From the
Factor VIII is the coagulation factor deficient in the
X-chromosome-linked bleeding disorder hemophilia A. Factor VIII is
homologous to blood coagulation factor V, both having a domain
structure of A1-A2-B-A3-C1-C2. Previous transfection studies
demonstrated that factor VIII is 10-fold less efficiently expressed
than the homologous coagulation factor, factor V. The inefficient
expression correlated with interaction of the factor VIII primary
translation product with the protein chaperonin BiP in the lumen of the
endoplasmic reticulum. In contrast, factor V was not detected in
association with BiP and was secreted efficiently. To determine whether
specific amino acid sequences within factor VIII inhibit secretion, we
have studied the secretion of factor VIII deletion and factor
VIII/factor V chimeric proteins upon transient transfection of COS-1
monkey cells. A chimeric factor VIII protein that contained the A1- and
A2-domains of factor V was secreted with a similar efficiency as
wild-type factor V, whereas the complementary chimera having the A1-
and A2-domains of factor VIII was secreted with low efficiency, similar
to wild-type factor VIII. These results suggested that sequences within
the A1- and A2-domains were responsible for the low secretion
efficiency of factor VIII. Secretion of A1-domain-deleted factor VIII
was increased approximately 10-fold compared to wild-type factor VIII
or A2-domain-deleted factor VIII. Expression of the factor VIII
A1-domain alone did not yield secreted protein, whereas expression of
the factor VIII A2-domain alone or the factor V A1-domain or A2-domain
alone directed synthesis of secreted protein,. Secretion of a hybrid in
which the carboxyl-terminal 110 amino acids of the A1-domain were
replaced by homologous sequences from the factor V A1-domain was also
increased 10-fold compared to wild-type factor VIII, however, the
secreted protein was not functional and the heavy and light chains were
not associated. These results localize a 110-amino acid region within
the A1-domain that inhibits factor VIII secretion. This region is
clustered with multiple short peptide sequences that have potential to
bind BiP.
Factor VIII functions in the intrinsic pathway of blood
coagulation as a cofactor to accelerate the activation of factor X by
factor IXa, a reaction that occurs on a negatively charged phospholipid
surface in the presence of calcium ions. Factor VIII is synthesized as
a single-chain polypeptide having the domain structure A1-A2-B-A3-C1-C2
(1, 2) . Upon secretion from the cell it is processed
within the B-domain to yield a heterodimer consisting of a
carboxyl-terminal-derived light chain of 80 kDa in a metal-ion
dependent association with a 200-kDa amino-terminal-derived heavy chain
fragment
(3) . The domain structure of factor VIII is identical
to that of the homologous coagulation factor, factor V
(4, 5) . The factor VIII A-domains are 330 amino acids
and have 40% amino acid identity with each other and to the A-domains
of factor V and the plasma copper-binding protein ceruloplasmin
(6) . Each C-domain is 150 amino acids and exhibits 40% identity
to the C-domains of factor V, and to proteins that bind glycoconjugates
and negatively charged phospholipids
(7) . The B-domain is
encoded by a single exon and exhibits little homology to the factor V
B-domain
(8, 9) . In plasma, the amino terminus of the
factor VIII light chain is bound by noncovalent interactions to a
primary binding site in the amino terminus of von Willebrand factor.
Although most evidence supports that the hepatocyte is the cell type
that produces factor VIII in vivo (10, 11, 12, 13) , there are no
known established or primary cell lines that express factor VIII. Thus,
our knowledge of factor VIII expression is derived from interpretation
of results from expression of the cDNA using expression vectors in
transfected mammalian cells. Expression of factor VIII in these
transfection systems is 2-3 orders of magnitude lower than that
observed with other genes using similar vectors and approaches. Studies
have identified three potential reasons that limit factor VIII
expression
(14) : 1) the factor VIII mRNA is inefficiently
expressed
(15, 16) , 2) the primary translation product
is inefficiently transported from the endoplasmic reticulum
(ER)
The inefficient secretion of factor VIII correlates with binding to
the protein chaperonin identified as the immunoglobulin binding protein
(BiP), also known as the glucose-regulated protein of 78 kDa (GRP78)
(18) within the lumen of the ER
(19) . BiP is a member of
the heat-shock protein family that exhibits a peptide-dependent ATPase
activity
(20) . BiP expression is induced by the presence of
unfolded protein or unassembled protein subunits within the ER
(21, 22) . High level factor VIII expression-induced BiP
transcription
(23) . In addition, factor VIII release from BiP
and transport out of the ER required high levels of intracellular ATP
(24) . In contrast, the homologous coagulation protein, factor
V, did not associate with BiP and did not require high levels of ATP
for secretion
(25) . Deletion of the factor VIII B-domain
yielded a protein that bound BiP to a lesser degree and was more
efficiently secreted
(17) . To evaluate whether the factor VIII
B-domain was responsible for BiP interaction, factor V and factor VIII
chimeric cDNA molecules were constructed in which the B-domain
sequences were exchanged
(26) . A factor VIII hybrid harboring
the B-domain of factor V was expressed and secreted as a functional
molecule, although the secretion efficiency of the hybrid was poor and
similar to wild-type factor VIII
(26) . This indicated that the
difference in secretion efficiency between factors V and VIII was not
directly attributable to specific sequences within the factor VIII
B-domain, the most divergent region between these homologous
coagulation factors. To further identify regions within factor VIII
that inhibit secretion, we have analyzed the expression of factor VIII
deletion and factor VIII/factor V chimeric molecules. The results
identify a 110-amino acid region within the factor VIII A1-domain that
inhibits secretion.
The factor VIII
deletion pVIII
The factor VIII deletion molecule pVIII
The factor VIII deletion plasmid p
The factor VIII chimera (pVIIIhcV) containing residues 1-708
from factor V was constructed by ligation of the 9,922-bp
XhoI/ MluI fragment from p740 MluI to the
2,260-bp SalI/ MluI fragment from p709 MluI.
The factor V hybrid molecule (pVhcVIII), harboring the heavy chain of
factor VIII (amino acid residues 1-739) was constructed by
ligating the 7,206-bp SalI/ MluI fragment from
p740 MluI to the 4,553-bp SalI/ MluI fragment
from p709 MluI. The hybrid pVIIIA1V containing the A1-domain of
factor V (amino acids 1-312) was constructed by ligation of the
11,026-bp XhoI/ MluI fragment from pVIII
The expression vector encoding the factor
VIII A1-domain alone (pVIIIA1) was constructed by ligation of the
6102-bp MluI/ SalI fragment from p372 MluI to
the following annealed phosphorylated oligonucleotide
MluI- SalI linker: 5`-CGCGTTGATGAG-3` and
5`-TCGACTCATCAA-3`, which contains two termination codons. The
expression vector encoding the factor VIII A2-domain (pA2) was
described previously
(28) . The factor V A1-domain alone was
assembled by ligating the 4924-bp XhoI/ MluI fragment
from pVIIIA1 to the 1067-bp SalI/ MluI fragment from
p313 MluI. The factor V A2-domain alone was constructed by a
3-way ligation of the 3859-bp ClaI/ MluI fragment from
pVIII
Chain association was measured by
co-immunoprecipitation experiments. The conditioned medium was
immunoprecipitated with the factor VIII heavy chain monoclonal antibody
(F8). The antibody-factor VIII complexes were collected by
centrifugation and the supernatant fluid was subsequently
immunoprecipitated with the light chain-specific monoclonal antibody.
The immunoprecipitated proteins were analyzed by SDS-polyacrylamide gel
electrophoresis as described above.
Factor VIII activity was
measured by a chromogenic assay
(33) or in a clotting assay
using factor VIII-deficient plasma
(34) . Factor V activity was
measured in a clotting assay using factor V-deficient plasma.
Total
RNA was isolated from transfected COS-1 cells at 60 h post-transfection
using the guanidine thiocyanate method
(35) . Northern blot
hybridization was performed using a dihydrofolate reductase cDNA probe
as described previously
(26) . The Northern blots were also
probed with a
Chimeric
molecules were constructed replacing factor VIII residues 1-740
with factor V residues 1-709 (designated VIIIhcV), factor VIII
residues 1-336 with factor V residues 1-313 (designated
VIIIA1V), and factor VIII residues 373-740 with factor V residues
314-709 (designated VIIIA2V). Transfected cells were
metabolically labeled with [
The results of these studies show that a 110-amino acid
region within the factor VIII A1-domain inhibits secretion of factor
VIII. Expression of independent heavy and light chains of factor VIII
previously demonstrated that the heavy chain was inefficiently secreted
compared to the light chain
(36) . To determine whether a
specific region within the heavy chain was responsible for the
inefficient secretion, analysis of in-frame deletions within the heavy
chain was performed. Secretion of A1-domain-deleted factor VIII was
increased approximately 10-fold compared to wild-type or
A2-domain-deleted factor VIII. In addition, deletion of the 36-amino
acid acidic region between the A1- and A2-domains of factor VIII did
not affect secretion efficiency (data not shown). Additional internal
deletions within the A1-domain were not secreted, likely due to
polypeptide misfolding. Thus, to further localize the region, chimeric
molecules were made between factor VIII and the homologous coagulation
protein, factor V, that is efficiently secreted. Whereas exchange of
factor VIII residues 1-226 for the homologous residues of factor
V did not increase the secretion efficiency, similar exchange of factor
VIII residues 227-336 improved secretion 5-10-fold in
different experiments. To determine if the A1-domain directly inhibits
secretion, we studied the secretion of individual domains. Compared to
the factor VIII A1-domain that was not detectably secreted, the factor
VIII A2-domain and the factor V A1-domain were effectively secreted.
Analysis of the data obtained from the different approaches by
characterizing the secretion of factor VIII deletion mutants, factor
VIII and factor V hybrid molecules, and expression of single A-domains
independent of the rest of the molecule implicated sequences between
residues 227 and 336 within the A1-domain as inhibiting secretion.
Previous studies indicated that the inefficient secretion of factor
VIII correlated with a stable interaction with BiP and a requirement
for high levels of intracellular ATP for BiP release and secretion
(17, 24) . In contrast, factor V was efficiently
secreted, was not detected in association with BiP, and did not require
high levels of ATP for secretion
(25) . We have now localized a
region that inhibits factor VIII secretion between residues 227 and 336
within the factor VIII A1-domain and propose that this represents a
high affinity BiP-binding site. Although it is likely that BiP can
interact with multiple sites within unfolded proteins, the
Escherichia coli chaperonin GroEL specifically interacts with
a distinct binding site on human granulocyte ribonuclease
(37) .
To date, a unique high affinity BiP-binding site has not been
identified within any protein transiting the secretory pathway. Two
studies
(38, 39) have reported on the amino acid
requirements for peptide binding to BiP. Both approaches show that
aliphatic amino acids are enriched in BiP-binding peptides. However,
one study using filamentous phage display
(38) also detected a
preponderance of aromatic amino acids (Trp and Phe), that were not
observed by direct sequencing of random-bound peptides
(39) .
Gething and co-workers
(38) , provided a statistical method to
predict the BiP binding potential of a particular 7-mer peptide. We
applied this statistical program to the 227-336 region of the
A1-domains of factor VIII and factor V. Residues Ile
At present it is not known what structural features are required for
the metal-ion dependent factor VIII heavy and light chain association
but it likely occurs through interactions between the A1-domain and
A3-domain. It is interesting that exchange of either factor VIII amino
acid residues 1-226 or 227-336 for homologous factor V
residues destroyed heavy and light chain association. This suggests
that chain association requires specific factor VIII residues between 1
and 226 as well as between 227 and 336. Analogous to the postulated
role of BiP in binding and retaining unassociated subunits of heavy and
light chain immunoglobulins
(40, 41) ,
We thank Dr. Mary-Jane Gething for applying the
BiP-score algorithm to the A-domains and Dr. Alnawaz Rehemtulla for
advice and discussion during the course of these studies.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
to the Golgi apparatus
(17) , and
3) high levels of von Willebrand factor are required in the conditioned
medium to promote stable accumulation of factor VIII
(3) .
Materials
Rabbit anti-factor V polyclonal
antibody was purchased from Dako Corp. (Carpinteria, CA). Factor V
monoclonal antibody E9 directed against the factor V light chain
(27) was kindly provided by Dr. K. G. Mann (University of
Vermont, Burlington, VT). Anti-factor VIII heavy chain monoclonal
antibody F-8 was described previously
(28) . Anti-factor VIII
light chain monoclonal antibody was obtained from Hybritech Corp. (La
Jolla, CA). Anti-factor VIII polyclonal antibody (R-305) was provided
by Genetics Institute, Cambridge, MA. Soybean trypsin inhibitor,
phenylmethylsulfonyl fluoride, and aprotinin were purchased from Sigma.
Human -thrombin and factor Xa were obtained from Hematological
Technology (Burlington, VT). [
S]Methionine
(>1000 Ci/mmol) was obtained from Amersham Corp. (Arlington Heights,
IL).
Plasmid Mutagenesis and Hybrid Construction
The
mammalian cell expression vector pMT2
(29) utilizes the
adenovirus major late promoter with SV40 enhancer and origin of
replication. The factor VIII expression vector pMT-VIII has
a unique XhoI site upstream of the initiation codon and a
unique SalI site at nucleotide 8379, where nucleotide 1 is at
the 5` end of the SV40 enhancer
(26) . The factor V expression
vector pMT
-V has two SalI sites flanking the
factor V cDNA at nucleotides 1088 and 7932
(26) . To facilitate
domain exchanges and deletions, site-directed oligonucleotide-mediated
mutagenesis was performed by the heteroduplex procedure
(30) using different sized (approximately 50 bases) mutagenic
oligonucleotides to introduce unique MluI restriction sites at
arginine residues 226, 336, 372, and 740 within factor VIII to yield
expression plasmids designated p226 MluI, p336 MluI,
p372 MluI, and p740 MluI. Plasmids p372 MluI
and p740 MluI were previously described
(28) . Unique
MluI restriction sites were introduced into the factor V cDNA
at arginine residues 198, 313, and 709 within factor V to yield
p198 MluI, p313 MluI, and p709 MluI. Double
MluI mutations were sequentially introduced into pMT2-V at
amino acids 313 and 709 to yield p313/709 MluI. Chimeric and
deletion molecules were constructed by restriction endonuclease
digestion, gel electrophoresis in low-gelling temperature agarose, and
ligation of the isolated fragments. The resulting plasmids were
purified over cesium chloride and characterized by extensive
restriction mapping and partial DNA sequencing.
A1 was constructed by digesting p372 MluI
with XhoI and MluI and ligation to complementary
phosphorylated oligonucleotides encoding the factor VIII signal peptide
and flanked by unique XhoI (5`) and MluI (3`)
restriction sites having the sequence:
5`-TCGAGAGCTTCGACCACCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTA-3`.
The factor VIII deletion molecule p
A1-ac deletes amino acids 1
through 372 and does not contain the factor VIII acidic region
(residues 336-372). A linker comprised of two complementary
synthetic oligonucleotides encoding the acidic region from 336 to 372
flanked by the 5` and 3` MluI restriction sites was ligated
into the unique MluI site of p
A1-ac to yield
pVIII
A1. The hybrid pVIIIA1V containing the A1-domain of factor V
(amino acids 1-312) was constructed by ligation of the 11,026-bp
XhoI/ MluI fragment from pVIII
A1 to the 1,067-bp
SalI/ MluI fragment from p313 MluI and
designated pVIIIA1V-ac. pVIIIA1V was generated by ligation of the
108-bp MluI fragment (336-372 acidic region) from
pVIII
A1 to MluI linearized pVIIIA1V-ac to yield pVIIIA1V.
A2 (336-739) has a
unique MluI site at the junction of the deletion and was
described previously
(28) . The factor VIII chimera containing
the factor V A2-domain (factor V amino acid residues 313-708) was
constructed by linearizing pVIII
A2 with MluI and ligating
it to the 1188-bp MluI fragment from p313/709 MluI.
1-225 was made by
ligation of the 3859-bp ClaI/ MluI fragment from
pVIII
A1 to the 7685-bp ClaI/ MluI fragment from
p226 MluI. The factor VIII chimera pV
,
replacing factor VIII amino acids 1-225 with residues 1-197
from factor V, was assembled by ligation of the 11,468-bp
XhoI/ MluI fragment from p
1-225 to the
722-bp SalI/ MluI fragment from p198 MluI. The
factor VIII deletion p
226-371 removes amino acids
226-371 and was constructed by ligation of the 4,531-bp
ClaI/ MluI fragment from p226 MluI to the
7,243-bp ClaI/ MluI fragment from p372 MluI.
The factor VIII chimera pV
, replacing factor
VIII amino acids 226-335 with factor V amino acids 198-312,
was constructed by a 3-way ligation of MluI linearized
p
226-371, the 106-bp MluI/ SacI fragment
from p198 MluI and the 238-bp SacI/ MluI
fragment from p313 MluI. Then the factor VIII acidic region
(336-372) was isolated from pVIII
A1 on a 108-bp
MluI fragment and ligated into the unique MluI site.
A1 to the
1,067 bp SalI/ MluI fragment from p313 MluI.
To construct the factor VIII chimera containing the A2-domain (amino
acids 313-709) of factor V(pVIIIA2V), pVIII
A2 was linearized
with MluI and ligated to the 1,188-bp fragment from
p313/709 MluI.
A1, the 1188-bp MluI fragment from
p313/709 MluI, and the 1141-bp ClaI/ MluI
fragment from pVIIIA1. The factor V heavy chain alone (94-kDa) was
constructed by ligation of the 4924-bp XhoI/ MluI
fragment from pVIIIA1 to the 2281-bp SalI/ MluI
fragment from p709 MluI.
DNA Transfection and Analysis
Plasmid DNA was
transfected into COS-1 cells by the DEAE-dextran procedure
(29) . Conditioned medium was harvested 60 h post-transfection
in the presence of 10% heat-inactivated fetal bovine serum for factor
VIII assay. Primary translation products were analyzed by pulse
labeling cells for 15 min with [S]methionine
(250 µCi/ml in methionine-free medium) and preparing cell extracts
in a Nonidet P-40 lysis buffer
(31) . Protein secretion was
monitored by metabolically pulse labeling cells with
[
S]methionine (250 µCi/ml for 15 min) and
chasing for increasing periods of time in medium containing excess
unlabeled methionine as described
(30) . Cell extracts and
conditioned medium were harvested as described previously
(30, 31) . The factor VIII was quantitatively
immunoprecipitated with an anti-factor VIII monoclonal antibody F-8
(28) coupled to CL-4B Sepharose or with anti-factor VIII light
chain monoclonal antibody coupled to Affi-Gel. Factor V was
immunoprecipitated with either monoclonal antibody E-9 specific to the
factor V light chain
(27) or a rabbit polyclonal antibody
coupled to Affi-Gel (Bio-Rad). The antibodies were tested prior to the
experiments to determine the amount of antibody required for
quantitative immunoprecipitation. In all cases excess antibody was
used. The immunoprecipitates were washed as described
(31) .
Immunoprecipitated proteins from the conditioned medium were
resuspended 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2.5
mM CaCl
, and 5% glycerol for electrophoresis on an
SDS-low bis-8% polyacrylamide gel
(32) . In addition to analysis
by immunoprecipitation, equal quantities of
[
S]methionine-labeled conditioned medium were
analyzed by SDS-PAGE. Proteins were visualized by autoradiography after
fluorography by treatment with En
Hance (Dupont, Boston,
MA). The band intensities were quantitated by scanning the lanes using
an LKB UltroScan XL laser densitometer (Pharmacia LKB Biotechnology
Inc., Uppsala, Sweden). The total amount of secreted protein was
estimated by adding the amount of single-chain to the amount of
processed fragments.
-actin cDNA probe to insure that the lanes were
equally loaded.
The A1-domain of Factor VIII Inhibits
Secretion
Previous studies in transfected mammalian cells
demonstrated that factor V was more efficiently secreted than factor
VIII
(25, 26) . In addition, secretion of the factor
VIII 90-kDa heavy chain fragment compared to the 80-kDa light chain
fragment, each fused to the factor VIII signal peptide, was
significantly reduced, although both proteins were translated at
similar levels in the cell
(36) .(
)
These
results suggested that the factor VIII light chain can be efficiently
secreted in the absence of the heavy chain and that the heavy chain
expressed alone is inefficiently secreted, similar to wild-type factor
VIII. To further localize a region(s) within the factor VIII heavy
chain that may inhibit secretion, the expression of A1-domain (residues
1-336) deleted factor VIII (
A1) and of A2-domain (residues
373-740) deleted factor VIII (
A2) was studied. In addition,
a series of chimeric proteins were constructed that replaced different
regions of factor VIII heavy chain with factor V residues. The factor V
and factor VIII cDNAs were engineered to contain unique MluI
restriction sites (encoding Thr-Arg) at the Arg residues 226, 336, 372,
and 740 in human factor VIII and at Arg residues 198, 313, and 709 in
human factor V. In this way, homologous sequences were exchanged by DNA
fragment exchanges and chimeric molecules were produced by transient
DNA transfection of COS-1 monkey kidney cells. These particular
residues were chosen because they are proteolytic cleavage sites that
are likely on the outer surfaces of these proteins and therefore
chimeric proteins that harbor novel junctions at these sites are more
likely to fold properly. Analysis of factor VIII expression upon
transient transfection of COS-1 monkey cells demonstrated that
introduction of the MluI restriction sites did not affect the
synthesis, secretion, functional activity, or the ability of the
molecules to be cleaved by thrombin (data not shown).
S]methionine and
cell extracts were analyzed for primary translation products by
immunoprecipitation with an anti-factor VIII polyclonal antibody. Total
radiolabeled conditioned medium was analyzed directly to rule out the
possibility that the mutant proteins were not efficiently recognized by
the antibody. In addition, total cellular RNA was prepared for analysis
by Northern blot hybridization to quantitate factor VIII mRNA levels.
Analysis of total conditioned medium from wild-type factor
V-transfected cells detected migration of factor V at 330 kDa, just
below a background polypeptide (Fig. 1 A, lane 4). In
contrast, wild-type factor VIII was barely detectable above background
bands (Fig. 1 A, lane 1). Deletion of the A1-domain
(Fig. 1 A, lane 2) or replacement of the factor VIII
heavy chain (Fig. 1 A, lane 6) or the A1-domain
(Fig. 1 A, lane 7) with corresponding sequences of factor
V increased the level of factor VIII polypeptides in the conditioned
medium significantly above the wild-type factor VIII. In addition, the
increased expression permitted direct detection of radiolabeled light
chain in the total conditioned medium from cells transfected with the
A1, VIIIhcV, or VIIIA1V (Fig. 1 A, lanes 2, 6, and
7). In contrast, deletion of the A2-domain (Fig. 1 A,
lane 3) or replacement of the factor VIII A2-domain with factor V
residues (Fig. 1 A, lane 8) did not increase the level of
factor VIII polypeptides detected in the conditioned medium. Secretion
of a factor V chimera that had factor V residues 1-709 replaced
for factor VIII residues was reduced compared to wild-type factor V
(Fig. 1 A, lane 5), suggesting the factor VIII residues
1-740 inhibit accumulation of the VhcVIII chimera in the
conditioned medium. In addition, a factor V chimeric molecule that had
the factor V light chain (residues 1546-2196) replaced by the
factor VIII light chain (residues 1649-2332) accumulated to
levels similar to wild-type factor V, indicating that the light chain
of factor VIII did not inhibit factor V accumulation in the conditioned
medium (data not shown). Analysis of mRNA (Fig. 1 B) and
primary translation products (Fig. 1 C) indicated that
the mRNAs encoding the deletion and chimeric factor VIII proteins were
expressed at similar levels and were translated with similar
efficiencies. Although there were minor variations in the mRNA levels
detected, they did not account for the major difference in the amount
of protein secreted into the conditioned medium. In addition,
differences in methionine content could not account for the increased
secretion associated with the absence of the A1-domain of factor VIII.
These results indicate that specific sequences within the factor VIII
heavy chain inhibit accumulation of factor VIII in the conditioned
medium.
Figure 1:
The A1-domain limits factor VIII
expression at a post-translational level. COS-1 cells were transfected
with the indicated plasmid DNAs encoding wild-type factor V (V),
wild-type factor VIII (VIII), or the indicated deletion or chimeric
molecules. At 60 h post-transfection cells were labeled for 6 h with
[S]methionine and equivalent amounts of total
conditioned medium were directly loaded onto a reducing 6.5%
polyacrylamide-SDS gel and analyzed by autoradiography ( panel
A). In parallel, RNA was isolated for Northern blot hybridization
analysis using a dihydrofolate reductase probe that reacts with all
vector-derived mRNA species ( panel B). Cells were also
pulse-labeled with [
S]methionine for 15 min and
lysates prepared for immunoprecipitation with an anti-factor VIII
polyclonal antibody ( panel C). Migration of factor V, factor
VIII related polypeptides ( VIII), and the factor VIII light
chain ( LC) is shown. Molecular weight markers are shown on the
left.
To determine whether the increased factor VIII accumulation
was due to increased secretion from the cell or increased stability in
the conditioned medium, more detailed
[S]methionine pulse labeling and chase analysis
was performed by immunoprecipitation with an anti-factor VIII light
chain-specific monoclonal antibody. The factor VIII wild-type
(Fig. 2 A, lane 1), the A1- (
A1; Fig. 2 B,
lane 1) and A2- (
A2; Fig. 2 B, lane 10) domain
deletion molecules, and a chimeric factor VIII replacing residues
1-740 for factor V amino acids (1-709) (VIIIhcV;
Fig. 2A, lane 10) were all translated at similar levels.
The majority of the translation products for the wild-type,
A1,
and VIIIhcV disappeared from the cell after 2 h, whereas the
A2
was detectable in the cell after 4 h. Analysis of the conditioned
medium detected factor VIII polypeptides secreted after 2 h as a
250-kDa single chain and a 80-kDa light chain doublet for wild-type,
A1, and VIIIhcV factor VIII molecules (Fig. 2). Secretion of
A2-domain-deleted factor VIII was slightly delayed, appearing at 4 h.
However, the secretion of both the
A1 and VIIIhcV were increased
approximately 5-10-fold, compared to wild-type or
A2 factor
VIII and there were also significant amounts of a 160-kDa
carboxyl-terminal light chain that was previously characterized to
extend to residue 1313
(25) . All proteins were relatively
stable in the conditioned medium over a 6-h period with some
degradation occurring after 24 h. Thrombin digestion of the
A1 and
A2 deletion molecules yielded the expected products migrating at
73, 50, and 43 kDa, whereas the 50- and 43-kDa fragments were absent in
their respective deletion molecules, indicating that neither the
A1-domain nor the A2-domain were not required for thrombin cleavage
(data not shown). The analysis of the A1- and A2-domain deletion
molecules show that neither the A1-domain or the A2-domain are required
for factor VIII secretion. Replacement of factor VIII heavy chain
residues 1-740 with homologous factor V residues increased the
secretion efficiency approximately 10-fold, to a level similar to the
A1 factor VIII. These results suggest that sequences within the
factor VIII A1-domain inhibit factor VIII secretion.
Figure 2:
The A-1 domain inhibits secretion of
factor VIII. Expression vectors encoding wild-type, A1-domain deleted
(A1), and A2-domain deleted (
A2), and the heavy chain
chimeric (VIIIhcV) factor VIII were transfected into COS-1 cells and
analyzed [
S]methionine pulse labeling and chase
as described under ``Experimental Procedures.'' Cell extracts
and conditioned medium were immunoprecipitated with a monoclonal
antibody reactive with the factor VIII light chain. Molecular weight
markers are shown on the left.
An Independently Expressed Factor VIII A1-domain Is Not
Secreted
The above results indicate that A1-domain within factor
VIII either actively retains factor VIII in the cell or otherwise
prevents efficient protein folding to inhibit factor VIII secretion.
Insight into whether the A1-domain contains a retention signal was
obtained by comparing expression of the factor VIII A1-domain alone to
the factor VIII A2-domain alone, each fused to the signal peptide of
factor VIII to direct transport into the ER. Whereas the A1-domain was
not secreted, most of the translated A2-domain was efficiently secreted
(Fig. 3 A). The A1-domain in the cell extract migrated as
a triplet that was converted to a single species by digestion with
N-glycanase, indicating the different species result from
heterogeneity in N-linked glycosylation (data not shown). The
secretion of the intact factor V 94-kDa heavy chain, the factor V
A1-domain, or the factor V A2-domain were also evaluated after a 15-min
pulse and a 6-h chase in complete medium (Fig. 3 B, lanes
1-8). Analysis of the conditioned medium after 6 h
(Fig. 3 B, lanes 9-11), demonstrated that the
factor V heavy chain or individual domains were all detectably secreted
compared to the factor VIII A1-domain. These results show that the
factor VIII A1-domain is uniquely not secreted from the cell and
suggest that the factor VIII A1-domain polypeptide is directly
responsible for inhibiting factor VIII secretion. Residues 227-336 in the A1-domain Inhibit Factor VIII
Secretion-To further localize the region that inhibits factor
VIII secretion, additional internal deletions within the A1-domain
(residues 1-226 or 227-336) were analyzed and found not to
be secreted, suggesting that smaller deletions affected folding of the
protein to inhibit secretion. Therefore, to preserve proper polypeptide
folding, we studied the secretion of factor VIII chimeric proteins
containing portions of the factor V A1-domain. Two factor VIII chimeric
molecules were constructed that had residues 1-226 or
227-336 replaced by homologous sequences within factor V
(residues 1-197 or 198-313, respectively). Analysis of
pulse-labeled cell extracts indicated that the wild-type and both
A1-domain chimeric proteins were translated with equal efficiencies
(Fig. 4, lanes 1-3). In contrast, analysis of the
conditioned medium demonstrated that the secretion of the chimeric
factor VIII protein that had factor VIII residues 227-336
replaced by factor V residues was increased approximately 5-fold
(Fig. 4, lane 7) compared to wild-type factor VIII
(Fig. 4, lane 5) or a chimera that had factor VIII
residues 1-226 exchanged for the homologous factor V sequences
(Fig. 4, lane 9). These results demonstrate that
residues 227-336 within the factor VIII A1-domain inhibit
secretion. Loss of Procoagulant Activity in the V Hybrid Correlates with Chain Dissociation-Analysis of
factor VIII activity in the conditioned medium indicated that all the
factor VIII and factor V chimeric proteins described in this report
were inactive, although they were all susceptible to thrombin cleavage
(data not shown). Further analysis by co-immunoprecipitation with
factor VIII light chain- and heavy chain-specific monoclonal antibodies
demonstrated reduced association of the heavy and light chains for the
A1-domain chimeric proteins. Immunoprecipitation with the heavy
chain-specific antibody detected single chain, heavy chain, and
associated light chain at 80 kDa for wild-type factor VIII
(Fig. 4, lane 5). Immunoprecipitation of the supernatant
with a light chain-specific monoclonal antibody also detected the the
single chain and some 80-kDa light chain, indicating that approximately
50% of the light chain was not associated (Fig. 4, lane
6), similar to previous observations
(3) . In contrast,
similar comparison of the amount of light chain co-immunoprecipitation
for either the V
(Fig. 4, lanes 9 and 10) or the V
hybrid
(Fig. 4, lanes 7 and 8) demonstrated the
majority of the light chain did not co-immunoprecipitate with the heavy
chain monoclonal antibody. The 160-kDa species reactive with the light
chain antibody for the 227-336 hybrid represents a
carboxyl-terminal light chain polypeptide extending to residue 1313.
These results show that both the V
and
V
chimeras display reduced chain association
that could contribute to the loss of procoagulant activity. Factor VIII Residues 227-336 Contain Multiple Peptides with
Potential to Bind BiP-Our results show that factor VIII contains
unique sequences between residues 227 and 336 that inhibit secretion
that are not observed in factor V. We hypothesize that this region
contains a high affinity BiP-binding site. Based on the ability of
phage displaying random peptides to bind BiP, Blond-Elguindi et al. (38) provided a statistical method to predict the
potential for any 7-mer peptide to bind BiP. We applied this algorithm
to the sequences within the 227-336 region within the A1-domain
of factors VIII and V (Fig. 5). The major difference between
factor VIII and factor V is in a region in the factor VIII A1-domain
that contains a hydrophobic cluster having several peptides between
residues Ile
and Phe
( arrows in
Fig. 5
) with scores greater than 10, a score with high
probability to bind BiP. Analysis of the factor VIII A2-domain or the
A3-domain did not identify a region with multiple BiP-binding peptides
(data not shown). These results suggest this hydrophobic pocket may
represent a high affinity BiP-binding site.
Figure 3:
Independently expressed factor VIII
A1-domain is not secreted. COS-1 cells were transfected with expression
vectors encoding the factor VIII A1-domain (residues 1-336) or
the A2-domain (373-740) each fused to the factor VIII signal
peptide ( panel A). The secretion of the factor V heavy chain
(94 kDa, residues 1-709), the factor V A1-domain (residues
1-313), or the factor V A2-domain (residues 314-709) each
fused to the factor VIII signal peptide was also evaluated ( panel
B). Cells were pulse-labeled with
[S]methionine for 15 min and chase performed for
the indicated periods of time ( panel A) or for 6 h ( panel
B) as described under ``Experimental Procedures.'' Cell
extracts and conditioned medium were analyzed using a factor VIII
polyclonal antibody for immunoprecipitation ( panel A) or a
factor V polyclonal antibody ( panel B). The mock sample
represents COS-1 cells that did not receive DNA. The molecular weight
markers are indicated on the left.
Figure 4:
Residues 226-336 inhibit factor
VIII secretion. Expression vectors encoding wild-type factor VIII
( wt) and the hybrids containing factor V exchanges of factor
VIII residues 1-226 (V) or 227-336
(V
) were transfected into COS-1 cells. Mock represents cells that did not receive DNA. After 60 h the cells
were pulse-labeled with [
S]methionine for 15 min
and chased in complete medium for 6 h, and then analyzed as described
under ``Experimental Procedures.'' Cell extracts and
conditioned medium were immunoprecipitated with the anti-factor VIII
heavy chain monoclonal antibody (F8) ( h). The supernatants
from the anti-factor VIII heavy chain antibody immunoprecipitation of
the conditioned medium were subsequently immunoprecipitated with
anti-factor VIII light chain monoclonal antibody ( l). The
migration of single chain ( s.c.), heavy chain ( h.c.),
light chain, and the 160-kDa extended light chain ( x-l.c.) are
shown. Molecular weight markers are shown on the
left.
Figure 5:
BiP scores for the 227-336 region of
factor VIII and the homologous region in factor V. The amino acid
sequence from residues 227-336 is shown for factor VIII (above)
and the homologous residues in factor V are shown below. The BiP scores
for all 7-mer peptides are shown as bars that occur at the
initial amino acid of the peptide. Black bars, factor VIII.
Gray bars, factor V. The solid bar indicates the
position of +10, a highly significant score for BiP binding. The
asterisk indicates the residues involved in type 1 copper
binding. The arrows indicate Ileand
Phe
.
to
Phe
have multiple BiP binding scores greater than 10,
indicating an extremely high probability of binding BiP
(38) that were not present in the factor V A1-domain or in the
A2- or A3-domains of factor VIII. Present studies are directed to
analyze the effect on secretion of specific mutations in this region.
and
chains of the major histocompatibility complex
(42) , and
nicotinic receptor subunits
(43) , we propose that BiP binds the
factor VIII A1-domain in the amino terminus until association occurs
with the A3-domain in the carboxyl terminus. It is particularly
intriguing that the A-domains of ceruloplasmin and factor VIII
(44) are structurally related to ancient copper-binding
proteins, such as azurin
(45, 46) , nitrite reductase
(47) , and ascorbate oxidase
(48) . These ancient
copper-binding proteins have a similar three-dimensional folding
pattern similar to the immunoglobulin variable domains
(46, 49) consisting of a double
-barrel, each of which is
composed of 7 anti-parallel
-strands. The structural homologies
between immunoglobulin variable regions and these copper-binding
proteins suggest similarities in folding intermediates and domain
association.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.