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Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1237-1244
Characterization of Two Naturally Occurring Mutations in the Second
Epidermal Growth Factor-Like Domain of Factor VII
By
Mathilde Hunault,
Arnaldo A. Arbini,
Josephine A. Carew,
Flora Peyvandi, and
Kenneth A. Bauer
From the Hematology-Oncology Section, Department of Medicine,
Brockton-West Roxbury Department of Veterans Affairs Medical Center,
West Roxbury, MA; Beth Israel Deaconess Medical Center, Harvard Medical
School, Boston, MA; A. Bianchi Bonomi Hemophilia and Thrombosis Center,
Department of Internal Medicine, IRCCS Ospedale Maggiore, Maggiore,
Italy; and the University of Milan, Milan, Italy
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ABSTRACT |
We investigated the mechanisms responsible for severe factor VII
(FVII) deficiency in homozygous Italian patients with either Gly97Cys or Gln100Arg mutations in the second
epidermal growth factor domain of FVII. Transient expression of
complementary DNA coding for the mutations in COS-1 cells showed
impaired secretion of the mutant molecules. Using stably transfected
Chinese hamster ovary (CHO) cells, we performed pulse-chase labeling
studies, immunohistochemistry, and experiments with inhibitors of
protein degradation, showing that FVII-Cys97 did not
accumulate intracellularly but was degraded in a pre-Golgi, nonlysosomal compartment by a cysteine protease. In stably transfected CHO cells expressing FVII-Arg100, the level of
intracellular FVII was not increased by several inhibitors of protein
degradation, but FVII-Arg100 was retained in the
endoplasmic reticulum for a longer period of time than wild-type FVII.
FVII-Arg100 had a lower apparent molecular weight than did
wild-type FVII under nondenaturing conditions, which is attributable to
misfolding due to abnormal disulfide bond formation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN FACTOR VII (FVII) is a vitamin
K-dependent glycoprotein that normally circulates in plasma at a
concentration of 0.5 µg/mL.1 FVII in association with
tissue factor initiates blood coagulation by activating factor IX and
factor X.2,3 The mature FVII molecule is a single chain of
406 amino acids and is comprised of several discrete domains including
the Gla domain, two epidermal growth factor (EGF)-like domains and a
large catalytic domain.4 It undergoes several
post-translational modifications before its secretion by the liver
including  -carboxylation of 10 glutamic acid residues in the Gla
domain, N-glycosylation of residues Asn145 and
Asn322,5 and O-glycosylation of residues
Ser52 and Ser60 in the first EGF
domain.6
Hereditary FVII deficiency is a rare autosomal recessive bleeding
disorder.7,8 Patients with FVII deficiency have been classified with respect to the plasma level of FVII antigen (VII:Ag) or
crossreacting material (CRM) as either CRM (low or
absent antigen), CRMR (reduced antigen), or
CRM+ (normal antigen). More than 30 different naturally
occurring mutations in the FVII gene have been reported.9
Most are point mutations that alter FVII function, but others interfere
with FVII biosynthesis. In this paper, we investigated the mechanisms responsible for FVII deficiency in two homozygous Italian patients who
were CRM and CRMR as a result of
mutations in the molecule's second EGF domain.
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MATERIALS AND METHODS |
Collection and processing of blood samples.
Blood was collected by atraumatic venipuncture into plastic tubes
containing 1/10th volume 0.129 mol/L buffered trisodium citrate. Plasma
was obtained by centrifugation at 2,500g for 15 minutes at
4°C, transferred into plastic tubes, and stored along with the
cellular elements at  80°C until use.
FVII assays.
FVII coagulant activity (VII:C) and VII:Ag were measured by one-stage
clotting assay using recombinant human tissue factor (RecombiPlastin,
Ortho Diagnostic Systems, Raritan, NJ) and an enzyme-linked
immunoabsorbent assay (ELISA) (American Bioproducts Co, Parsippany,
NJ), respectively. A normal pool was constructed by mixing equal
volumes of plasma from 30 healthy control subjects.
DNA isolation and in vitro amplification using polymerase chain
reaction (PCR).
DNA was purified by standard techniques from leukocyte nuclei obtained
from whole blood.10 Oligonucleotides and PCR conditions used to amplify the entire coding sequence of the FVII gene have been
described in detail.11 PCR amplifications12
were performed using a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk,
CT). PCR products were generated in 20 µL reaction mixtures that
contained 200 ng of genomic DNA, 0.4U of Taq DNA polymerase (Perkin
Elmer Cetus), oligonucleotide primers at a concentration of 0.5 µmol/L each, dNTPs at a concentration of 100 µmol/L each,1 mmol/L
MgCl2, 10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, and 0.01 mg/mL of autoclaved gelatin.
Cloning and sequencing of PCR fragments.
Amplified PCR fragments were purified and ligated into PT7Blue
T-vectors (Novagen, Madison, WI). Cloned inserts were sequenced by the
dideoxy chain termination method on an Applied Biosystems 373A DNA
Sequencer (Foster City, CA). All sequence alterations were
confirmed in at least two independent clones. Sequence analyses were
performed using the GCG Sequence Analysis Software Package (Genetics
Computer Group, Inc, Madison, WI) from the Molecular Biology Computer
Research Resource (Boston University, Boston, MA).
Restriction enzyme analysis.
Restriction enzyme digestion of PCR fragments was used to detect
mutations that introduced or abolished a restriction site. Ten µL of
PCR-amplified product were digested with 0.5 U of enzyme in a final
volume of 20 µL for 16 hours. Restriction fragments were subjected to
electrophoresis in nondenaturing 8% (wt/vol) polyacrylamide gels.
After completion of the electrophoretic procedure, gels were stained in
0.5 µg/mL ethidium bromide for 5 minutes and photographed under
ultraviolet (UV) transillumination.
Antibodies.
The production and purification of Mab1476,13 a murine
monoclonal antibody (MoAb), which recognizes an epitope in the
aminoterminal region of FVII, has been described.14 This
antibody was used to detect FVII in pulse-chase experiments and for immunohistochemistry.
Construction of expression vectors and site-directed mutagenesis.
A 2.4 Kb complete human FVII complementary DNA (cDNA)4 with
EcoRI/BamHI linkers at each end was provided by Dr Earl
W. Davie (Seattle, WA). This cDNA was cloned into the EcoRI
site of the PT7-BlueT vector to obtain the plasmid
PT7EcoRIFVIIwtEcoRI and
subsequently modified as previously reported to obtain
PT7Sal IFVIIwtEcoRI.14
The FVII cDNA was then isolated and cloned into pED-mtxr
provided by Dr Randal J. Kaufman15 to obtain the plasmid
pEDFVIIwt, a dicistronic messenger RNA mammalian expression vector
carrying the dihydrofolate reductase (DHFR) gene at the 3' open
reading frame. To investigate the influence of the Gly97Cys
and Gln100Arg substitutions on FVII levels,
pEDFVIICys97 and pEDFVIIArg100 were
obtained by site-directed mutagenesis of
PT7Sal IFVIIwtEcoRI using a
commercially available kit (Transformer Site-Directed Mutagenesis Kit,
Clontech, Palo Alto, CA). Oligonucleotides
(5'-GAACGGCTGCTGTGAGCAGTACTGCAGTGATCACACG-3') and
(5'-GAACGGCGGCTGTGAGCGGTACTGCAGTGATCACACG-3')
spanning nucleotides 7817 to 7853 of the human FVII cDNA were used to
introduce a G to T at position 7824 and an A to G at position 7834 (bold letters) coding for Gly97Cys or
Gln100Arg, respectively. These primers also introduced a
BclI restriction site (underlined), arising from a silent
GAC to GAT mutation at nucleotide 7847, to
facilitate screening for clones carrying the mutations. The mutant FVII
cDNA were then inserted into the pED expression vector to obtain
pEDFVIICys97 and pEDFVIIArg100.
Several restriction enzyme digests were performed to confirm that we
had produced vectors containing the mutations. A Sal I-EcoRI digest released a 2.4 kb fragment containing the
complete FVII cDNA and 1.2 kb of the gene's 3' untranslated
region, thereby confirming that the entire cDNA had been introduced
into the vector. A HindIII digest of the vector generated three
fragments of 955, 2735, and 4000 bp resulting from three sites in pED
and none in the FVII cDNA. EcoRI digestion linearized the 7.7 kb construct (5.3 kb pED + insert 2.4 kb cDNA) by cleavage at a single
site in pED. To confirm the presence of the mutations, we amplified from the construct a 893 bp fragment with the oligonucleotides 5'-CCCGGTCGACTCAACAGGCAGGGGCAGCACT-3'
(position 94 to 74) introducing a Sal I site (bold
letters) and 5'-CAGGCGGAGCAGCG-3' (position 10648-10661 in
exon 8). This PCR product coded for the first 248 amino acids of FVII
excluding exon 1b. Because the mutagenic primers introduced a Bcl
I site in addition to the mutation, a Bcl I digest of the
product gave two fragments of 695 and 198 bp for the FVIIwt construct,
whereas the FVIICys97 and FVIIArg100 constructs
yielded fragments of 458, 237, and 198 bp confirming that the mutations
had been introduced. Moreover, because the G to T transversion at
position 7824 resulting in Gly97Cys introduces a Bbv
I site, Bbv I digestion of the 893 bp fragment from
pEDFVIICys97 led to the generation of a 713 bp product that
was cleaved into pieces of 445 and 268 bp in the presence of the
mutation. As the A to G transversion at position 7834 resulting in
Gln100Arg introduces a BsrBI site, BsrBI
digestion of the 893 bp product from pEDFVIIArg 100 generates fragments of 447 and 446 bp fragments in the presence of the mutation.
Cell culture and transfection assays.
For transient transfection experiments, Monkey COS-1 cells (ATCC
CRL1650; American Type Culture Collection, Rockville, MD) were maintained in Dulbecco's Modified Eagle's Medium (DMEM)
supplemented with 10% (vol/vol) fetal bovine serum (FBS), 2 mmol/L
L-glutamine, 10 mmol/L HEPES pH 7.2, 100 units/mL of penicillin G, 100 µg/mL streptomycin, and 5 µg/mL of vitamin K1
(AquaMEPHYTON, Merck & Co Inc, West Point, PA) in a 5% CO2
atmosphere at 37°C. Twenty hours before transfection, COS-1 cells
were plated on 60 mm culture dishes at a density of 1 × 106 cells/dish. Five micrograms of the pEDFVII constructs
was transfected into cells by Lipofectamine (GIBCO-BRL, Gaithersburg,
MD) according to the manufacturers' instructions. After 16 hours, medium was changed, and 36 hours later, supernatants and cell
lysates were harvested and assayed for VII:C and VII:Ag. Results of
transient assays are expressed as the percentage of FVIIwt and
represent the mean ± SE.
To obtain stable cell lines expressing recombinant FVIIwt,
FVIICys97, and FVIIArg100, we used
DHFR-deficient Chinese ovary (CHO) cells (CHO-DUKX-B11)16 provided by Dr Barbara C. Furie (Boston, MA). These cells were grown in
alpha-modified essential medium (AMEM) supplemented with 10% FBS, 2 mmol/L L-glutamine, 10 mmol/L HEPES pH 7.2, 100 units/mL of penicillin
G, 100 µg/mL streptomycin, 5 µg/mL of vitamin K1, 10 µg/mL
adenosine, 10 µg/mL deoxy-adenosine, and 10 µg/mL thymidine. CHO-DUKX-B11 cells were plated on 100-mm culture dishes at a density of
3 × 106 cells/dish. Transfections were performed as
described above with 20 µg of plasmid and 45 µL of Lipofectamine.
Two days after transfection, cells were divided at a 1 to 8 ratio and
selected for DHFR expression using medium deficient in ribonucleosides
and deoxyribonucleosides. Twelve days after transfection, 24 colonies
were picked at random and isolated in 12-well (24 mm) plates. At day
20, when the cells achieved confluence, each well was split into two 35 mm dishes. Two days later, cell lysates of the wells were harvested and
assayed for VII:Ag. A single clone stably transfected with each
construct and expressing high levels of FVII was selected for further
experiments. The rates of intracellular FVII synthesis for the selected
clones expressing FVIIwt, FVIICys97, and
FVIIArg100 were 33.3 ± 2.1, 52.5 ± 5.8, and 47.7 ± 5.5 ng/mL/h (mean ± SEM, n = 9), respectively.
Metabolic labeling studies.
Nearly confluent 60 mm dishes of CHO cells stably expressing
recombinant FVII were used for pulse-chase experiments. Fresh media
with FBS was added 4 hours before cells were deprived of methionine for
45 minutes and labeled for 15 minutes with 0.4 mL of methionine-free
AMEM (GIBCO-BRL, Gaithersburg, MD) containing 110 µCi
Expre35S Protein Labeling Mix (~73%
L-[35S]methionine and ~22%
L-[35S]cysteine; DuPont NEN Research Products, Billerica,
MA) in a 5% CO2 atmosphere at 37°C. A chase was then
performed in 1 mL medium containing an excess of unlabeled L-methionine
(GIBCO-BRL, Gaithersburg, MD) for various time periods. At each time
point, medium was harvested and phenylmethyl sulfonyl fluoride (PMSF) added to a final concentration of 1 mmol/L. Cell extracts were prepared
in 350 µL ice-cold NP-40 lysis buffer (50 mmol/L NaCl, 50 mmol/L Tris
pH 8.0, 1% (wt/vol) NP-40) supplemented with 1 mmol/L PMSF. The cell
lysates were precleared overnight at 4°C with 50 µL of 20%
(vol/vol) fixed Staphylococcus aureus Cowan I (SAC) coupled
with a rabbit antimouse IgG (Sigma, St Louis, MO) in NP-40 lysis
buffer. Immunoprecipitation of FVII was accomplished by incubating
precleared cell lysates and conditioned media with 4 µg of MoAb
MC1476 for 2 hours at 4°C. The resulting immune complexes were
adsorbed with 30 µL of 20% (vol/vol) Protein A Sepharose FF (Sigma)
coupled 5:1 (vol/vol) with rabbit antimouse IgG antiserum in NP-40
lysis buffer. Pellets were washed four times in NP-40 lysis buffer and
resuspended either in buffer for further enzymatic digestion (see
below) or in polyacrylamide gel electrophoresis (PAGE) sample buffer
with or without reducing agents, and denatured by heating to 95°C
for 5 minutes. The immunoprecipitated proteins were resolved by sodium
dodecyl sulfate (SDS)-PAGE in 8% (wt/vol) gels, and analyzed by
fluorography on X-OMAT-AR film (Eastman-Kodak Co, Rochester, NY) after
treatment with En3Hance (DuPont NEN Research Products,
Billerica, MA). To quantitate the relative amount of FVII
immunoprecipitated at each time, the radioactivity incorporated into
bands containing FVII was analyzed with the Umax PowerlookII Imaging
Analyzer (Umax Data System Inc, Taiwan).
Immunohistochemistry.
CHO cell lines expressing FVII were grown overnight at 150,000 cells/glass coverslip (Baxter Scientific products, McGawpark, IL). The
coverslips were washed once in phosphate-buffered saline (PBS) and
fixed for 1 hour in 3% (vol/vol) paraformaldehyde (Fisher Scientific,
Pittsburgh, PA) in PBS. The cells were sequentially washed,
permeabilized for 3 minutes in 0.1% (vol/vol) Triton X-100 (Sigma),
and washed three more times. They were then incubated in 0.15%
(wt/vol) glycine containing 0.1% (wt/vol) bovine serum albumin (BSA)
for 15 minutes followed by anti-FVII Mab (4 µg in 0.5 mL of PBS
containing 1% BSA) for 30 minutes. The cells were again washed three
times and then incubated with fluorescein isothiocyanate (FITC)-labeled
goat-antimouse IgG (1/1000 in PBS containing 1% BSA) (Cappel, Durham,
NC) for 30 minutes. After further washing, the coverslips were mounted
on glass slides with Airvol (Air Products, Allentown, PA).
Immunofluorescence microscopy was performed on a Zeiss axioplan
fluorescence microscope at 630× magnification (Zeiss, Thornwood, NY).
Effect of protein degradation inhibitors on FVIIwt,
FVIICys97, and FVIIArg100 levels.
To study the effect of various protein degradation inhibitors on FVII
biosynthesis, confluent stably transfected CHO cells grown in 60 mm
dishes were incubated with media containing either lactacystin (10 µmol/L) (Calbiochem, La Jolla, CA), ammonium chloride (50 mmol/L),
leupeptin (100 µmol/L), N-acetyl-Leu-Leu-Norleucinal (50 µg/mL), or
brefeldin A (10 µg/mL) (Sigma) dissolved according to the
manufacturers' recommendations and used at previously published concentrations.17-21 Four hours later, cell lysates were
harvested and assayed for VII:Ag.
Analysis of N-linked glycosylation and sialation.
Immunoprecipitated proteins were incubated with 100 mU/mL
Endo- -N-acetylglucosaminidase H (Endo H) or 9.4 U/mL
Peptide-N4-(N-acetyl--glucosaminyl) asparagine amidase
(N-glycanase) for 18 hours at 37° C according to the
manufacturer's instructions. For sialation analysis,
immunoprecipitated proteins were incubated for 1 hour at 37°C with
4 U/mL neuraminidase in 1 mmol/L PMSF, 20 mmol/L Tris-maleate pH 6.0, 10 mmol/L calcium acetate, and 1.75% (vol/vol) NP-40. All enzymes were
from Genzyme (Cambridge, MA). The reactions were terminated by adding
5× PAGE sample buffer, and were analyzed by SDS-PAGE.
Informed consent.
Informed consent to perform research studies was obtained from the
patients. The study was approved by the Human Studies Committee of the
Brockton-West Roxbury VA Affairs Medical Center.
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RESULTS |
Patients and genomic studies.
We investigated the molecular defects in two Italian patients with FVII
deficiency. Patient 1 is a 28-year-old male with a mild hemorrhagic
diathesis manifested by epistaxis and excessive bleeding after dental
extractions.22 Patient 2 is a 53-year-old woman with severe
bleeding evidenced by recurrent hemarthrosis with chronic arthropathy
and an episode of cerebral hemorrhage.22 The levels of
VII:Ag and VII:C in the two patients were 2% and less than 1% of
normal and 12% and less than 1% of normal, respectively (Table 1).
For each patient, we subcloned and sequenced the entire coding region
and the exon/intron boundaries of the FVII gene as well as its
5'-flanking region. In patient 1, a G to T transition at position
7824 (GGC to TGC) and a C to T transition at
position 7880 (CAC to CAT) in exon 5 were
identified resulting in Gly97Cys and a neutral dimorphism
in the codon for His115, respectively. Two previously
described FVII polymorphisms known to influence FVII levels, a C to A
substitution at position 10976 (CGG to CAG) in exon
8 resulting in Arg353 Gln and the insertion of a
decanucleotide at position 323 in the 5'-flanking region
of the FVII gene, were also present. In patient 2, an A to G
transversion at position 7834 (CAG to CGG) in exon
5 resulting in Gln100Arg was identified. No alleles with
wild-type sequence were identified in either patient. To definitively
establish that the patients were homozygous, we tested for the presence
of the various sequence alterations using restriction enzyme analysis.
The G to T transversion at position 7824, resulting in
Gly97Cys, introduces a single Bbv I site. Digestion
of a 313 bp PCR product spanning exon 5 with Bbv I generates
fragments of 170 and 143 bp in the presence of the mutation. The A to G
transversion at position 7834, resulting in Gln100Arg,
introduces a BsrBI site and digestion with this enzyme
generates fragments of 147 and 166 bp in the presence of the mutation.
Msp I restriction analysis was used to identify the
Arg353Gln polymorphism,23 whereas the
decanucleotide insert at position 323 was assessed by
visualization of a 10 bp difference in fragment size after
EcoRI digestion of a PCR fragment spanning this
region.24 Based on the results of these restriction
analyses (data not shown), patient 1 was homozygous for
Gly97Cys and the two polymorphisms whereas patient 2 was
homozygous for only Gln100Arg (Table 1).
Transient transfection assays in COS-1 cells.
To investigate the influence of the Gly97Cys and
Gln100Arg substitutions on FVII biosynthesis, transient
transfections were performed in COS-1 cells using the dicistronic pED
vectors containing either wild-type or mutant FVII cDNAs. Assays of
VII:Ag in cell lysates showed that FVIICys97 and
FVIIArg100 were reduced to 38% and 54% of FVIIwt,
respectively, whereas the levels in the conditioned media were
decreased to 6.6% and 16.7% of FVIIwt, respectively (n=8). The small
amount of FVIICys97 released into the media was
functionally active as assessed by VII:C assay (6.1% ± 0.3% of
wt) because the level was similar to VII:Ag (6.6% ± 0.5%). In
contrast, the Gln100Arg mutation impaired the molecule's
function because the VII:C level in the media (2.8% ± 0.2%) was
considerably lower than VII:Ag (16.7% ± 2.1%)
(Table 2).
Expression studies in stably transfected cell lines.
Based on the results of the transient transfection experiments, it
appeared that the Gly97Cys and Gln100Arg
mutations led to impaired FVII biosynthesis. To study these defects,
pEDFVIIwt, pEDFVIICys97, and pEDFVIIArg100
were transfected into DHFR-deficient CHO cells to obtain stably transfected cell lines. It can be observed that the amounts of FVII in
cell lysates of the stable cell lines transfected with pEDFVIICys97 and pEDFVIIArg100 were actually
greater than FVIIwt. This is attributable to the selection of
high-level FVII producers. After a 15 minute pulse with
[35S] methionine, the recombinant FVIIwt in cell lysates
was maximal at 30 to 60 minutes and decreased as the protein was
secreted (Fig 1, top). FVIICys97 was synthesized at a rate
similar to FVIIwt with maximal accumulation at 60 minutes, but remained
in the cell for a longer period of time. Approximately, 40% of the
maximal amount of intracellular FVIICys97 was still present
at 240 minutes as compared with 12% for FVIIwt (Fig 1, top).
FVIIArg100 was retained intracellularly even longer with
maximal accumulation at 60 to 120 minutes, and 55% of the protein was
retained in the cell at 240 minutes. In the conditioned media, FVIIwt
was barely detectable at 30 minutes and then proceeded to accumulate
(Fig 1, bottom). The levels of FVIICys97 and
FVIIArg100 were much lower than that of FVIIwt at all time
points. At 240 minutes, the levels of FVIICys97 and
FVIIArg100 in the media were 30% and 16% of FVIIwt,
respectively (Fig 1, bottom).
Using immunohistochemical techniques to detect intracellular VII:Ag, we
found different patterns of localization for FVIIwt, FVIICys97, and FVIIArg100. Staining of FVIIwt
(Fig 2A and B) and FVIICys97 (Fig 2C) was mostly
perinuclear suggesting that these molecules were localized primarily in
the Golgi apparatus. In contrast, FVIIArg100 (Fig 2D)
staining was predominantly diffuse without perinuclear enhancement,
suggesting that it was retained for a longer duration of time in
the endoplasmic reticulum (ER) than the other recombinant proteins.
Whereas the acquisition of Endo H resistance is frequently employed to
monitor protein transit to the Golgi complex from the ER, the
radioactive bands that were observed for FVIICys97 and
FVIIArg100 in pulse-chase experiments were generally of low
intensity that precluded interpretation of these experiments (data not shown).
Effects of protein degradation inhibitors on FVII biosynthesis.
We next analyzed the effects of various inhibitors of protein
degradation on intracellular FVII levels in stably transfected cells
expressing FVIIwt, FVIICys97, and
FVIIArg100. Inhibitors of lysosomal degradation,
including NH4 Cl, which inactivates lysosomal enzymes by pH
modification, and leupeptin, which inhibits cathepsins B, D, H, and L,
did not increase intracellular FVII levels
(Table 3) suggesting that
FVIICys97 and FVIIArg100 were not degraded in
the lysosome. Lactacystin, a potent specific inhibitor of proteasome
degradation, also did not increase FVII levels (Table 3). However,
ALLN, a neutral inhibitor of the cysteine proteases, calpain, and
cathepsin D, induced a significant increase in FVIICys97
levels (147% with ALLN versus 100% without ALLN, P = .0004)
without altering the levels of FVIIwt or FVIIArg100. This
data therefore suggested that FVIICys97 is degraded
intracellularly.
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Table 3.
Effect of Various Inhibitors of Protein Degradation and
Brefeldin A on Intracellular Levels of FVIIwt, FVIICys97,
and FVIIArg100
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To localize the site of degradation, we investigated the effect of
brefeldin A on intracellular levels of FVII in the three cell lines.
Brefeldin A blocks protein transport from the ER to the Golgi complex
and causes translocation of Golgi components back to the ER. After
incubation with brefeldin A, FVIIwt and FVIIArg100
increased significantly within the cells to 190% and 151% of control
levels, respectively, consistent with normal translocation of these two
proteins from the ER to the Golgi in the absence of the drug. In
contrast, the intracellular level of FVIICys97 increased
only modestly in the presence of brefeldin A to 125% of control
levels, thereby suggesting that the degradation of this molecule
occurred primarily in a pre-Golgi compartment. Analysis of
immunoprecipitates obtained after a 45 minute pulse with 110 µCi
[35S] methionine and 2 hours of chase in the presence and
absence of 10 µg/mL of brefeldin A confirmed that FVIIwt and
FVIIArg100 levels in cell lysates increased to 188% and
180% of control levels respectively as measured by imaging analysis of
radioactive bands, whereas FVIICys97 levels were unaffected
(105% versus 100% in control cells) (data not shown).
Analysis of altered mobility of FVIIArg100.
In
Fig
1, FVIIwt and FVIICys97 from conditioned media are
visualized by nondenaturing PAGE as a single band with an apparent mol
wt of 48 kD. This is slightly greater than that for the intracellular FVII species and results from post-transcriptional modifications of the
protein. However, FVIIArg100 from the conditioned media
appeared as a single band with a faster electrophoretic mobility than
FVIIwt on nonreducing 8% SDS-PAGE. A smaller difference in
electrophoretic mobility between FVIIwt and FVIIArg100 was
also observed in the cell lysates. We hypothesized that this difference
in electrophoretic mobility resulted from either a change in
glycosylation or alteration of the molecule's secondary structure.
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| Fig 1.
Pulse-chase labeling experiments in CHO cells transfected
with pEDFVIIwt, pEDFVIICys97, and
pEDFVIIArg100. After a 15-minute pulse with
[35S] methionine, the cells were chased for 30, 60,120, 180, and 240 minutes. Equivalent amounts of cell lysate (top) or
conditioned media (bottom) for FVIIwt (wt), FVIICys97
(97) and FVIIArg100 (100) were immunoprecipitated using a
Mab against FVII and analyzed by 8% SDS-PAGE under nonreducing
conditions. The location of molecular weight markers in kD is denoted
at the left-hand side of the figure. In the conditioned media (bottom),
the 77 kD band was nonspecific as it was observed with equal intensity
in untransfected CHO cells (data not shown).
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| Fig 2.
Immunohistochemical localization of wild-type and mutant
FVII molecules in stably transfected CHO cells. FVIIwt (A, B) and
FVIICys 97 (C) were mostly localized in the perinuclear
area, whereas FVIIArg100 (D) was present diffusely
throughout the cytoplasm without perinuclear enhancement. Untransfected
CHO cells did not react with either the anti-FVII Mab or the
fluorescent second antibody indicating that the observed labeling was
specific for FVII (data not shown).
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| Fig 3.
Analysis of the altered electrophoretic mobility of
FVIIArg100. After a 15-minute pulse with
[35S] methionine, stably transfected cells expressing
FVIIwt and FVIIArg100 were chased for 60 minutes and 120 minutes, respectively, to analyze FVII in cell lysates. In the
conditioned media, FVIIwt and FVIIArg100 were both
investigated after 180 minutes of chase. FVIIwt (wt) and
FVIIArg100 (100) were immunoprecipitated using a MoAb
against FVII. (A) Equivalent amounts of cell lysate were analyzed by
8% SDS-PAGE before (-) or after treatment with Endo H and N-Glycanase
(N-Gly). (B) Conditioned media were analyzed before (-) or after
treatment with neuraminidase (Neura) by 10% SDS-PAGE. (C) Conditioned
media were evaluated under nonreducing conditions (control) or after
reduction by 100 mmol/L Dithiothreitol (DTT). The 77 kD band was
nonspecific because it was also observed in conditioned media from
untransfected CHO cells (data not shown). Molecular weight markers in
kD are indicated on the left.
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To investigate N-linked glycosylation, radiolabeled FVII was
immunoprecipitated and incubated with Endo-H or N-Glycanase which respectively cleaves either high-mannose and certain hybrid-type carbohydrates at the GlcNAc1-4GlcNAc linkage to leave a single GlcNAc residue attached to asparagine25 or all N-linked
carbohydrates by hydrolyzing the asparginyl-oligosaccharide
bond.26 As the secretion of FVIIwt and
FVIIArg100 occur at different rates, we evaluated lysates
from the two stably transfected cell lines at different chase times.
After a 15-minute pulse with [35S] methionine and 120 minutes of chase time, FVIIwt migrated more rapidly after digestion
with either Endo H or N-Glycanase, reflecting the removal of N-linked
high mannose oligosaccharides from the nascent 46 kD protein (Fig 3A).
FVIIArg100 behaved similarly to FVIIwt at 180 minutes,
suggesting that N-linked glycosylation of this molecule was occurring
normally. To detect the presence of sialic acid on the glycans of
FVIIwt and FVIIArg100, we performed digestion with
neuraminidase. Recombinant FVIIwt and FVIIArg100 both
migrated more rapidly after digestion with neuraminidase, but the
difference in electrophoretic mobility of FVIIArg100 as
compared with FVIIwt remained (Fig 3B). This suggested that modification of the glycans with sialic acid was similar for the two
molecules. However, after reduction of the disulfide linkages with 100 mmol/L dithiothreitol, FVIIwt and FVIIArg100 migrated with
a similar electrophoretic mobility (Fig 3C), thereby suggesting that
disulfide bond formation of the FVII molecule was affected by the
Gln100 Arg substitution. Similar results were obtained
after reduction with 5% (vol/vol) -Mercaptoethanol (data not shown).
| |
DISCUSSION |
We investigated the mechanisms responsible for FVII deficiency in two
unrelated Italian patients with a bleeding diathesis. Sequencing of the
coding sequences and intron/exon boundaries of the FVII gene showed
that they were homozygous for different mutations within the coding
region for exon 5. Patient 1 had a G to T mutation at position 7824 and
patient 2 had an A to G mutation at position 7834 resulting in
Gly97Cys and Gln100Arg, respectively. These two
mutations were previously reported in association with other mutations
in doubly heterozygous patients.27-29 Patient 1, who was
homozygous for Gly97Cys, was also homozygous for two
polymorphisms known to reduce FVII levels, Arg353Gln in
exon 8 and the decanucleotide insert in the 5' flanking region of
the FVII gene.23,30,31
To study the mechanism by which the two mutations reduce FVII levels,
we performed transient expression studies in COS-1 cells with cDNA
encoding FVIIwt, FVIICys97, and FVIIArg100 and
showed that the secretion of the two mutant proteins was impaired. The
levels of VII:Ag and VII:C in the conditioned media were reduced
concordantly for FVIICys97, whereas a higher level of
VII:Ag relative to VII:C was observed for FVIIArg100. The
results were similar to those in the patients' plasmas. We also
observed that the intracellular levels of VII:Ag were reduced in
transient transfection assays performed with the mutant cDNA.
To study the intracellular processing of the mutant proteins, we
performed pulse-chase experiments and immunohistochemical staining of
stably transfected CHO cells expressing FVIICys97 and
FVIIArg100 in comparison to FVIIwt. Our results showed that
the proteins did not accumulate intracellularly in spite of major
secretion defects. A potential mechanism to account for reduced amounts of FVII protein in the cells is degradation. Intracellular degradation of abnormal proteins can result from lysosomal proteolysis or from
pre-Golgi or ER degradation, which has been termed the "quality control function" of the ER.32,33 Protein degradation
within the ER is a complex, poorly understood process and occurs either within the ER lumen or on the cytoplasmic side. Some of the proteolytic events are adenosine triphosphate (ATP)-dependent and several of the
enzymes involved in this process are sensitive to serine protease
inhibitors. Some proteins with an abnormal conformation are degraded by
a soluble ATP-dependent pathway in which conjugation occurs
between the abnormal protein and multiple molecules of ubiquitin
followed by hydrolysis by a 26S proteolytic enzyme complex. The
proteolytic core of this structure is the 20S proteasome, which
has been localized to the cytoplasmic surface of the ER by
immunoelectron microscopy.34
To investigate potential mechanisms of intracellular degradation, we
examined the biosynthesis of FVIICys97 using inhibitors of
lysosomal pH (NH4Cl), lysosomal proteolytic enzymes
(leupeptin), and the 20S-proteasome (lactacystin). We also evaluated
the effect of ALLN, a common inhibitor of neutral Ca2+-dependent cysteine proteases, which inhibits the
ubiquitin-proteasome pathway.35 The intracellular level of
FVIICys97 did not change in the presence of inhibitors of
lysosomal function (ie, NH4Cl and leupeptin) or
lactacystin, whereas a significant increase was observed with ALLN.
These data indicate that FVIICys97 degradation does not
occur in lysosomes and involves a cysteine protease, which is, however,
independent of the proteasome pathway.
Brefeldin A inhibits protein secretion in a pre-Golgi compartment such
that proteins destined for secretion remain in the ER.36
Whereas the level of FVIIwt rose by 90% of baseline in the presence of
brefeldin A, the intracellular level of FVIICys97 increased
by less than 30%. The Gly97Cys mutation is in the second
EGF domain of FVII and results in substitution of a small nonpolar side
chain by a polar side chain. Based on the crystal structure of
activated FVII bound to the extracellular domain of tissue
factor,37 Gly97 helps position the
Cys98-Cys112 loop to facilitate intramolecular
disulfide bonding. We postulate that the resulting alteration in
protein conformation causes FVIICys97 to undergo
degradation in a pre-Golgi compartment.
For FVIIArg100, pulse-chase experiments showed that the
mutant protein was retained for a longer time interval in stably
transfected CHO cells than FVIIwt and the level was not increased by
the various inhibitors of protein degradation. In the presence of
brefeldin A, the levels of FVIIArg100 increased by 51% as
compared to FVIIwt that increased by 90%. This data coupled with the
immunohistochemical demonstration that FVIIArg100 is
present diffusely throughout the cytoplasm is consistent with retention
of a substantial portion of the mutant protein in the ER.
The more rapid electrophoretic mobility of FVIIArg100 as
compared with FVIIwt suggested that FVIIArg100 might be
incompletely glycosylated or have an abnormal tertiary structure. To
investigate alterations in N-linked glycosylation and sialation,
radiolabelled FVIIwt and FVIIArg100 from conditioned media
were treated with Endo H, N-Glycanase, and neuraminidase. The
difference in electrophoretic mobility remained between FVIIwt and
FVIIArg100 after digestion with the three enzymes, implying
that N-linked glycosylation and sialation of FVIIArg100
were unaffected. We did not evaluate whether O-linked sugars on
residues Ser52 and Ser60 of
FVII6,38 were altered because FVIIAla52, a
mutant molecule lacking glycosylation at this residue, has been
reported to have the same electrophoretic mobility as
FVIIwt.38 Because the second EGF domain contains three
intramolecular disulfide bonds (Cys91-Cys102,
Cys98-Cys112, and
Cys114-Cys127), we compared the electrophoretic
mobility of FVIIwt and FVIIArg100 under reducing as well as
nonreducing conditions. After reduction, the difference in
electrophoretic mobility between the two molecules was no longer
present, leading us to speculate that FVIIArg100 does not
undergo normal disulfide bonding during its biosynthesis. To confirm
this, chemical determination of the disulfide bonding pattern of
FVIIArg100 would need to be performed and the results
compared with those for FVIIwt. The mutation replaces a neutral Gln
residue with a larger positively-charged Arg, which may sterically
interfere with Tyr118 and electrostatically interfere with
the positively-charged side chain of His115.37
We therefore postulate that the Gln100Arg mutation in FVII
results in a protein with an abnormal conformation that remains in the
ER for an extended period during its biosynthesis and reduces its secretion.
Whereas defective biosynthesis is a major defect conferred by the
Gln100Arg mutation, a small amount of dysfunctional protein
is secreted as evidenced by the low specific activity of the FVII (ie,
ratio of VII:C to VII:Ag) that is present in the patient's plasma as well as conditioned medium of transfected COS-1 cells. Our data is in
agreement with studies of FVIIArg100 reported by
Kemball-Cook et al,39 who showed that the activated form of
the mutant recombinant protein had markedly diminished affinity for
tissue factor and, in complex with soluble tissue factor, had less than
5% of the ability of FVIIwt to activate factor X. Orning et
al40 have also provided data that the peptide sequence,
Glu99-Gln100-Tyr101, in the second
EGF domain of FVII, inhibits the ability of FVIIwt to mediate tissue
factor-dependent factor X activation. Based on the crystal structure
of the FVIIa-tissue factor complex, Gln100 is located at
the interface of the second EGF and the protease domain, but is not in
contact with tissue factor.
Whereas Gly97Cys and Gln100Arg have previously
been reported in double heterozygotes with FVII deficiency, this is the
first report of homozygous patients with these mutations. The severe
clinical phenotype of patient 2 with Gln100Arg is
consistent with studies showing a major secretion defect as well as
markedly impaired function of the small amount of FVII that is released
from cells. It is unclear why patient 1 with Gly97Cys does
not have a similarly severe bleeding diathesis. Because it is not
possible to obtain sufficient FVIICys97 protein to perform
detailed biochemical studies, we cannot evaluate whether a very small
amount of circulating FVII protein posseses sufficient biologic
function to attenuate the bleeding diathesis.
| |
ACKNOWLEDGMENT |
The authors thank Dr Michael Yaffee (Beth Israel Deaconess Medical
Center, Boston, MA) for his assistance in the analysis of the
coordinates of FVIIa from the FVIIa-soluble tissue factor crystal structure.
| |
FOOTNOTES |
Submitted May 13, 1998; accepted October 8, 1998.
Supported by the Medical Research Service of the Department of Veterans Affairs.
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.
Address reprint requests to Kenneth A. Bauer, MD, Department of
Veterans Affairs Medical Center, 1400 VFW Pkwy, West Roxbury, MA 02132;
e-mail: bauer_md.kenneth_a+{at}brockton.va.gov.
| |
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Abstract
Introduction