The Propeptides of the Vitamin K-dependent Proteins
Possess Different Affinities for the Vitamin K-dependent
Carboxylase*
Thomas B.
Stanley
,
Da-Yun
Jin,
Pen-Jen
Lin, and
Darrel W.
Stafford§
From the Department of Biology, Center for Thrombosis and
Hemostasis, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599-3280
 |
ABSTRACT |
The vitamin K-dependent
-glutamyl
carboxylase catalyzes the modification of specific glutamates in a
number of proteins required for blood coagulation and associated with
bone and calcium homeostasis. All known vitamin K-dependent
proteins possess a conserved eighteen-amino acid propeptide sequence
that is the primary binding site for the carboxylase. We compared the
relative affinities of synthetic propeptides of nine human vitamin
K-dependent proteins by determining the inhibition
constants (Ki) toward a factor IX
propeptide/
-carboxyglutamic acid domain substrate. The
Ki values for six of the propeptides (factor X,
matrix Gla protein, factor VII, factor IX, PRGP1, and protein S) were
between 2-35 nM, with the factor X propeptide having the
tightest affinity. In contrast, the inhibition constants for the
propeptides of prothrombin and protein C are ~100-fold weaker than
the factor X propeptide. The propeptide of bone Gla protein
demonstrates severely impaired carboxylase binding with an inhibition
constant of at least 200,000-fold weaker than the factor X propeptide.
This study demonstrates that the affinities of the propeptides of the
vitamin K-dependent proteins vary over a considerable
range; this may have important physiological consequences in the levels
of vitamin K-dependent proteins and the biochemical mechanism by which these substrates are modified by the carboxylase.
 |
INTRODUCTION |
The vitamin K-dependent carboxylase catalyzes the
post-translational modification of specific glutamates to
-carboxyglutamate (Gla)1
in a number of proteins. Most vitamin K-dependent proteins
are involved in the hemostatic process (prothrombin, factors VII, IX,
and X, and proteins C, S, and Z), whereas two others (bone Gla protein
and matrix Gla protein) are associated with bone (1-4). Two new
putative vitamin K-dependent proteins of unassigned
function, proline-rich Gla proteins (PRGP1 and PRGP2), were identified
by sequence homology searches and are believed to be membrane proteins (5).
A conserved eighteen-amino acid sequence essential for substrate
recognition is found in all vitamin K-dependent proteins and was first identified by Pan and Price (6) based on sequence comparisons of the blood and bone vitamin K-dependent
proteins. The conserved region is present as a propeptide sequence
amino-terminal to the highly conserved Gla domains of the vitamin
K-dependent blood proteins and is proteolytically removed
to form the mature protein. With bone Gla protein, this sequence is
also present as a propeptide amino-terminal to the mature form of the
protein, whereas with matrix Gla protein the vitamin
K-dependent propeptide-like sequence is part of the mature
form of the protein (7). Confirmation of the importance of the
propeptide sequence in carboxylation is demonstrated by experiments
where deletion of the propeptide abrogates carboxylation of factor IX
or protein C expressed in cell culture (8, 9). In addition, mutagenesis
studies have identified a number of highly conserved amino acids
(e.g. Phe
16, Ala
10, Leu
6) as well as less conserved
positions (
17 and
15) whose mutation affects carboxylation. (8,
10-12). Glutamate-containing peptides with covalently linked
propeptides have 1000-fold lower Km values than
similar peptides without the propeptide sequence and are competitively
inhibited by free propeptide (13). A peptide containing the propeptide
and Gla domain of factor IX (FIXproGLA) has a sub-micromolar
Km for the carboxylase and can be fully carboxylated
in a processive manner (14, 15).
There is significant evidence that the vitamin K-dependent
propeptide sequence is the primary binding site for the carboxylase. The decarboxylated mature forms of the vitamin K-dependent
blood proteins (i.e. without the propeptide sequence) (16,
17) or the Gla domains themselves (18) are poor substrates for the carboxylase. In addition, the propeptide attached to normally uncarboxylated glutamate-containing peptides are multiply carboxylated both in vivo and in vitro (18, 19). Peptides
containing the factor IX propeptide followed by factor IX Gla domain,
the rest of the factor IX sequence, or a random glutamate containing
sequence have similar Km values for the carboxylase
(18). Therefore, the propeptide of the vitamin K-dependent
proteins appears to confer the perceived affinity of the carboxylase
for its substrate with little or no contribution from other domains. An
exception to this may be with the vitamin K-dependent bone
Gla protein in which an attached propeptide is not necessary for
efficient binding to the carboxylase (18, 20-22).
Since the importance of the propeptide sequence in vitamin
K-dependent carboxylation was first identified based on the
presence of highly conserved residues at specific positions, it has
been assumed that the propeptides form similar structures and therefore bind the carboxylase with similar affinities. A previous study has
questioned this assumption (23), but a systematic comparison of the
relative affinities has not been reported. Therefore, we determined the
relative affinities of the propeptides of nine vitamin
K-dependent proteins by competitive inhibition of a factor IX propeptide/Gla domain substrate. Previously, these comparisons could
not be performed because of the presence of endogenous substrates in
mammalian microsomal preparations of the enzyme or contamination by
free propeptide used to elute the affinity purified carboxylase. Therefore, these studies were performed using carboxylase purified by a
metal-ion dependent antibody from recombinant insect cells which do not
contain vitamin K-dependent proteins and are free of
endogenous substrates (24). We find that the inhibition constants of
most of the propeptides vary over a 100-fold range and the propeptide
of bone Gla protein has severely reduced carboxylase binding affinity.
Because variations in amino acid sequences of the highly conserved
propeptide sequences must confer the varied affinities, such studies
should provide insights into which amino acids within the propeptide
are important for carboxylase recognition. In addition, the variation
in affinities of the propeptides may have important physiological
consequences in affecting the levels of vitamin K-dependent
proteins in vivo and the biochemical mechanism by which
these proteins are modified by the carboxylase.
 |
EXPERIMENTAL PROCEDURES |
Materials--
All chemicals were reagent grade. Peptides based
on the propeptide sequences (Fig. 2) of the human vitamin
K-dependent proteins were chemically synthesized, purified
by reverse phase high performance liquid chromatography and verified to
be correct by ion spray mass spectrometry by Chiron Technologies
(Clayton Victoria, Australia). The concentrations of all peptides were
determined by amino acid analysis. The FIXproGLA (R-4Q, R-1S) peptide
was prepared as described previously (14). The insect cell expression
vector (pVL1392) was purchased from Pharmingen (San Diego, CA), and the
baculovirus viral DNA (BacVector 3000) was purchased from Novagen
(Madison, WI). The Sf9 cells (Spodoptera frugiperda)
were obtained from the Lineburger Cancer Center at the University of
North Carolina-Chapel Hill, and the High Five cells (Trichoplusia
ni) were a gift from Dr. Thomas Kost of Glaxo-Wellcome Inc.
(Research Triangle Park, NC). The HPC4 antibody affinity resin was
provided by Dr. Charles T. Esmon (Oklahoma Medical Research Foundation,
Oklahoma City, OK). The FLAG antibody and FLAG peptide standards were
purchased from Sigma.
Expression of r-Carboxylase in High Five Cells--
The cDNA
encoding the human vitamin K-dependent carboxylase was
sub-cloned into the EcoRI site of the pVL1392 vector. The sequence coding for the FLAG antibody epitope (DYKDDDDK) was introduced at the amino-terminal end of the carboxylase, and a HPC4 antibody tag
(EDQVDPRLIDGK) (26) was added at the carboxyl-terminal end. The
engineered vector was cotransfected with baculovirus BacVector 3000 triple-cut virus DNA into Sf9 cells. Recombinant virus was isolated by plaque purification, amplified, and titered by plaque assay
according to the instructions of the manufacturer (27). Expression of
carboxylase was done by infection of ~2 × 106/ml
High Five cells with the recombinant virus at a mutiplicity of
infection of ~1. Cells were collected after 48 h by
centrifugation and stored at
80 °C.
Preparation of Microsomes from High Five Cells--
A total of
1.8 × 109 cells from 1.2 liters of culture expressing
the recombinant human carboxylase were washed twice with Buffer A (20 mM phosphate (pH 7.4), 150 mM NaCl, 1×
protease inhibitor mixture (28) and 10% glycerol) and resuspended in
50 ml of Buffer A. The sample was homogenized by 15 strokes with a
Dounce homogenizer and then sonicated with four 5-s pulses using a
Ultrasonic Heat Systems sonicator. Cellular debris was removed by
centrifugation at 4000 × g for 15 min, and the
supernatant was centrifuged at 105,000 × g for 1 h. The microsomal pellet was resuspended in 20 mM phosphate
(pH 7.4), 500 mM NaCl, 1× protease inhibitor mixture, and
10% glycerol and stored at
80 °C.
Purification of r-Carboxylase Using HPC4 Antibody
Resin--
Microsomes were diluted to a final protein concentration of
12 mg/ml and solubilized by the addition of an equal volume of solubilization buffer (50 mM Tris (pH 7.4), 0.15 M NaCl, 1% CHAPS and 0.2% phosphatidylcholine, 10%
glycerol, and 1× PIC mixture) at 4 °C for 1 h. The solubilized
microsomes were centrifuged at 105,000 × g for 1 h, and the pellet was discarded. A total of 10 ml of HPC4 resin was
equilibrated with wash buffer (20 mM Tris (pH 7.4), 0.15 M NaCl, 0.5% CHAPS and 0.2% phosphatidylcholine, 1×
protease inhibitor mixture) and added to the solubilization supernatant
along with a final concentration of 5 mM CaCl2
and incubated overnight with gentle stirring. The resin was centrifuged and poured into a column, washed with 50 ml of wash buffer plus 5 mM CaCl2, and eluted with wash buffer plus 10 mM EDTA. Carboxylase samples were collected, aliquoted, and
stored at
70 °C. Concentration of enzyme was estimated from dot
blots of carboxylase using the anti-FLAG antibody and known
concentrations of FLAG peptide standards.
Carboxylase Assays--
The inhibition constants
(Ki) for the various propeptides were determined
from the ability of the propeptides to inhibit carboxylation of the
FIXproGLA substrate. Purified recombinant carboxylase (~40
nM final concentration) was mixed on ice with a final
concentration of 25 mM MOPS (pH 7.4), 0.5 M
NaCl, 0.28% CHAPS, 0.12% phosphatidylcholine, 222 µM
vitamin K hydroquinone, 6 mM dithiothreitol, and 5 µCi of
NaH14CO3 (specific activity, 54 mCi/mmol; ICN
Corp). Aliquots of this mixture were added to an indicated final
concentration of propeptide and FIXproGLA peptide. Reactions were
transferred to a 20 °C water bath and incubated for 1 h.
Reactions were stopped by the addition of 75 µl of 1 N NaOH, and the
total amount of 14CO2 incorporation was
determined as described previously (23). The background
14CO2 incorporation in the absence of the
substrate was subtracted from each assay point. This background
averaged <1 nM/min compared with 0.6 nM/min
observed in the absence of vitamin K and was unaffected by the
propeptide concentrations used in our assays except as noted with the
factor VII propeptide. Data for inhibition of varied concentrations of
the FIXproGLA substrate by set concentrations of the factor IX, factor
X, and prothrombin propeptides were fit by numerical integration using
the program Dynafit2 (29) to
determine the most likely inhibition mechanism and estimates of the
kinetic parameters. Values for the inhibition constant
(Ki) for each propeptide were determined by fitting
the data for inhibition of a set concentration of the FIXproGLA
substrate (0.5 µM) with each propeptide by nonlinear regression to the equation for tight binding competitive inhibition (Equation 1) where: Ki* is the apparent inhibition
constant, It is the total propeptide
concentration, Et is the total carboxylase concentration, and Km is the Michaelis constant for
the FIXproGLA peptide (30).
|
(Eq. 1)
|
All Ki values are reported as the average of
at least three independent determinations ± S.D. For the factor
VII propeptide, an equation (Equation 2) was derived using the rapid equilibrium assumption based on the mechanism in Scheme 1 which accounts for carboxylation of the propeptide itself.
|
(Eq. 2)
|
 |
RESULTS AND DISCUSSION |
The purpose of this study was to compare the relative affinities
of the propeptides of nine vitamin K-dependent proteins for the vitamin K-dependent
-glutamyl-carboxylase. These
studies were conducted with recombinant human vitamin
K-dependent carboxylase expressed in insect cells and
purified by a metal ion-dependent antibody to an engineered
epitope tag. Therefore this carboxylase preparation is free of
endogenous substrates or free propeptide required for purification of
carboxylase from liver.
We first looked at the ability of the factor IX, factor X, and
prothrombin propeptides to inhibit the carboxylation of varied concentrations of a factor IX propeptide/Gla domain substrate. As can
be seen in Fig. 1, each of the
propeptides inhibits carboxylation of the FIXproGLA peptide, although
significantly different concentrations of propeptide are required to
achieve similar levels of inhibition. Because the apparent inhibition
constants for the factor IX and factor X propeptides are within the
range of the enzyme concentration used in the assays, these propeptides
appear to be tight-binding inhibitors of the carboxylase, and the
inhibition constants cannot be determined by simple inhibition
kinetics. Therefore, data were fit by numerical integration using the
program Dynafit (29) to various plausible mechanisms for inhibition by
the propeptides (i.e. competitive, non-competitive, mixed
etc). For all three propeptides (Fig. 1), best-fits were achieved by a
competitive inhibition mechanism with affinity constants for factor X
(Ki = 1.3 ± 0.4 nM), factor IX
(Ki = 16 ± 3.1 nM), and
prothrombin (Ki = 270 ± 50 nM).
The inhibition data are consistent with free propeptide being a
competitive inhibitor of FIXproGLA carboxylation as seen previously
with attached propeptide substrates (13, 18). The significant
differences in the Ki values for the factor X,
factor IX, and prothrombin propeptides indicate that these propeptides
possess very different apparent affinities for the carboxylase.

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Fig. 1.
Effect of factor X, factor IX, and
prothrombin propeptides on FIXproGLA carboxylation. A,
effect of 0 nM ( ), 50 nM ( ), and 200 nM ( ) factor IX propeptide on FIXproGLA carboxylation.
Lines were drawn according to Equation 1 with Et = 38 nM, Km = 62 ± 14 nM, kcat = 0.30 ± 0.02 min 1, and Ki = 16 ± 3.1 nM. B, effect of 0 nM ( ), 20 nM ( ), and 80 nM ([trif) factor X
propeptide on FIXproGLA carboxylation. Lines were drawn according to
Equation 1 with Et = 38 nM,
Km = 57 ± 14 nM,
kcat = 0.3 ± 0.02 min 1, and
Ki = 1.3 ± 0.4 nM. C,
effect of 0 nM ( ), 5000 nM ( ), 2500 nM ( ), and 625 nM ( ) prothrombin
propeptide on FIXproGLA carboxylation. Lines were drawn according to
Equation 1 with Et = 38 nM,
Km = 74 ± 19 nM,
kcat = 0.33 ± 0.02 min 1, and
Ki = 270 ± 50 nM.
|
|
To compare the relative affinities of the vitamin
K-dependent propeptides for the carboxylase, we determined
the inhibition constants for nine propeptides by competitive inhibition
of a propeptide/Gla domain substrate (FIXproGLA) carboxylation with increasing concentrations of a given propeptide. The sequence and
Ki value for each peptide are summarized in Fig. 2. As noted previously, the propeptides
appear to be tight binding inhibitors of the carboxylase, therefore
data (Fig. 3) were fit to equations
(Equation 1) appropriate for this kind of inhibition (30). As can be
seen in Figs. 2 and 3, the relative affinities of the vitamin
K-dependent propeptides vary over a significant range. The
propeptide of factor X has the tightest affinity (Ki = 2.6 nM) followed by matrix Gla protein
(Ki = 5.8 nM). The propeptides of
protein S (Ki = 12.3 nM) and PRGP1 (Ki = 12.8) are in the same range followed by factor IX (Ki = 33.6 nM). PRGP1 is a putative
vitamin K-dependent protein that was identified by homology
searches based on the highly conserved Gla domain sequence motifs of
the vitamin K-dependent proteins (5). However, it is not
known whether this protein is actually carboxylated. Because the
Ki of the PRGP1 propeptide is within the range
observed for the known carboxylated vitamin K-dependent
proteins and the propeptide is all that is required to direct
carboxylation of an adjacent glutamate containing region (18, 19), the
PRGP1 precursor likely is able to bind and be modified by the vitamin
K-dependent carboxylase.

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Fig. 2.
Sequences of synthetic peptides and their
inhibition constants. Sequences are based on the propeptides of
the indicated human vitamin K-dependent proteins.
Inhibition constants toward FIXproGLA carboxylation are determined as
described under "Experimental Procedures" and are reported as the
average of at least three independent determinations ± S.D.
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Fig. 3.
Inhibition of FIXproGLA carboxylation by
propeptides of various vitamin K-dependent
propeptides. The effect of the various propeptides on the
carboxylation of 0.5 µM factor IX propeptide/Gla domain
peptide are shown. Data were normalized to the carboxylation of the
FIXproGLA peptide in the absence of propeptide. Propeptides used are as
follows: factor X ( ), matrix Gla protein ( ), protein S (×),
PRGP1 ( ), factor IX ( ), prothrombin ( ), protein C ( ), and
bone Gla protein ( ).
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|
An unusual phenomenon is observed with the factor VII propeptide when
it is used as an inhibitor in the competition assays. As seen in Fig.
4A, increasing concentrations
of the factor VII propeptide reduce the carboxylase activity to a
point, but at higher concentrations the activity increases. When the
inhibitor (factor VII propeptide) is varied in the absence of the
FIXproGLA substrate, a concentration-dependent increase in
carboxylation is observed (Fig. 4A). Small
glutamate-containing peptides without covalently linked propeptides are
commonly used as substrates for the carboxylase and are
carboxylated at much higher rates than substrates with attached
propeptides (31). The factor VII propeptide, unlike the other vitamin
K-dependent propeptides, contains a di-glutamate pair and
appears to be acting as a glutamate substrate for the carboxylase.
Apparent carboxylation of the factor IX propeptide, which contains a
single glutamate, is also observed, albeit at a significantly lower
level than that of the factor VII propeptide. However, at the
concentrations of propeptides used in our assays, incorporation of
radioactive CO2 into these propeptides did not affect our
Ki determinations (Fig. 4A,
inset). There is no apparent incorporation of radioactive CO2 into the propeptides of prothrombin and matrix Gla
protein, which do not contain glutamates (Fig. 4A,
inset). These observations suggest that amino acids
surrounding a particular glutamate may greatly influence its affinity
for the carboxylase active site and that glutamate pairs in particular
have higher affinity for the carboxylase active site as has been noted
previously (32). Because these results indicate that the propeptide
binding site and glutamate active site of the carboxylase can be
occupied by separate factor VII propeptide molecules, we analyzed the
data for inhibition by the factor VII propeptide using an equation (Equation 2) derived from a mechanism (Scheme
1) that accounts for the carboxylation of
the propeptide itself. This analysis yields an estimate for the
Ki = 11.1 ± 0.8 nM. To further validate the estimate of the Ki for the factor VII
propeptide, we also analyzed the effect of the factor VII propeptide on
varying concentrations of the FIXproGLA propeptide (Fig.
4B). The concentrations of the factor VII propeptide used in
this experiment are well below the concentrations at which it
demonstrates background activity. The value of the
Ki from this experiment is 5.7 ± 0.8 nM, similar to the value obtained by analysis of varying
propeptide at one substrate concentration (Fig. 4A).
Although it is possible that the factor VII propeptide is carboxylated
in vivo, this observation is likely a consequence of the
high concentrations of propeptide used in these inhibition assays.
Nevertheless, this phenomena does demonstrate the concept that amino
acids surrounding a particular glutamate can significantly affect its
carboxylation.

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Fig. 4.
Inhibition of FIXproGla carboxylation by
factor VII propeptide. A, the effect of varying
concentrations of factor VII propeptide ( ) or matrix Gla protein
propeptide ( ) on the carboxylation of 0.5 µM FIX
proGLA is shown. The line for factor VII propeptide in the presence of
FIXproGLA was drawn according to Equation 2 using
Vmax1 = 11 ± 1.3 nM/min,
Vmax2 = 733 ± 47 nM/min,
Km = 60 nM, Kglu = 2900 µM, and Ki = 11 ± 0.8 nM. The carboxylation of the factor VII propeptide ( ) in
the absence of the FIXproGLA peptide is also shown. Inset,
background carboxylation with varying concentrations of propeptides of
matrix Gla protein ( ), prothrombin ( ), factor IX ( ), and
factor VII ( ) in the absence of FIXproGLA substrate. B,
inhibition of various concentrations of FIXproGLA by 0 nM
( ), 20 nM ( ), and 80 nM ( ) factor VII
propeptide. Lines were drawn using Equation 1 with
kcat = 0.29 ± 0.01 min 1,
Et = 38 nM, Km = 60 ± 7.6 nM, and Ki = 5.7 ± 0.8 nM.
|
|
In contrast to the other propeptides, we were unable to measure
significant inhibition using the bone Gla protein propeptide. We
observe less than 10% inhibition by 400 µM bone Gla
protein propeptide, therefore it must have significantly weaker
affinity (Ki > 500 µM) than that
observed for the other propeptides. The bone Gla propeptide is the only
known vitamin K-dependent propeptide sequence that contains
a glycine at the highly conserved position
10. A previous study has
shown that substitution of glycine in place of alanine at position
10
in a factor IX propeptide/Gla domain peptide increases its
Km for the carboxylase 30-fold (23). Therefore this
alteration may be at least partially responsible for the severely
reduced affinity of the bone Gla propeptide. Studies have shown that
decarboxylated bone Gla protein without a covalently attached
propeptide is an excellent substrate for the carboxylase (18, 20-22)
although a covalently attached propeptide is required for efficient
carboxylation of the vitamin K-dependent blood clotting
proteins (8, 9, 16, 17). Therefore, the bone Gla protein substrate has
a very weak propeptide but appears to have a strong binding site within
its Gla domain, whereas the vitamin K-dependent blood
proteins have strong propeptides but weak binding sites within their
Gla domains This suggests the possibility that bone Gla protein and the
blood coagulation proteins may have separate mechanisms for substrate
recognition. Interestingly, the Ki for the
propeptide-like sequence in the other known vitamin
K-dependent bone protein, matrix Gla protein, appears to be
in the same range as the blood clotting proteins. Therefore the weak
affinity of the bone Gla protein propeptide is not characteristic of
the vitamin K-dependent bone proteins. Whether the
propeptide plays a role in the carboxylation of bone Gla protein will
require further investigation.
The comparisons of the relative affinities of the vitamin
K-dependent proteins in this study show that the affinities
of most of the vitamin K-dependent propeptides vary over a
10-fold range. Two propeptides (prothrombin and protein C) have
significantly weaker affinities, and the propeptide of bone Gla protein
is at least 200,000-fold weaker than the factor X propeptide. The
physiological importance and consequences of the broad range of
propeptide affinities are unclear. For the vitamin
K-dependent blood coagulation proteins, the propeptide is
essential for substrate recognition and binding of the carboxylase.
Vitamin K-dependent carboxylation proceeds through a
processive mechanism in which multiple glutamic acid residues can be
modified in a single binding event (15). Therefore vitamin
K-dependent carboxylation proceeds through a multi-step pathway in which multiple binding interactions and/or catalytic steps
may contribute to the perceived affinity of the substrate for the
carboxylase; nevertheless, the critical role of the propeptide in
substrate recognition suggests that the binding affinity of the
propeptide for the carboxylase should significantly affect the binding
of the entire substrate.
Two in vivo studies provide evidence for variation in the
affinities of the vitamin K-dependent blood coagulation
proteins for the carboxylase. De Metz et al. (34) have found
that liver microsomes from warfarin-treated cows accumulate
uncarboxylated precursors of prothrombin, factor IX, and factor X. Although the prothrombin precursor would be expected to be present at
higher concentrations, 69% of the carboxylase was complexed with
factor X precursors, whereas 21% was associated with prothrombin and 8% with factor IX (34). Further evidence for the high affinity of the
propeptide of factor X for the carboxylase is that warfarin treatment
of HepG2 cells increases the level of factor X associated with the
carboxylase with a concomitant decrease in the amount of prothrombin
associated with the carboxylase (25). In a cell line that does not
express factor X, no such competition was observed, and the authors
ascribe this phenomena to a reduced affinity of the prothrombin
precursor compared with that of factor X precursors. Both of these
studies are consistent with our observation that factor X propeptide
has a significantly higher affinity for the carboxylase than the
prothrombin propeptide, and therefore the observed variations in
affinities of the propeptides can impact the binding of the entire
vitamin K-dependent protein substrates.
Patients with mutations in the factor IX propeptide demonstrate marked
clinical effects, which demonstrates the physiological importance of
propeptide affinity. Propeptide mutations in factor IX that increase
the Km of the substrate for the carboxylase cause an
unusual sensitivity to warfarin in several patients (23, 33). Without
warfarin treatment, these patients demonstrate normal or near-normal
factor IX activity, but the factor IX activity is depressed below 1%
upon warfarin administration, well below the range observed for the
other vitamin K-dependent factors. This indicates that
reduced propeptide binding affects the level of a particular vitamin
K-dependent protein. Regulation of the levels of vitamin
K-dependent proteins should be subordinate to a number of
factors such as transcription levels and relative protein stability.
Nevertheless, the observed competition of the factor X and prothrombin
substrates for the carboxylase and the clinical consequences of factor
IX propeptide mutations suggest that the significant variations in the
affinities of the vitamin K-dependent propeptides may also
have a significant role in determining the levels of these proteins.
This work provides the first extensive study of the relative affinities
of the vitamin K-dependent propeptides for the carboxylase. Despite the sequence similarities observed for all the propeptides, there is a wide variation in the observed affinities of the propeptides for the carboxylase. Further work will be required to ascertain the
structural determinants of these variable affinities and the physiological consequences of these observations.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant HL06350 (D. W. S.).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.
Present address: Dept. of Molecular Sciences,
Glaxo-Wellcome Inc., Research Triangle Park, NC 27709.
§
To whom correspondence should be addressed. Tel.: 919-962-0597;
Fax: 919-962-9266; E-mail: dws{at}emailunc.edu.
2
Available on the World Wide Web at
http://www.biokin.com.
 |
ABBREVIATIONS |
The abbreviations used are:
Gla,
-carboxyglutamic acid;
CHAPS, 3-[(3-cholamidopropyl)]dimethylammonio-1-propanesulfonate;
MOPS, 3-(N-morpholino)propanesulfonic acid;
FIXproGLA, 59-amino
acid peptide containing the human factor IX propeptide and first 41 residues of factor IX Gla domain (sequence position 18 to 41).
 |
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