific
MoAbs tested, suggesting that the proper folding of the rFVII (N57D)
EGF-1 module was compromised. In most cases, proteins that fail to fold
correctly are not released from the endoplasmic reticulum
(ER)37-39 and are cleared rapidly from the ER by way of
lysosomal or ER-mediated protein degradation pathways. This
interpretation is compatible with the reduced secretion levels observed
with rFVII (N57D).
Molecular modeling of the effect of the N57D substitution on the
structure of the EGF-1 domain of FVII was performed initially utilizing
the x-ray crystallographic coordinates of the FIX EGF-128
module as a template. The high structural homology between the EGF
domains of the blood coagulation proteins40-43 combined
with the greater than 60% sequence homology between the FIX and FVII
EGF-1 modules make the FIX EGF-1 module an excellent prototype for the
FVII model. This initial model predicted that the nitrogen of the
secondary amino group of residue N57 would be .298 nm from
the carbonyl atom of residue C81, a distance ideal for intramolecular
hydrogen bonding (data not shown). The prediction was confirmed by an
updated model of the FVII EGF-1 domain (Fig
7) constructed using the x-ray
crystallographic coordinates of FVIIa.14 In this model the
atomic distance between the side chains of N57 and C81 was .283 nm (Fig
7). Notably, the importance of an interface between the amino and
carboxyl regions of human EGF for its three dimensional structure has
recently been established.44 Furthermore, the integrity of
this interface is required for receptor binding by both human EGF and
transforming growth factor .44 Thus, a nuclear magnetic
resonance model of the structure of human EGF is compatible with the
concept that an interface between the amino and carboxyl regions of
EGF-1, mediated by a hydrogen bond between N57-C81, is of structural
and functional significance in human coagulation FVII.
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| Fig 7.
Molecular modeling of the human FVII EGF-1 module. The
figure depicts the FVII EGF-1 loop containing N57 and shows the .283-nm
distance from the side chain nitrogen (blue) of N57 to the main chain
carbonyl oxygen (red) of C81. Carbon atoms are colored green and
sulphur atoms are colored yellow. The main chain is shown as a solid
orange ribbon.
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|
In conclusion, we have shown that there are three important
consequences of the N57D mutation in the FVII EGF-1 domain. Firstly,
there is a marked decrease in secretion of the FVII (N57D) protein.
Secondly, the FVII (N57D) protein that is secreted possesses no
procoagulant activity and does not bind either TF or a
conformation-dependant, EGF-1-specific MoAb. Thirdly, molecular
modeling data support the hypothesis that the effects of this mutation
are a result of misfolding of the EGF-1 module of FVII caused by the
loss of an important intramolecular hydrogen bond. Although recent
crystallization and molecular modeling of the FVIIa/sTF complex by
Banner et al14 has indicated that N57 is not located at the
FVIIa/sTF binding interface, the critical role of a structurally intact
EGF-1 domain for FVII function is confirmed11,16-18 and
extended by this report.
| |
FOOTNOTES |
Submitted February 28, 1997;
accepted August 25, 1997.
Address reprint requests to Bryan J. Clarke, PhD,
Department of Pathology, HSC 4N65, McMaster University, 1200 Main St
West, Hamilton, Ontario, L8N 3Z5, Canada.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Robert Kelley, Genentech Inc, for generously
providing recombinant human TF apoprotein, Dr David Stuart for the
x-ray crystallography coordinates of the isolated factor IX EGF-1
domain, and Dr David Banner for both the x-ray crystallography
coordinates of factor VII and helpful discussions. The authors also
gratefully acknowledge the Heart and Stroke Foundation of Ontario
(B.J.C.) and the Canadian Red Cross Society (F.A.O. and M.A.B.) for
their support.
| |
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Abstract
Introduction
Abstract
