From the
The A2 domain (residues 373-740) of human blood
coagulation factor VIII (fVIII) contains a major epitope for inhibitory
alloantibodies and autoantibodies. We took advantage of the
differential reactivity of inhibitory antibodies with human and porcine
fVIII and mapped a major determinant of the A2 epitope by using a
series of active recombinant hybrid human/porcine fVIII molecules.
Hybrids containing a substitution of porcine sequence at segment
410-508, 445-508, or 484-508 of the human A2 domain
were not inhibited by a murine monoclonal antibody A2 inhibitor, mAb
413, whereas hybrids containing substitutions at 387-403,
387-444, and 387-468 were inhibited by mAb 413. This
indicates that the segment bounded by Arg
Inhibitory antibodies (inhibitors) to factor VIII (fVIII)
Deletion mapping studies by
immunoblotting have limited the A2 epitope to residues 373-536 in
the NH
Citrated hemophilia A and normal pooled human plasmas were
purchased from George King Biomedical, Inc. Heparin-Sepharose was
purchased from Sigma. Fetal bovine serum, G418, penicillin, and
streptomycin were purchased from Life Technologies, Inc. Unilamellar
phosphatidylcholine/phosphatidylserine (75:25) (w/w) vesicles were
prepared as described previously(12) .
Published procedures were used to isolate porcine factors
IXa, X, and Xa and thrombin(13, 14) . Inhibitor IgG was
prepared as described previously(9) . Recombinant
desulfatohirudin was a gift from Ciby-Geigy. Albumin-free pure
recombinant human fVIII was a gift from Baxter Biotech. FVIII heavy
chains (A1, A2, and B domains covalently linked) were purified by
dissociating heterodimeric fVIII chains with EDTA, followed by
chromatographic isolation. EDTA was added to a final concentration of
50 mM to fVIII in 0.6 M NaCl, 50 mM HEPES,
0.01% Tween 80, pH 6.0. After 18 h, the preparation was subjected to
cation exchange chromatography on Resource S (Pharmacia Biotech Inc.),
which binds light chains but not heavy chains. Prior to application to
the column, the fVIII solution was diluted with 20 mM MES, 10
mM EDTA, 0.01% Tween 80, pH 6.0, and applied to the column
equilibrated in 0.25 M NaCl, 20 mM histidine
chloride, 5 mM CaCl
Affinity columns were prepared by coupling purified fVIII
heavy chains to Affi-Prep 10 (Bio-Rad). Coupling was done at 0.82 mg/ml
resin with 93% efficiency. The column was equilibrated in 0.5 M NaCl, 50 mM Tris-Cl, pH 7.4. Purified inhibitor IgG was
dialyzed into the equilibration buffer and loaded onto the column, the
flow through was collected in loading buffer and concentrated, and the
column was washed further with 50-100 volumes of the same buffer.
The elution buffer was 50 mM Tris, 0.5 M
MgCl
The expression vector ReNeo, which contains a neomycin
resistance gene and B-domainless human fVIII cDNA, designated
HB
All transfected cell lines were maintained in
Dulbecco's modified Eagle's medium-F12 containing 10% fetal
bovine serum, 50 units/ml penicillin, 50 µg/ml streptomycin. Fetal
bovine serum was heat-inactivated at 56 °C for 1 h prior to use.
HB
FVIII and fVIII hybrids were partially purified and concentrated
from the growth medium by heparin-Sepharose chromatography. A 10-ml
column of heparin-Sepharose was equilibrated with 0.075 M NaCl, 10 mM HEPES, 2.5 mM CaCl
The activity of recombinant fVIII proteins was measured by
one-stage clotting assay (17) and by a plasma-free
assay(11) . FVIII concentrations were determined by
enzyme-linked immunoassay(11) . One unit of fVIII is defined as
the activity in 1 ml of normal citrated human plasma. The standard was
recombinant heterodimeric fVIII that contained variable amounts of B
domain. An extinction coefficient at 280 nm of 1.0
(mg/ml/cm)
Two COOH-terminal carboxamide peptides corresponding to
residues 484-509 in human and porcine fVIII were synthesized and
purified using published methods(19) . The identity of both
peptides was confirmed by sequence analysis.
cDNAs for HP4 and HP5 were constructed by
using convenient restriction sites as described under
``Experimental Procedures.'' The expressed hybrids were
tested in a coagulation assay for inhibition by mAb 413, a murine
monoclonal antibody that competes with human A2 inhibitors for binding
to A2(9) . mAb 413 inhibited neither HP4 nor HP5, indicating
that it does not bind these hybrids and that a major determinant of the
A2 epitope is in the sequence bounded by residues 387-508 (Fig. 1).
To further localize the epitope, constructs HP6
through HP11 were made by the SOE method(16) . The SOE
constructs are complementary pairs defined by a splice site within the
387-508 substitution in HP5. Because of sequence identity between
human and porcine fVIII, we define boundaries of porcine substitutions
by the first amino acids that differ between human and porcine fVIII at
the NH
There are nine
differences between human and porcine fVIII in the 484-508
segment (). We made two more complementary constructs, HP12
and HP13, which contain porcine substitutions at residues 489-508
and 484-488 and which differ from human fVIII by four and five
residues, respectively. Neither HP12 nor HP13 was inhibited by mAb 413 (Fig. 1). This indicates that reactive side chains of the A2
epitope for mAb 413 are on both sides of the
Ser
We also measured the
inhibition of the recombinant fVIII proteins by mAb 413 using a
plasma-free assay, which measures the ability of fVIIIa to support
factor X activation by factor IXa. The IC
By systematic replacement of human fVIII sequences with
homologous porcine fVIII sequences, we have shown that a major
determinant of the A2 inhibitor epitope lies in a 25-residue segment
bounded by Arg
Antibody competition
binding experiments have indicated that A2 inhibitors bind to a common
epitope or a narrowly clustered set of epitopes(9) . Comparison
of inhibitor activity against porcine substitution mutants in the
484-508 segment (HP9, HP12, and HP13) offer a higher resolution
analysis of inhibitor specificity than competition binding experiments.
We observed decreased binding to HP9, HP12, and HP13 by all four
inhibitors tested, although RC antibody showed significant reactivity
with HP13 (Fig. 4). Thus, residues with the 489-508 segment
are important for recognition by RC antibody, with a minor contribution
due to residues with the 484-488 segment. Overall, the results
with all four human inhibitors and with mAb 413 indicate that there is
little variation of the A2 epitope in the human inhibitor response.
However, further mutational analysis in this region may reveal fine
specificity that is not yet apparent.
Although the 484-508
segment contributes substantially to the binding of fVIII to A2
inhibitors, a synthetic 26-residue peptide corresponding to the human
sequence in this region only weakly blocked inhibition by A2 antibodies (Fig. 3). For several reasons, synthetic peptides frequently are
poor antigen mimics(20, 21) . In this case, incomplete
antibody blocking was observed at saturating concentrations of peptide.
This partial competitive behavior is consistent with partial formation
of the native fold of the 484-508 segment by the peptide. Thus,
at saturating concentrations, it would bind only part of the inhibitor
idiotope, thereby reducing the affinity of the inhibitor for A2.
Protein antigens typically have 15-20 side chains in contact
with antibody(22) . Side chain interactions, as opposed to main
chain interactions, dominate protein-protein interactions. Recent
studies indicate that only a few of these side chains contribute most
of the binding energy(23, 24, 25, 26) .
An extensive analysis of growth hormone epitopes for several murine
monoclonal antibodies revealed the following hierarchy for side chain
contributions to the binding energy: Arg > Pro > Glu
It is clear from our study that a significant
amount of binding energy for A2 inhibitors is present in the
Arg
Because epitopes are usually made up of more than one fold because
they consist of either discontinuous segments or a continuous segment
in a loop region(20, 22, 29) , we cannot exclude
the possibility that part of the A2 epitope lies outside the
484-508 region. Ware et al.(10) found that a
triple mutation (Glu
We have shown that hybrid human/porcine
fVIII molecules can be used to locate a major determinant of the A2
inhibitor epitope. The region we have identified is sufficiently small
that site-directed mutagenesis may be used to characterize the
contribution of individual side chains to the binding of fVIII to A2
inhibitors. Additionally, hybrid molecules may also be useful for
characterizing the remaining inhibitor epitopes in fVIII. Further
characterization of the inhibitory epitopes in fVIII may lead to the
construction of recombinant fVIII molecules to deal with the inhibitor
problem in hemophilia A.
Identical residues are marked by asterisks.
We gratefully acknowledge the technical assistance of
Renard Rawls, Chris Skoog, and Dr. Hiba Tamim and Dr. Kenneth
Kaushansky (University of Washington) for advice on SOE mutagenesis.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and Ile
contains a major determinant of the A2 epitope. mAb 413 did not
inhibit two more hybrids that contained porcine substitutions at
residues 484-488 and 489-508, indicating that amino acid
side chains on both sides of the Ser
-Arg
bond within the Arg
-Ile
segment
contribute to the A2 epitope. The 484-508, 484-488, and
489-508 porcine substitution hybrids displayed decreased
inhibition by A2 inhibitors from four patient plasmas, suggesting that
there is little variation in the structure of the A2 epitope in the
inhibitor population.
(
)develop either in about 25% of hemophilia A patients
following fVIII infusions or, rarely, as autoantibodies in
nonhemophiliacs. Most inhibitors are directed against epitopes located
in the A2 domain (residues 373-740) or the C2 domain (residues
2173-2332) of fVIII(1, 2, 3) .
Additionally, an epitope of unknown significance has been identified
within residues 1690-2172 in the A3 or C1
domain(3, 4, 5) . C2 inhibitors act by blocking
the binding of fVIII to phospholipid(6) . Additionally, they
inhibit the binding of fVIII to von Willebrand factor(7) ,
although the clinical significance of this is not known. A2 inhibitors
bind noncompetitively to the intrinsic pathway factor X activation
complex (fVIIIa/factor IXa/phospholipid) and block factor X
activation(8) . Competition antibody binding assays indicate
that the A2 antigenic region is either a single epitope or a narrowly
clustered set of epitopes(9) .
-terminal region of the A2 domain and the C2 epitope
to residues 2170-2327(2, 10) . Deletion mapping
studies are informative when immunoreactivity is retained in the
deletion mutant. However, it is difficult to interpret results from
nonreactive mutants because of the possibility of misfolding and loss
of antigenic structure. Recently, we eliminated the A2 epitope in a
recombinant fVIII molecule by substituting porcine residues
387-604 for the homologous human sequence(11) . Because
this hybrid was active, the substituted region is likely to have a
native conformation. This finding was consistent with the deletion
mapping studies and demonstrated the feasibility of using hybrid
human/porcine fVIII molecules for detailed structural analysis of fVIII
inhibitor epitopes. In this study, we constructed a series of active
hybrids that contained progressively smaller segments of porcine fVIII
to localize a major determinant of the A2 epitope to residues
Arg
-Ile
.
Materials
Proteins
, 0.01% Tween 80, pH 7.4. The
flow through containing heavy chains was adjusted to pH 7.4 with 1 M Tris-Cl, pH 8.5, and applied to a Resource Q column
equilibrated in 0.15 M NaCl, 20 mM HEPES, 5
mM CaCl
, 0.01% Tween 80, pH 7.4. Heavy chains were
eluted with a NaCl gradient (0.15-1 M). Typical recovery
of heavy chains was 60-70%. Purity was confirmed by
SDS-polyacrylamide gel electrophoresis.
Affinity Purification of Inhibitor Antibodies
, 50% ethylene glycol, pH 7.5. Yields of inhibitor
activity ranged from 20 to 75%. Bovine serum albumin was added to 0.5
mg/ml as a stabilizing agent to the pooled, eluted IgG.
Construction and Expression of Hybrid Human/Porcine FVIII
Molecules
, were gifts from Biogen, Inc. HB
lacks the entire B domain, which is defined as the
Ser
-Arg
cleavage fragment produced
by thrombin. All DNA constructs containing modified sequences were
prepared using Bluescript II K/S- (Stratagene) as the cloning
vector. Modified sequences were transferred into the ReNeo expression
vector using SpeI/ApaI fragment replacement (see Fig. 1). ReNeo plasmid DNA was purified using a Qiagen Plasmid
Maxi kit (Qiagen, Inc.) and was used for subsequent transfections into
baby hamster kidney cells. All novel sequences of DNA generated by the
polymerase chain reaction were confirmed by dideoxy
sequencing(15) .
Figure 1:
Hybrid B-domainless
human/porcine fVIII constructs and their inhibition by mAb 413. Black regions correspond to areas of porcine substitution
whose boundaries are defined by residues of human/porcine nonidentity. Hatched regions correspond to regions of identity between
human and porcine fVIII that are contained within splice sites. The 5`
and 3` arrows show positions of a and d primers used for SOE
mutagenesis. BU, Bethesda units of anti-fVIII inhibitory
activity.
Construction of fVIII Hybrids HP4 and HP5 by Restriction
Fragment Insertion
HP4 and HP5 were prepared by restriction
fragment insertions using HB and HP1, a fVIII cDNA
containing a complete substitution of the human A2 domain by the
porcine A2 domain (11) (see Fig. 1). A SpeI/BglII fragment from HP1 was inserted into SpeI/BglII-cut HB
to produce the
HP4 cDNA, which encodes a porcine substitution of human residues
387-541. Similarly, an AlwN1/AlwN1 fragment
from HP1 was inserted into AlwN1/AlwN1-cut
HB
to produce the HP5 cDNA, which encodes a porcine
substitution of human residues 387-508. Prior to this, the single AlwN1 site in Bluescript II K/S- was removed, and
HB
was inserted into the NotI site in the
plasmid polylinker region.
Construction of fVIII Hybrids HP6 through HP13 by
Splicing-by-overlap Extension (SOE) Mutagenesis
SOE mutagenesis
of fVIII was performed as described by Horton et
al.(16) . This method consists of two rounds of polymerase
chain reactions using outer primer pairs (``a'' and
``d''), which are common to reactions with all constructs,
and inner primer pairs (``b'' and ``c''), which
define the splice site. HP6, HP7, HP8, HP9, HP10, and HP11 were
prepared using HB and HP5 as the templates for the
two first round polymerase chain reactions. HP12 and HP13 were prepared
using HB
and HP9 as the templates for the two first
round polymerase chain reactions. The 5` outer primer, a,
5`-GGCATGGAAGCTTATGTCAAAGTAN-3`, contains the AccI restriction
site in the A1 domain (see Fig. 1). The 3` outer primer, d,
5`-CCTCTTTGGTCTACAGATTCTTTG-3`, lies outside the BglII
restriction site in the A2 domain. The inner primers were i)
5`-ATAAAGTAAAGGTCCCA-3` and 5`-TGGGACCTTTACTTTAT-3` for both HP6
(porcine substitution 387-444) and HP7 (porcine substitution
468-508), ii) 5`-TGAGGGTAGATGTTATATGGTC-3` and
5`-GACCATATAACATCTACCCTCA-3` for HP8 (porcine substitution
387-468) and HP9 (porcine substitution 484-508), iii)
5`-GACTTTTATAACTTCTGTCA-3` and 5`-TGACAGAAGTTATAAAAGTC-3` for both HP10
(porcine substitution 387-403) and HP11 (porcine substitution
410-508), and iv) 5`-TGAATACAAAGGACGGACATC-3` and
5`-GATGTCCGTCCTTTGTATTCAGGGAGACTTCTAAAAGG-3` for HP12 (porcine
substitution 489-508) and 5`-TGGGTGCAAAGCGCT-3` and
5`-AGCGCTTTGCACCCAAGGAGATTACCAAAAGGTG-3` for HP13 (porcine substitution
484-488). HP6 and HP7, HP8 and HP9, HP10 and HP11, and HP12 and
HP13, represent complementary pairs (see Fig. 1). Complementary
pairs are generated by reversing the templates in the first round
polymerase chain reactions. Inner primers are identical between pairs
if sequences are identical at the splice site (HP6/HP7 and HP10/HP11).
In all constructs, AccI/BglII fragments were inserted
into HB
in Bluescript that had been cut with AccI and BglII to generate the final Bluescript cDNA
products.
Expression of HB
and Hybrid
Proteins
and hybrid proteins initially were transfected
into COS-7 cells to confirm that active protein could be expressed.
Then they were stably transfected into baby hamster kidney cells and
selected for G418 resistance as described previously (11) except
that expressing cells were maintained in growth medium containing 400
µg/ml G418. Maximum expression was obtained using Nunc triple
flasks. Cells from Corning T-75 flasks grown to 80-90% confluence
were transferred to Nunc triple flasks in medium containing 100
µg/ml G418 and grown to approximately 90% confluence. The medium
was removed and replaced with fresh medium without G418. FVIII
expression was monitored by coagulation assay and 100-150 ml of
medium was collected twice daily for 4-5 days. Typical expression
levels were 60 µg (300 units) of fVIII/200 ml of medium/24 h.
Pooled medium was stored at 4 °C in 0.05% (w/v) sodium azide.
,
0.005% Tween 80, 0.02% sodium azide, pH 7.40. Medium (100-200 ml)
from expressing cells was applied to the column, which then was washed
with 30 ml of equilibration buffer without sodium azide. FVIII was
eluted with 0.65 M NaCl, 20 mM HEPES, 0.01% Tween 80,
pH 7.40, and was stored at -80 °C. The yield of fVIII
coagulant activity was typically 50-75%.
FVIII Assays
was used to calculate the concentration
of fVIII and to determine a specific coagulant activity of 5000
units/mg. Inhibitor titers were measured by the Bethesda assay (18), in
which recombinant fVIII was added to hemophilia A plasma to a final
concentration of 1 unit/ml. In the plasma-free assay, the activity of
the intrinsic pathway factor X activation complex (intrinsic fXase),
consisting of factor IXa, activated fVIII, and phospholipid, was
measured under conditions in which the intitial velocity of factor X
activation was directly proportional to the concentration of activated
fVIII. fVIII preparations (0.025-0.2 nM) were activated
by 40 nM porcine thrombin in the presence of 0.5 nM factor IXa, 425 nM factor X, 50 µM phosphatidylcholine/phosphatidylserine vesicles in 0.15 M NaCl, 20 mM HEPES, 5 mM CaCl
, 0.01%
Tween 80, pH 7.40. After 5 min at room temperature, the reaction was
stopped with 0.05 M EDTA and 100 nM desulfatohirudin.
Factor Xa generated by the reaction was determined using a chromogenic
substrate assay(14) .
Synthetic Peptides
Mapping the Human A2 Epitope for Monoclonal Antibody
413
Previously, we eliminated the A2 epitope in fVIII by making
a recombinant hybrid human/porcine molecule, HP2, that contains a
substitution of a porcine sequence corresponding to residues
387-604 (11) (Fig. 1). This region had previously been
shown to contain an inhibitor epitope(2, 10) . In the
present study, we made a series of hybrid fVIII proteins containing
smaller substitutions of porcine fVIII in the NH-terminal
region of the A2 domain (Fig. 1). After initial screening by
transient protein expression in COS-7 cells, these constructs were
stably expressed in baby hamster kidney cells and partially purified
for assay with A2 inhibitors. The specific coagulant activity of the
hybrid proteins was indistinguishable from that of B-domainless and B
domain-containing fVIII.
-terminal and COOH-terminal ends of the insertions.
The regions of identity are shown in Fig. 1as hatched regions. Thus, the spice sites for the HP6-HP7, HP8-HP9,
HP10-HP11, and HP12-HP13 pairs are at residues 444-445,
483-484, 403-404, and 488-489, respectively. mAb 413
did not inhibit HP7, HP9, and HP11, but it inhibited their
complementary pairs, HP6, HP8, and HP10, as well as wild-type
HB
(Fig. 1). The lack of inhibition of HP9
indicates that amino acid side chains in the 484-508 region
contribute significantly to the binding of human fVIII to mAb 413.
Additionally, the inhibition of HP8 by mAb 413 suggests that the
373-483 region is not part of the A2 epitope.
-Arg
bond.
of mAb 413 for
HP9, HP12, and HP13 was greater than 0.8 µM (Fig. 2), whereas the IC
for HB
and HP8 was
3 nM. These results are consistent with
the Bethesda assay and they indicate that mAb 413 binds HP9, HP12, and
HP13 more than 3 orders of magnitude less tightly than HB
and HP8.
Figure 2:
Inhibition of control, B-domainless fVIII,
and hybrid human/porcine fVIII molecules by mAb 413. fVIII activity was
measured in a plasma-free intrinsic fXase assay as a function of mAb
413 concentration as described under ``Experimental
Procedures'' and expressed as the percentage of activity observed
in the absence of mAb 413. FVIII constructs: ,
HB
;
, HP8;
, HP9; ▾, HP12;
, HP13.
Peptide Inhibition of mAb 413
Synthetic
COOH-terminal carboxamide peptides corresponding to HP9 region residues
Arg-Phe
in human fVIII and
Ser
-Phe
in porcine fVIII were tested
in the plasma-free assay for their ability to block the inhibition by
mAb 413. Fig. 3shows the concentration dependence of inhibition
of fVIIIa by mAb 413 in the presence and the absence of 25 µM peptide. The human peptide shifts the inhibition curve to the
right (
IC
= 6 nM). The porcine
peptide also reproducibly shifts the inhibition curve to the right, but
its effect is significantly weaker (
IC
= 1
nM). There was no further inhibition by either peptide at
concentrations up to 250 µM (data not shown). These
results indicate that mAb 413 can specifically recognize the human
peptide but that the peptide can only partially block mAb 413
inhibitory activity.
Figure 3:
Inhibition of mAb 413 inhibition by human
and porcine segment 484-509 peptides. Purified human fVIII was
incubated with increasing concentrations of mAb 413 for 10 min at room
temperature in the presence and the absence of 25 µM peptide prior to activation with thrombin and measurement of fVIII
activity by plasma-free assay. , mAb 413 only;
, mAb 413
plus porcine peptide;
, mAb 413 plus human
peptide.
Inhibition of Hybrid fVIII by Heavy Chain-specific
Patient IgG
Usually inhibitor plasmas consist of a mixture of
antibodies against both A2 and light chain epitopes(3) . We
isolated heavy chain-specific (A1, A2, and B domains covalently linked)
antibodies from four patients by chromatography on human heavy chain
Affi-Gel. Patients SC, JM, and NS are nonhemophiliacs with anti-fVIII
autoantibodies, and RC is a hemophiliac with alloantibodies. All four
affinity-purified antibodies were shown to be more than 95% neutralized
by recombinant A2 domain(9) , indicating that they contain only
anti-A2 inhibitors. The concentration-dependent inhibition of
HB, HP8, HP9, HP12, and HP13 by these antibodies was
compared with mAb 413 by fVIII coagulation assay (Fig. 4). All
antibody preparations inhibited HB
and HP8 similarly,
as expected. There was less than 50% inhibition of HP9, HP12, and HP13
by mAb 413 and antibodies from SC, JM, and NS at the highest antibody
concentrations used. RC antibody inhibited HP12 and HP13 by 50 and 90%,
respectively, at the highest concentrations of antibody used, but they
were inhibited more weakly than HB
at lower
concentrations. These results indicate that mAb 413 and SC, JM, NS, and
RC antibody A2 inhibitors recognize a very similar epitope. The
inhibition of HP13 by RC antibody indicates that its major binding
determinant is located within residues 489-508.
Figure 4:
Inhibition of fVIII and hybrid
human/porcine fVIII by A2 inhibitors. Residual fVIII coagulant activity
was measured after incubation with antibody as defined by the Bethesda
assay (18). Abscissa values refer to final antibody (Ab)
concentrations in the assay. FVIII constructs: ,
HB
;
, HP8;
, HP9; ▾, HP12;
, HP13.
-Ile
(Fig. 1).
Although there is always concern when using recombinant proteins that
the observed differences in binding may be due to misfolding of the
protein, the hybrids used in this study had normal procoagulant
activity, which minimizes this possibility.
Asp
Phe
Ile, with Trp, Ala, Gly, and Cys not
tested(24) . Our results are consistent with this, because 12 of
the 25 residues in the 484-508 A2 segment contain these side
chains ().
-Ile
sequence. The dissociation
constant for the binding of mAb 413 to fVIIIa A2 is
0.1
nM(8) , which corresponds to standard free energy of
binding of -13.6 kcal/mol (from
G
= RT ln K
). HP12
and HP13 have nonoverlapping substitutions of porcine sequence and do
not have a measurable affinity for mAb 413. From the inhibition curves
in Fig. 2, we estimate that the dissociation constant is greater
than 0.8 µM. This corresponds to a
G
of greater than 5.3 kcal/mol for each mutant. Thus, greater than
10 kcal/mol binding free energy is lost by substitution with porcine
side chains in the 484-508 region. The sum of free energy changes
due to individual side chain replacement is greater than the standard
free energy of binding because of side chain
interactions(27, 28) . Therefore, changes in standard
free energy due to substitutions or mutations can only be used
semiquantitatively to study the overall interaction. Additionally, it
is possible that the substituted porcine side chains themselves
adversely affect inhibitor binding. Nevertheless, the results with HP12
and HP13 suggest that most of the A2 epitope binding energy of mAb 413
and the four human inhibitors is in the 484-508 segment.
Gln, Glu
Gln, and Glu
Gln) of an A2 inhibitor reactive
fVIII deletion polypeptide expressed as a fusion protein in Escherichia coli abolished A2 inhibitor reactivity. This
suggests that the Glu
-Glu
segment,
which is conserved in human and porcine fVIII(30) , contains
part of the A2 epitope. Alternatively, loss of antigenic structure due
to misfolding of this substitution mutant could also account for the
loss of inhibitor binding.
Table: Sequence of human and porcine FVIII from
residues 484-508
, B-domainless
fVIII (lacking residues 741-1648); SOE, splicing-by-overlap extension;
MES, 2-(N-morpholino)ethanesulfonic acid; mAb, monoclonal
antibody.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.