From the Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455
Received for publication, November 14, 2002, and in revised form, December 20, 2002
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ABSTRACT |
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Site-directed mutagenesis of the 40 N-terminal residues ( Interaction between the vitamin K-dependent
plasma proteins and a membrane surface is essential for hemostasis (1,
2). This interaction is mediated through contact of the
The Gla domains of the different vitamin K-dependent plasma
proteins show striking sequence homology, yet have quite different affinities for phospholipid membranes (8). If the Gla domain constitutes the membrane contact region, individual amino acid residues
within this domain should contribute to these different affinities.
This suggestion has been supported by mutations of the Gla domain that
affect membrane affinity. For example, the P10H mutant of bovine
protein C shows an ~10-fold enhancement in membrane affinity, whereas
the H10P mutant of human protein C shows an ~3-fold decline in
membrane affinity (9). Although a major difference among the vitamin
K-dependent proteins is a Gla residue versus
another amino acid at position 32, the Q32E mutation of protein C has
no impact on membrane affinity (10, 11). Similarly, replacement of
Glu32 in factor X has little impact on protein function
(12), and Glu32 of human prothrombin was described as only
moderately important (13), having little impact on either
Km or Vmax of the
prothrombinase reaction. However, a double mutant of human protein C,
S11G/Q32E, shows an ~10-fold enhancement in membrane affinity (11),
and a double mutant of factor VII, P10Q/K32E, has a 25-fold enhancement
in membrane affinity (14). Under appropriate conditions, these proteins
show similar improvement in function (11, 14-16).
Although previous studies showed that proteins with enhanced membrane
affinity can be created, the contribution of individual amino acid
residues was not determined, and possible improvement by changes in
other sites was not shown. This study used human factor VII (FVII) as a
model Gla domain to investigate these questions and presents a simple
functional assay that allows evaluation of minute quantities of impure
protein as an initial screen of mutant protein activity. Single-site
mutants as well as those with the highest affinity were purified and
characterized. The results indicate that nearly all functional
improvements arose from membrane binding affinity. The largest
enhancements in membrane affinity resulted from mutation in the region
of residue 32, a position that is located quite far from the N-terminal
end of the protein, where membrane contact is generally suggested (39). The mutant with highest function, (Y4)P10Q/K32E/D33F/A34E, showed 150-296-fold improvement over wild-type FVII. Overall, the proteins described in this study contribute to a better understanding of the
membrane contact site and the binding forces involved while providing
novel reagents to probe coagulation reaction mechanisms that may be of
use in therapy.
Protein Expression and Purification--
Cloning and mutagenesis
were performed by ATG Inc. (Eden Prairie, MN) following standard
procedures (17). Human FVII cDNA was cloned from a human liver
cDNA library and then subcloned into the vector pRc-CMV.
Mutagenesis was verified by sequencing of the entire Gla domain of all
variant FVII proteins, including untranslated pre- and propeptide
segments. Proteins were expressed in fetal human kidney 293 cells that
were stably transfected using the agent LipofectAMINETM
2000 (Invitrogen) following the manufacturer's instructions. Following
previously outlined procedures (9), Geneticin-resistant colonies were
selected, and high producing clones were grown to confluence in three
layered flasks (Nunc) containing Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 1.0 mM
nonessential amino acids, 50 units/ml penicillin, 50 µg/ml streptomycin, 10 µg/ml vitamin K1, and 100-200 µg/ml
Geneticin. Confluent cells were rinsed with and then cultured in
serum-free Dulbecco's modified Eagle's medium containing 1.0 mM nonessential amino acids, 10 µg/ml vitamin
K1, and 0.5 mM benzamidine hydrochloride. EDTA
(pH 7.4) and benzamidine hydrochloride were added to conditioned medium
intended for purification to concentrations of 5.0 and 2.0 mM, respectively. The conditioned medium was
vacuum-filtered twice through Whatman double-filter paper (qualitative
No. 1) and then through a 0.22-µm polyether sulfone membrane (Corning Inc.). If not immediately purified, the filtered medium was stored at
The filtered medium was diluted 1:1 with distilled and deionized water
containing 4.0 mM EDTA (pH 7.4) and 2.0 mM
benzamidine hydrochloride and then applied to a High Q
Macro-Prep anion-exchange column (Bio-Rad). The column was
equilibrated prior to loading and washed extensively with Tris buffer
(50 mM Tris, 100 mM NaCl, and 0.02% (w/w)
NaN3 (pH 7.4)) containing 2.0 mM EDTA and 2.0 mM benzamidine hydrochloride. The column was eluted
isocratically with the same buffer containing 400 mM NaCl
and no EDTA.
Eluted fractions containing FVII activity were pooled and diluted 1:1
with Tris buffer containing 30 mM CaCl2 and 2 mM benzamidine hydrochloride. The pooled and diluted
fractions were subjected to immunoaffinity chromatography using a
calcium-dependent anti-human FVII monoclonal antibody
(CaFVII22, provided by Dr. Walter Kisiel) coupled to
Affi-Prep®Hz support (Bio-Rad). The column was washed with
Tris buffer containing calcium and then eluted with Tris buffer
containing 15 mM EDTA and 2.0 mM benzamidine
hydrochloride. Fractions containing FVII activity were pooled and
subjected to a final ion-exchange chromatography step using a
Mono Q HR5/5 anion-exchange column (Amersham Biosciences). The column
was equilibrated and washed extensively with Tris buffer. Proteins were
eluted with a linear gradient of 100-500 mM NaCl over 30 min (flow rate of 1.0 ml/min). Eluted protein was concentrated by
centrifugation filtration (Millipore Ultrafree,
Mr 10,000 cutoff) and then stored at FVII Protein Determination--
A commercial preparation
of FVIIa (NovoSeven, Nordisk) was used as the standard for all
measurements. Protein concentrations were determined by the
Bradford assay (40). The amidolytic activity for the chromogenic
substrate S-2288 (Chromogenix) was also used as a standard of
comparison. Plasma clotting assays, described previously (15), were
used to determine FVIIa concentration. To ensure that FVIIa was the
limiting component, the assay was conducted in the presence of excess
TF (Innovin). TF concentrations in the Innovin preparation were
determined from the concentration of NovoSeven needed to generate
maximum activity. The TF concentration in the preparation used in this
study was 2.7 nM. FVII was activated by mixing either crude
or purified protein with 20 µl of Innovin solution, followed by
incubation at 37 °C until activation was complete. (Typically, 1.0 µl of solution resulted in a final concentration of ~0.3
nM FVII.) Activation of FVII was monitored by adding 2.65 µl of the activation mixture to 112.5 µl of prewarmed standard Tris
buffer (0.05 M) containing 100 mM NaCl,
1.0 mg/ml bovine serum albumin, and 6.67 mM
CaCl2. To initiate coagulation, 37.5 µl of prewarmed
FVII-deficient plasma (Sigma) was added, and clotting time was
determined by the hand tilt-test method. Full activation occurred
within 1 h, and concentrations of FVIIa were determined by
comparison with the NovoSeven standard. The results for the different
assay comparisons gave the same protein concentration within the
estimated error of the assays (±20%).
Active site-blocked wild-type FVIIa (WT-FVIIai) was produced as
previously described (15) using the active-site inhibitor dansyl-glutamylglycylarginyl chloromethyl ketone (Dr. Walter Kisiel) and NovoSeven as the source of WT-FVIIa. WT-FVIIai concentrations were determined using the Bradford assay (40).
Preparation of Small Unilamellar Vesicles--
Highly pure
phospholipids in organic solvent (bovine brain phosphatidylserine (PS)
and egg phosphatidylcholine (PC), Sigma) were mixed at proper ratios
and thoroughly dried, first under a stream of argon gas and then under
vacuum for 14 h. Dried phospholipids were solubilized in Tris
buffer and sonicated on ice for 3-s bursts every 10 s for 3 min
(Heat Systems-Ultrasonics W-385). This was repeated four times. The
sonicated vesicles were applied to a Sepharose 4B size-exclusion column
(Amersham Biosciences). Eluted fractions containing vesicles with
average diameters of 32-38 nm (LSA2 photon correlation spectrometer,
Langley Ford Co.) were pooled for use in membrane binding studies. The
concentrations of phospholipid were determined by phosphorus analysis
(18) using a phospholipid/phosphorus weight ratio of 25:1.
Light Scattering Measurements--
Protein-membrane binding was
determined by relative light scattering at 90° using methods
previously described (19). Briefly, for a constant particle
concentration and for particles that are small compared with the
wavelength of light, the ratio of the light scattering intensity of a
protein·vesicle complex (I2) to that of the
vesicles alone (I1) is related to the ratio of
the molecular weight of the protein·vesicle complex
(M2) to that of the vesicles alone
(M1) by the relationship in Equation 1,
Matrix-assisted Laser Desorption Ionization Time-of-Flight
(MALDI-TOF) Analysis--
MALDI-TOF mass spectrometry was used to
confirm the identity of the variant FVII proteins tested and to assess
the extent of carboxylation. Purified proteins were incubated at
37 °C for 30 min in the presence of either chymotrypsin or trypsin
at a ratio of 500:1 (w/w) FVII protein/protease. Reaction solutions were dried by vacuum centrifugation and solubilized in a 5:95 acetonitrile/water solution containing 0.1% trifluoroacetic acid. The
solutions were desalted using reverse-phase chromatography (ZipTip,
Millipore Corp.), mixed 1:1 with a saturated matrix solution (5-methoxysalicylic acid in 50:50 ethanol/water solution), spotted on
the spectrometer target, allowed to dry, and then subjected to
MALDI-TOF mass spectrometry (BiflexTM III, Bruker). Minimum
laser power was used to obtain spectra. Moderate increases in power
above this minimum did not result in changes in the distribution of the
various carboxylated species observed. The percentage of each
carboxylation species was determined by measurement of peak areas using
integration software provide by the spectrometer manufacturer.
Factor X Activation by FVIIa--
The relative activities of
FVIIa variants were determined by a method outlined previously
(15). Full activation of FVII was first ensured by incubation
with TF (18 pM; Innovin) in 50 µl of Tris buffer
containing 5.0 mM CaCl2 and 1.0 mg/ml bovine serum albumin. The mutant FVII proteins with higher membrane affinity (K32E, P10Q/K32E, P10Q/D33E, and (Y4)P10Q/K32E/D33F/A34E) were added to
a final protein concentration of 3.0 nM. WT-FVIIa, P10Q, and K32E were added to a final concentration of 10 nM. Full
activation of the FVIIa preparation was achieved after incubation for
1 h. A range of WT-FVIIai concentrations was added, and the
mixture was allowed to equilibrate for another 2 h at 37 °C.
Factor X (Enzyme Research Laboratories) was added to a concentration of 200 nM to initiate the reaction. After 10 min, the reaction
was stopped by addition of excess EDTA (15 mM). The
concentration of factor Xa was measured as activity for chromogenic
substrate (0.32 mM S-2222, Chromogenix) by monitoring the
absorbance change at 405 nm in a Beckman DU-70 UV-visible
spectrophotometer. The amount of FVIIa that was bound to tissue factor
(FVIIa·TF) was estimated from the activity observed relative to that
of a standard with no WT-FVIIai. The concentration of WT-FVIIai bound
to TF (WT-FVIIai·TF) was estimated from the fraction activity that
was lost. Results are presented in a Hill-type plot represented by Equation 3 (15),
Clotting Activity of FVII Proteins from Purified and Crude
Sources--
Pure FVII preparations or conditioned medium
containing FVII were added to Innovin (0.1 ml) in an amount to generate
~0.3 nM FVII. This concentration of pure FVII provided a
final coagulation time of 25 s for all samples. Because all FVII
variants have the same clotting time in the absence of inhibitor (see
pure protein analysis below and Ref. 15), use of a constant clotting
time allowed the determination of the FVII concentration in an
unpurified sample. It was important that TF was maintained in excess
over FVII. Activation of FVII was allowed to proceed to completion (60 min at 37 °C). Activated FVII solution (containing 2.5 µl of
Innovin) plus varying amounts of WT-FVIIai were mixed with Tris buffer
containing 6.67 mM CaCl2 and 1.0 mg/ml bovine
serum albumin to create 112.5-µl aliquots, which were incubated for 1 h at 37 °C to achieve equilibrium binding between TF,
WT-FVIIai, and FVIIa. Coagulation was initiated by addition of 37.5 µl of prewarmed FVII-deficient plasma. Clotting times (CT) were
determined, and results were evaluated by a plot of log(CT/CTo)
versus log[WT-FVIIai], where CTo is the clotting
time without inhibitor. Relative function of the different FVIIa
variants was estimated by offset of the plots for the two proteins.
Analysis of Protein Carboxylation States by MALDI-TOF Mass
Spectrometry--
The identities of purified recombinant FVII proteins
were verified by mass spectrometry of Gla domains released from the
intact proteins by limited protease digestion. The Gla domains
consisted of either 1-40 (containing all Gla residues) or 1-32 (less
one carboxylation site at residue 35) N-terminal residues. Use of the
methoxysalicylic acid matrix and the lowest practical laser power
resulted in a low level of undercarboxylated peptide species (Fig.
1). In most cases, the fully carboxylated
peptide (theoretical m/z of 5235 for the +1
charge state of K32E) was the most abundant peak (Fig. 1A).
However, in-source decarboxylation of Gla residues occurred during
MALDI-TOF analysis and reached quantitative levels when the sinapinic
acid matrix was used (20).
Undercarboxylation was detected by peaks separated by 44 mass units and
a small peak corresponding to the fully decarboxylated peptide
(m/z 4751) (Fig. 1A). The quantitative
distribution among the different species was very consistent for
replicate samples as well as for many plasma-derived versus
recombinant proteins (20). This consistency suggested that comparison
of quantitative MALDI-TOF data could be used to detect relative
differences in the carboxylation states of various proteins. For
example, repeated measurement of different preparations of
plasma-derived bovine factor X gave 77 ± 4% signal intensity in
the fully carboxylated peptide. This level of fully carboxylated
peptide corresponded to 97-98% carboxylation of all Glu residues.
Similar results were obtained for bovine prothrombin and human protein
C. Consequently, the somewhat lower yield of the fully carboxylated
peptide of recombinant WT-FVII (46%) suggested undercarboxylation of
the parent protein (Fig. 1B). In fact, undercarboxylation at
position 35 of recombinant WT-FVIIa has been previously observed (21, 22). That position 35 of recombinant WT-FVII was the major site of
undercarboxylation was also suggested by MALDI-TOF analysis of peptide
1-32, which gave a high yield of the fully carboxylated state (70%)
(Fig. 1B). Undercarboxylation at position 35 of FVII (and a
corresponding residue in factor IX (23)) had no detected impact on the
activity of the mature proteins (21, 22).
The total carboxylation levels of WT-FVII and P10Q/K32E, determined by
this procedure, were 9.3 (of 10 theoretical) and 10.4 (of 11 theoretical) residues per Gla domain, respectively. These estimates
were nearly identical to the values of 9.6 ± 0.9 and 10.7 ± 0.8 obtained by amino acid analysis after base hydrolysis (14).
Comparative analysis by MALDI-TOF was used to estimate the
carboxylation states for the FVII mutants produced in this study. Five
of the seven proteins showed >93% of theoretical Gla levels (Fig.
1B, last column), suggesting a carboxylation
state of the parent protein similar to that of commercial FVIIa. Two
mutants showed substantially lower levels of carboxylation, 8.9 of 11 theoretical residues for P10Q/D33E and 10.9 of 12 theoretical residues
for (Y4)P10Q/K32E/D33F/A34E. The latter mutants contained additional
Glu residues beyond position 32. Given that undercarboxylation occurs
at position 35 of recombinant WT-FVIIa, it was possible that the
additional Glu residues at positions 33 and 34 were undercarboxylated as well. If correct, the functional impact of Gla residues at positions
33 and 34, detected in the following studies, may underestimate the
true impact of Gla at these positions.
Interaction of Purified FVII Variants with Phospholipid
Vesicles--
When small unilamellar vesicles of 25:75 PS/PC were used
to measure protein binding, the variant proteins displayed increasing membrane affinity in the order WT-FVII < P10Q < K32E (Fig.
2A). Mutants with higher
affinity (K32E) bound at the theoretical limit (Fig. 2A), so
equilibrium binding constants could not be estimated. Because binding
affinity is dependent on PS content of the membrane, use of a lower PS
content (10:90 PS/PC) (Fig. 2B) provided an equilibrium of
bound and free protein for most mutants. Affinity increased in the
order WT-FVII < P10Q < K32E < P10Q/D33E < P10Q/K32E. Dissociation constants estimated from these results are
reported in Table I. The
KD value obtained for P10Q/K32E (0.16 ± 0.08 µM) compared well with the value of 0.22 µM
for small unilamellar vesicles of 10:90 PS/PC reported by Shah et
al. (14). Even lower PS content was needed to estimate the binding
constant for the highest affinity mutant (5:95 PS/PC) (Fig.
2C). Estimated KD values indicated a
3-fold enhancement in membrane affinity for the (Y4)P10Q/K32E/D33F/A34E
mutant over the P10Q/K32E variant.
Activation of Factor X--
Functional evaluation of the FVIIa
mutants was carried out in a purified system that detected factor X
activation. Experiments were performed under equilibrium competition
conditions in which the FVIIa variants must compete with inhibitor,
WT-FVIIai, for TF (described in Ref. 15). To allow comparison of
results for different proteins, the FVIIa species and WT-FVIIai
concentrations were maintained in great excess over TF, so the total
FVIIa and FVIIai concentration approximated the respective free
concentration. The ability of the lowest affinity FVIIa variants (10 nM) to displace WT-FVIIai increased in the order
WT-FVIIa < P10Q < K32E (Fig. 3A). Mutants with higher
function were evaluated at 3 nM and gave increasing
affinity in the order K32E < P10Q/D33E < P10Q/K32E < (Y4)P10Q/K32E/D33F/A34E (Fig. 3B). The WT-FVIIai
concentrations required to reach 50% inhibition
(FVIIai·TF/FVIIa·TF = 1.0) are reported in Table I. In
agreement with the higher affinity of WT-FVIIai for TF (15, 24, 25),
its concentration at 50% inhibition was lower than that of
WT-FVIIa.
The data in Fig. 3 are presented in the manner of a Hill plot. The
plots have slopes of ~1.0, as anticipated. Validity of this analysis
was supported by titration at different FVIIa levels. For example, a
3.4-fold difference in inhibitor concentration (Table I) was observed
for titration of K32E at 3 nM (Fig. 3B) versus 10 nM (Fig. 3A). The
concentration of WT-FVIIai at the midpoint of each titration curve was
used to estimate the relative binding affinity of FVIIa variants. The
K32E, P10Q/K32E, and (Y4)P10Q/K32E/D33F/A34E mutants displayed 13.8-, 45-, and 149-fold increases in activity over WT-FVIIa, respectively. If
the sites make independent contributions to membrane affinity, the
13.8-fold enhancement for K32E and the 3.1-fold enhancement for P10Q
would suggest a 42.8-fold enhancement for P10Q/K32E. This was very
nearly the value that was observed. Thus, this functional assay
suggested that the P10Q and K32E modifications functioned in a manner
that was independent of each other.
Overall, the results of the functional assay in the purified system
under conditions in which the protein ligands (FVIIa and WT-FVIIai)
were in large excess over TF mirrored the differences in membrane
affinity observed in the phospholipid binding studies (Fig. 2). It
appeared that all improvements in function arose from changes in the
membrane contact site.
Functional Evaluation by Coagulation--
Coagulation assays were
conducted under conditions in which TF was more abundant than FVIIa. To
compare the relative function of different FVIIa variants
versus inhibition by WT-FVIIai, the concentration of free
FVIIai needed to approximate the concentration of total FVIIai. Low
affinity variants such as WT-FVIIa were displaced at low inhibitor
concentration, at which most of WT-FVIIai was bound to TF.
Consequently, comparison of protein function by this assay was limited
to FVIIa variants with high affinity. This included K32E and better.
These variants were displaced at WT-FVIIai concentrations that greatly
exceed the TF concentration, so total WT-FVIIai approximated free
WT-FVIIai in the solution.
The coagulation assay showed the same sequence of function observed by
other measurements: K32E = P10Q/D33E < P10Q/K32E < (Y4)P10Q/K32E/D33F/A34E (Fig. 4,
A and B). Comparison of inhibitor levels needed
to increase clotting time by 60% (log(CT/CTo) = 0.2)
suggested that (Y4)P10Q/K32E/D33F/A34E had up to 6.9-fold higher
function than P10Q/K32E. Use of this value and the enhancement of
P10Q/K32E over WT-FVIIa (43-fold) resulted in a total enhancement for
(Y4)P10Q/K32E/D33F/A34E of 296-fold (Table I).
This coagulation assay appeared useful for screening mutants in
conditioned media (Fig. 4B). In fact, results obtained using this crude protein source were very reproducible for different batches
of media and were indistinguishable from the results obtained for
purified proteins (Fig. 4A). Screening of FVIIa variants in conditioned media was used as a facile first method to estimate protein
function to identify the best proteins for purification. This assay
appeared amenable to high throughput analysis and might be used in
future studies to identify beneficial mutants from large libraries of
cells expressing proteins with different modifications. A logical
modification would be to quantify FVIIa activity by measuring the
product, factor Xa, by amidolytic assay.
The Tyr4, D33F, and A34E mutants were introduced
sequentially into P10Q/K32E to estimate their individual impact. These
proteins were evaluated only in the preliminary screening test. The
result showed that the Tyr4 insertion conferred a 2-fold
functional enhancement (data not shown) and that this was independent
of other changes in the Gla domain. Introduction of D33F also increased
the function of P10Q/K32E by 2-fold. Incorporation of D33F into
WT-FVIIa was not attempted, so it was not possible to determine whether
this change was independent of other changes. Introduction of A34E into
proteins that did not contain K32E had no detected influence on protein
function, but showed a 1.5-fold increase in function when introduced
into P10Q/K32E/D33F. The order of observed enhancements of function generally followed changes in membrane affinity, but appeared to
slightly exceed changes in KD values (Table I).
Human FVII displays one of the lowest affinities for phospholipid
membranes among the vitamin K-dependent plasma proteins (8). Modifications within the Gla domain of FVII (P10Q/K32E) have been
shown to enhance membrane affinity (14) and, under appropriate
conditions, to increase function of the protein (15). This study
identified several new sites that also enhance membrane affinity and
protein activity. Mutations were designed to test the independence and
relative contribution of each site. A simple assay, amenable to
screening large numbers of variant proteins, that used unpurified FVIIa
was shown to faithfully report changes in activity due to mutagenesis
of the Gla domain.
The interactions responsible for binding of the Gla domain of vitamin
K-dependent plasma proteins to phospholipid membranes are
not fully understood. The location of amino acids that enhance affinity
as well as the magnitude of the changes can offer information about the
membrane contact site and binding mechanism. All but one of the
beneficial changes reported here were clustered on the same surface of
the Gla domain (Fig. 5), suggesting a
possible membrane contact surface.
-carboxyglutamic acid domain) of blood clotting
factor VII was carried out to identify sites that improve membrane
affinity. Improvements and degree of change included P10Q (2-fold),
K32E (13-fold), and insertion of Tyr at position 4 (2-fold). Two other
beneficial changes, D33F (2-fold) and A34E (1.5-fold), may exert their
impact via influence of K32E. The modification D33E (5.2-fold) also
resulted in substantial improvement. The combined mutant with
highest affinity, (Y4)P10Q/K32E/D33F/A34E, showed 150-296-fold
enhancement over wild-type factor VIIa, depending on the assay used.
Undercarboxylation of Glu residues at positions 33 and 34 may result in
an underestimate of the true contributions of
-carboxyglutamic acid
at these positions. Except for the Tyr4 mutant, all
other beneficial mutations were located on the same surface of the
protein, suggesting a possible membrane contact region. An initial
screening assay was developed that provided faithful evaluation of
mutants in crude mixtures. Overall, the results suggest features of
membrane binding by vitamin K-dependent proteins and
provide reagents that may prove useful for research and therapy.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carboxyglutamic acid
(Gla)1 domains of these
proteins with membranes containing acidic phospholipids (3, 4). The Gla
domain consists of ~40 N-terminal residues of which 9-12 glutamic
acid residues are post-translationally modified to Gla (5-7). In the
presence of calcium, the Gla domain adopts structure and binds to
membranes by a mechanism that is not fully understood.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
70 °C.
SDS-PAGE analysis indicated that the proteins were highly pure and
contained only zymogen FVII and FVIIa in both nonreducing and reducing
gels. The percentage of FVIIa ranged from 40 to 95%. Prior to assay
for enzyme activity, the proteins were fully activated using a
commercial source of tissue factor (TF; Innovin, Dade Behring
Corp.).
where
(Eq. 1)
n/
c is the change in
refractive index as a function of concentration of the light scattering
species and was estimated as described (19). Light scattering
contributions of the buffer and protein were subtracted to obtain
I1 and I2. Values of
M2/M1 were estimated at
various amounts of added protein and were plotted versus the
protein (P)/phospholipid (PL) ratio (w/w). Dissociation constants
(KD) for protein-membrane binding were estimated from the relationship in Equation 2.
The amount of free protein ([P]free) was estimated
from the known weight concentrations of phospholipid and protein in the solution and the difference between
M2/M1 and the theoretical value of M2/M1 if all of
the added protein were bound. Weight concentrations of bound and free
FVII were converted to molar concentration with a Mr
of 50,000 for all FVII variants. The concentration of P·PL was
estimated from the known concentration of phospholipid and the ratio
M2/M1.
P·PLmax was calculated by assuming saturation binding at
a ratio of 1:1 protein/phospholipid in the complex
(M2/M1(max) = 2.0)
(19).
(Eq. 2)
(Eq. 3)
where KD(VIIai) is the
dissociation constant for the WT-FVIIai·TF complex and
KD(VIIa) is the dissociation constant
for the FVIIa·TF complex. Comparison of a plot of
log([WT-FVIIai·TF]/[FVIIa·TF]) versus
log[WT-FVIIai] for two FVIIa variants at an identical and constant
FVIIa concentration will allow estimation of their relative affinities
for TF. Equation 3 represents free protein concentrations. Therefore,
conditions were selected to ensure that the total protein concentration
was in large excess over TF, so total protein was approximately equal
to free protein.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
MALDI-TOF mass spectrometry of Gla
domains. Recombinant FVII proteins were digested with
either chymotrypsin or trypsin (500:1, w/w) at 37 °C for 30 min. The
peptides were desalted by the ZipTip procedure, mixed with matrix, and
subjected to MALDI-TOF mass spectrometry. A, a
representative spectrum for K32E; B, amino acid sequences
and yield of different carboxylation states. Shaded residues
indicate the sites of mutation and the amino acids that have been
incorporated. Gla residues, converted from glutamic
acid, are indicated by X. The percentages of the fully
carboxylated peptides or peptides minus one carboxyl group
(m/z 44) or minus two carboxyl groups are also shown.
Observed carboxylation versus theoretical number of Gla
residues is shown in the last column. Q10, P10Q;
E32, K32E; Q10E32, P10Q/K32E; Q10E33,
P10Q/D33E; Y4Q10E32F33E34, (Y4)P10Q/K32E/D33F/A34E.
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Fig. 2.
Protein binding to phospholipid
vesicles. A, binding to vesicles of 25:75 PS/PC;
B, binding to vesicles of 10:90 PS/PC; C, binding
to vesicles of 5:95 PS/PC. Titrations are shown for WT-FVII
( ), P10Q (
), K32E (
), P10Q/K32E (
), P10Q/D33E (
),
and (Y4)P10Q/K32E/D33F/A34E (
). Experiments were performed in
triplicate in Tris buffer containing 5.0 mM
CaCl2 and 62.5 µg/ml phospholipid. Means ± S.D. are reported. The vertical axis shows the ratio
of M2 (weight of the protein·vesicle complex)
to M1 (weight of the vesicles alone). The
dashed lines represent the theoretical maximum. The
least-squares method was used to fit linear equations to the data using
the program KaleidaGraph (Synergy Software).
Impact of mutagenesis on FVII activity and membrane affinity
View larger version (21K):
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Fig. 3.
Factor X activation by FVIIa variants.
Analysis of low (A) and higher (B) affinity
proteins. In A, 10 nM WT-FVIIa ( ),
P10Q (
), or K32E (
) was added to a solution containing 18 pM TF (Innovin), 5.0 mM CaCl2, and
1.0 mg/ml bovine serum albumin in Tris buffer. In B, 3.0 nM K32E (
), P10Q/K32E (
), P10Q/D33E (
), or
(Y4)P10Q/K32E/D33F/A34E (
) was added to the same solution. After an
incubation time that assured full activation of FVIIa in the solution
(30 min), WT-FVIIai was added at the concentrations shown, and the
reactions were allowed to equilibrate for 2 h at 37 °C. Factor
X was added to a final concentration of 200 nM, and the
incubation was continued for 10 min. The reaction was halted by
addition of excess EDTA (15 mM, 7.4 pH), and factor Xa
activity for the chromogenic substrate S-2222 was measured. Activity
was converted to the ratio of FVIIai·TF to FVIIa·TF and
plotted. All experiments were performed in triplicate and S.D. are
shown.
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Fig. 4.
Clotting activity of FVII variants from
purified (A) and unpurified (B)
sources. FVIIa proteins K32E ( ), P10Q/K32E (
),
P10Q/D33E (
), and (Y4)P10Q/K32E/D33F/A34E (
) were activated by
incubation with Innovin for 1 h. The activation mixture was
diluted to ~5.5 pM FVIIa and 45 pM TF, and
various levels of WT-FVIIai were added (112.5-µl total volume). After
sufficient time to allow equilibrium binding (1 h at 37 °C), 37.5 µl of prewarmed FVII-deficient plasma was added to initiate the
clotting reaction. Experiments were performed in triplicate, and all
data points plotted.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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[in a new window]
Fig. 5.
Gla domain of FVIIa showing the sites of
mutated amino acid residues. A space-filling model
(RasMol) is presented for the 1-50 amino acid residues of the light
chain of human FVIIa (Protein Data Bank code 1DAN). Residues 10 and
32-34 are presented in stick format. The locations of residues 3 and 4 are indicated to designate the location of the Tyr4 insert.
Bound calcium ions are presented in orange.
The exception was the Tyr4 insertion, which is located on
nearly the opposite surface in the protein, adjacent to the N-terminal structure referred to as the -loop (26). Insertion of an
additional hydrophobic residue at position 4 was motivated by sequence
comparison with bovine protein Z, a vitamin K-dependent
protein with very high membrane affinity. The
-loop of the Gla
domain provides three solvent-exposed hydrophobic side chains
(Phe4, Leu5, and Leu8 for human
FVII) (Fig. 5) (27). Although this region in bovine prothrombin
fragment 1 is a contact point in the crystal (26) and may be subject to
some distortion, these hydrophobic groups have been suggested to
constitute a central feature of the membrane-binding site (28-30).
Insertion of tyrosine (or phenylalanine; data not shown) at this
position resulted in a 2-fold increase in protein function. One
possible explanation for enhanced membrane binding is contact of the
Tyr4 side chain with the hydrophobic region of the
phospholipid membrane. However, hydrophobic insertion by the
-loop
has been questioned on many grounds (3, 31-35). The impact of the
Tyr4 insertion was small (2-fold;
G = 0.4 kcal/mol) compared with the free energy for transfer of a large
hydrophobic group such as the phenyl side chain from aqueous solution
(to octanol;
G =
3.6 kcal/mol for
KD = 400-fold) (36). The small impact of
the Tyr4 insertion suggested an indirect role in membrane association.
Replacement of Pro10, a residue present only in vitamin
K-dependent proteins displaying low membrane affinity, also
increased protein affinity by 2-3-fold. Assay by the preliminary
screen showed that any of a number of amino acid substitutions (Gln, Tyr, Glu, Leu, or Asn) at this position gave similar enhancement. The
small effect of Pro10 replacement and the large range of
permissible residues suggested a relatively peripheral effect. Thus,
this level of affinity enhancement may result from increased mobility
within this region and improved packing of hydrophobic groups near
H-bonding networks. Complete removal of a H-bond from an aqueous
environment results in a change in free energy of approximately 2.4
to
3.6 kcal/mol (37).
The largest single benefit was provided by introduction of glutamic
acid at position 32, producing a 13-fold enhancement as a single
independent site (G =
1.6 kcal/mol). Basic
residues occur at this position in human and bovine FVII. This change
in membrane affinity, which resulted in a large improvement in protein function, equates with a free energy change of ~15% of the total binding energy for the protein (KD ~ 10
7 M;
G ~
10
kcal/mol). Although not measured as independent changes, it was likely
that the D33F (2-fold enhancement) and A34E (1.5-fold enhancement)
mutations exerted their impact by indirect means such as by increasing
the pKa of Gla32, thereby increasing
affinity for ion pairs such as calcium binding. Gla32 has
been proposed to associate with an extra calcium ion during membrane
binding (8). It was reasoned that another Gla residue at position 34 may enhance this interaction, whereas addition of a large hydrophobic
side chain in this vicinity may serve to isolate this charge complex
from water molecules. The total impact of changes at positions 32-34
(39-fold;
G =
2.2 kcal/mol) may be sufficient
to suggest a direct role for this region in membrane association. This
conclusion is necessarily tentative and must be confirmed by additional study.
Completely independent contribution as a result of change at positions 10 and 32 was surprising. In the case of human protein C, simultaneous changes at residues 11 and 32 are required to realize enhancement (11). It is possible that Ser11 of human protein C hinders development of a structure, allowing contact of position 32 with calcium or the membrane.
Another surprising benefit was the D33E mutation. This change
introduced a 5.2-fold increase in function over P10Q. Taking into
consideration the observed undercarboxylation of the P10Q/D33E mutant,
possibly at position 33 (see below), it is possible that Gla33 offers a significantly larger enhancement and may
rival the impact of K32E. This was surprising for several reasons.
First of all, the charge difference and electrostatic effect of the
D33E mutation (charge =
1) were much smaller than those of
the K32E mutation (
charge =
3). Furthermore, the D33E
mutation would leave a cationic group at position 32 and would move the
Gla residue relatively far from any of the suggested membrane contact
regions. Clearly, additional effort is needed to identify the mechanism
and binding site of vitamin K-dependent proteins.
The highest affinity mutant that has been identified to date,
(Y4)P10Q/K32E/D33F/A34E, provided 146-296-fold enhancement over WT-FVIIa (G =
3.0 to
3.4 kcal/mol). The range
of functional enhancement arose from use of different assays. The
benefit of P10Q/D33E also differed with these assay methods. It is
possible that these method-dependent differences arose from
undercarboxylation of these two mutants, producing preparations with
different functional states. Assays that used excess FVIIa would detect
an average function for the entire preparation, resulting in an
overestimate of the function of the low affinity species and an
underestimate of the function of the high affinity species.
The coagulation assays also suggested heterogeneity of these preparations. Titration of K32E and P10Q/K32E gave plots with parallel slopes, indicating similar differences between these proteins at all levels of inhibition. Parallel plots would arise if both protein preparations were homogeneous but had different functional levels. Nearly all of the much larger number of mutants that were assayed by the initial screening test (Fig. 4B) gave similar curve shapes and slopes. The exceptions were mutants that contained Glu residues beyond position 32. These proteins, including P10Q/D33E and (Y4)P10Q/K32E/D33F/A34E, showed substantial inhibition at low WT-FVIIai concentrations, but great resistance to complete inhibition at high WT-FVIIai concentrations. This could arise from initial displacement of a low affinity FVIIa species (possibly an undercarboxylated protein), followed by competition with a high affinity FVIIa species (the fully carboxylated protein). MALDI-TOF analysis suggested undercarboxylation of these proteins, as well. Although further studies are needed, it is possible that the 5.2-fold increase in function arising from the D33E mutation (Table I) and the 149-fold increase in function of (Y4)P10Q/K32E/D33F/A34E actually underestimate the value for the fully carboxylated protein. The best analysis of the fully carboxylated protein (296-fold enhancement) (Table I) may be obtained by the coagulation test at high levels of FVIIai. Several future approaches may used to address this matter. For example, although complete separation of proteins by charge has proven difficult, cell lines may be found that provide more complete carboxylation of FVII polypeptides to generate proteins with the highest function.
Overall, this study has outlined a number of sites that improve
function of FVIIa and, by inference, suggested approaches that may
enhance the membrane affinity of other vitamin K-dependent proteins. Mutations within the Gla domain have allowed for
investigation of surfaces within this domain that may contribute to the
protein-membrane interactions. In contrast to the view of membrane
interaction centering around hydrophobic residues near the N terminus
(39), we have found that amino acid changes within the region of the Gla domain encompassing residues 32-34 resulted in the greatest enhancements in membrane affinity (Fig. 5). Access to proteins with a
range of function may prove useful in investigation of blood clotting
reactions both in vitro and in vivo. The large enhancements obtained may also provide benefit for therapies that are
based on the vitamin K-dependent proteins (38).
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ACKNOWLEDGEMENT |
---|
We thank Dr. Walter Kisiel for providing the calcium-dependent anti-human FVII antibody and WT-FVIIai used in this study.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
Molecular Biology, and Biophysics, 420 Washington Ave. SE, Minneapolis,
MN 55455. Tel.: 612-624-3622; Fax: 612-625-2163; E-mail:
nelse002@tc.umn.edu.
Published, JBC Papers in Press, December 27, 2002, DOI 10.1074/jbc.M211629200
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ABBREVIATIONS |
---|
The abbreviations used are:
Gla, -carboxyglutamic acid;
FVII, factor VII;
WT, wild-type;
WT-FVIIai, active site-blocked wild-type factor VIIa;
TF, tissue factor;
dansyl, 5-dimethylaminonaphthalene-1-sulfonyl;
PS, phosphatidylserine;
PC, phosphatidylcholine;
MALDI-TOF, matrix-assisted laser desorption
ionization time-of-flight.
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