From the Division of Thrombosis Research, Department of Medicine, and Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030 and the Cox Laboratory for Bioengineering, Rice University, Houston, Texas 77005
Received for publication, February 2, 2001, and in revised form, February 23, 2001
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ABSTRACT |
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Glycoprotein Ib The platelet receptor for von Willebrand factor
(vWF),1 the glycoprotein (GP)
Ib-IX-V complex, is composed of four polypeptide subunits (GP Ib (GP Ib
), the ligand binding
subunit of the platelet glycoprotein Ib-IX-V complex, is sulfated on
three tyrosine residues (Tyr-276, Tyr-278, and Tyr-279). This
posttranslational modification is known to be critical for von
Willebrand factor (vWF) binding; yet it remains unclear whether it
provides a specific structure or merely contributes negative charges.
To investigate this issue, we constructed cell lines expressing GP
Ib
polypeptides with the three tyrosine residues converted to either
Glu or Phe and studied the ability of these mutants to bind vWF in the
presence of modulators or shear stress. The mutants were expressed
normally on the cell surface as GP Ib-IX complexes, with the
conformation of the ligand-binding domain preserved, as judged by the
binding of conformation-sensitive monoclonal antibodies. In contrast to their normal expression, both mutants were functionally abnormal. Cells
expressing the Phe mutant failed to bind vWF in the presence of either
ristocetin or botrocetin. These cells adhered to and rolled on
immobilized vWF only when their surface receptor density was increased
to twice the level that supported adhesion of cells expressing the
wild-type receptor and even then only 20% as many rolled and rolled
significantly faster than wild-type cells. Cells expressing the Glu
mutant, on the other hand, were normal with respect to
ristocetin-induced vWF binding and adhesion to immobilized vWF but were
markedly defective in botrocetin-induced vWF binding. These results
indicate that GP Ib
tyrosine sulfation influences the interaction of
this polypeptide with vWF primarily by contributing negative charges
under physiological conditions and when the interaction is induced by
ristocetin but contributes a specific structure to the
botrocetin-induced interaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, GP
Ib
, GP IX, and GP V (1, 2)) encoded by four separate genes (3-6).
GP Ib
is the largest subunit within the complex and so far the only
subunit implicated in ligand binding, being capable of binding vWF (7),
-thrombin (7), P-selectin (8), and leukocyte Mac-1 (9). The
ligand-binding region resides within ~300 amino acids at the GP
Ib
N terminus and is held high above the cell membrane by a stiff,
highly O-glycosylated mucin-like domain (Fig.
1) (3, 10). The ligand-binding region can
be divided into three distinct structural subdomains that are all
implicated in vWF binding (7): seven tandem leucine-rich repeats,
disulfide loops flanking the leucine-rich repeats, and a highly
negatively charged sequence spanning residues Asp-269 to Asp-287 (3,
10). Three tyrosine residues (Tyr-276, Tyr-278, and Tyr-279) are
embedded in this negatively charged sequence (2, 7) and each is fully
sulfated, a modification critical for the binding of vWF and
-thrombin (11-13).
View larger version (27K):
[in a new window]
Fig. 1.
Schematic depiction of the N-terminal
ligand-binding region of GP Ib . The
box shows the amino acid sequence of GP Ib
from residues
269 to 287. Specific epitopes for four
conformation-dependent GP Ib
antibodies are
indicated.
Tyrosine sulfation is a widespread posttranslational modification of
proteins (14-20), but few studies have addressed its effects on
protein function. Tyrosine sulfation could affect protein function in
at least two ways; the sulfates could simply serve as an additional means of providing negative charges for electrostatic interactions or
they could provide a specific structure for intra- or interpolypeptide interactions. To address the role of tyrosine sulfation in the interaction between vWF and GP Ib, we established CHO cell lines that express GP Ib
mutants in which the three sulfated tyrosine residues were replaced by either Phe or Glu residues. In the Phe mutant, the negative charges contributed by sulfation are eliminated, whereas in the Glu mutant, the side chain is changed but the charge is
maintained. Here we report findings on the effects of the mutations on
cell surface expression of the GP Ib-IX complex, modulator-induced vWF
binding, and on cell adhesion to immobilized vWF under fluid shear stress.
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MATERIALS AND METHODS |
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Site-directed Mutagenesis--
Polymerase chain reaction-based
site-directed mutagenesis was performed using a commercial kit
(QuickChangeTM, Stratagene, La Jolla, CA) directly on the mammalian
expression vector pDX containing the GP Ib cDNA. By this method,
codons for tyrosines 276, 278, and 279 were converted to codons for
either phenylalanine or glutamic acid. The mutant constructs were
sequenced in their entirety to verify targeted mutations. The
sequencing reaction was performed using a dye terminator kit, and the
results were analyzed on an ABI model 737A automated sequencer (ABI,
San Leandro, CA).
Cell Lines--
The mutant and wild-type GP Ib constructs
were transfected into CHO cells stably expressing GP Ib
and GP IX
(CHO
IX cells) (21) by a commercial method using liposomes as DNA
carriers (LipofectAMINE, Life Technologies, Inc.) (11, 22). The plasmid pREP4 (Invitrogen, Carlsbad, CA), which carries a hygromycin-resistant marker, was cotransfected with the GP Ib
mutants. The cotransfection allowed for selection of mutant-expressing cells by growth in hygromycin. Transfected cells were first grown in
-minimal essential medium (
-MEM, Life Technologies, Inc.) without fetal bovine serum for 12-18 h and then grown in complete
-MEM medium supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.). For transient expression, which was used to determine whether the mutations affect the surface expression of GP Ib
, cells were harvested 72 h after transfection and processed for flow cytometry analysis. To establish stable cell lines, transfected cells were grown in complete
-MEM containing 500 µg/ml hygromycin
(Calbiochem). The cells were also sorted for GP Ib
expression by
incubating a suspension of cells in 1 µg/ml monoclonal GP Ib
antibody WM23 (provided by Dr. Michael C. Berndt, Baker Medical
Research Institute, Prahran, Victoria, Australia) and selecting the
antibody-coated cells with magnetic beads coated with sheep anti-mouse
IgG (Dynabeads, Dynal Biotech, Inc., Lake Success, NY).
Flow Cytometry--
Surface expression of the GP Ib mutants
was determined by flow cytometry using WM23. This antibody binds to the
macroglycopeptide region of GP Ib
, a region unaffected by the
mutations (23, 24). Flow cytometry was also used to determine whether
the mutations had a global effect on the conformation of the N-terminal
ligand-binding region of GP Ib
. The mutants were evaluated for their
capacity to bind five monoclonal GP Ib
antibodies: AK2, SZ2 (both
from Research Diagnostics, Inc., Flanders NJ), AN51 (DAKO Corp.,
Carpinteria, CA), C34 (from Vth International Workshop on Leukocyte
Antigens), and TM60 (kindly provided by Dr. Noamasa Yamamoto of Tokyo
Metropolitan Institute of Medical Science, Japan), with WM23 serving as
the control. Flow cytometry analysis was also performed prior to each binding or adhesion experiment to determine the surface expression level of GP Ib
.
Flow cytometry analysis was performed as previously described (25). Briefly, cells were detached with 0.53 mM EDTA, washed with phosphate-buffered saline (PBS), and incubated with the appropriate antibody at saturating concentrations (0.5-1 µg/ml) in PBS containing 1% bovine serum albumin for 60 min at room temperature. The cells were then incubated with a fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG (Zymed Laboratories, Inc., South San Francisco, CA) for 30 min at room temperature. The fluorescence intensity of the labeled cells was measured using a FACScan flow cytometer (BD Bioscience, San Jose, CA), and the plasma membrane receptor level was expressed as the geometric mean fluorescence.
Modulator-induced vWF Binding-- Purified human vWF (a gift from Dr. Michael Berndt) was iodinated as described previously (11). The specific activity of the 125I-labeled vWF was 0.24 mCi/mg of protein.
The binding of vWF to GP Ib-IX-expressing cells was induced with either
ristocetin or botrocetin, as described previously (11, 21). Cells were
detached with 0.53 mM EDTA and washed with
Ca2+- and Mg2+-free Tyrode's buffer (138 mM NaCl, 5.5 mM glucose, 12 mM
NaHCO3, 0.36 mM
NaH2PO4, 2.9 mM KCl, 1% bovine
serum albumin, pH 7.4). Washed cells were resuspended in Tyrode's
buffer to a final cell density of 4 × 107 cells/ml.
125I-Labeled vWF was added to 25-µl aliquots of the cell
suspension at concentrations ranging from 0.4 to 6.4 µg/ml, along
with either 1.0 mg/ml ristocetin (Sigma) or 20 µg/ml purified
botrocetin (from Dr. Michael Berndt). The reaction volume was brought
to 100 µl with Tyrode's buffer. The mixture was incubated for 30 min
at room temperature and the cells were then spun through a 20% sucrose cushion to remove unbound vWF. The capillary tips containing the cell
pellets were cut off, and radioactivity associated with cell pellets
was counted in a counter. Specific binding was determined by first
subtracting the nonspecific counts obtained from CHO
IX cells
(lacking GP Ib
) and then correcting for differences in receptor
surface density as determined by flow cytometry.
Cell Adhesion to Immobilized vWF under Flow-- Cell adhesion to immobilized vWF was studied using a parallel-plate flow chamber (26). The bottom of the chamber was made up of a glass coverslip that had been coated with a vWF solution of 40 µg/ml for 45 min, as previously described (26). Specific wall shear stress was generated by drawing PBS through the chamber at defined flow rates with a syringe pump. The shear stress is proportional to the height and the width of the chamber, the fluid viscosity, and flow rate (27). The assembled flow chamber was mounted onto an inverted-stage microscope (DIAPHOT-TMD; Nikon, Garden City, NY) connected to a silicon-intensified target video camera (model C2400, Hammatus, Waltman, MA) and a video cassette recorder. The parallel-plate flow chamber was maintained at 37 °C by a thermostatic air bath during experiments.
Cells were injected into the chamber (0.6 ml at a cell density of 500,000 cells/ml) and incubated with immobilized vWF for 1 min before PBS was perfused through the chamber. The interaction of cells with the matrix within a single view field was recorded in real time for 3-5 min on videotape and was analyzed off-line using Inovision imaging software (IC-300 Modular Image Processing, Inovision Corp., Durham, NC). A rolling cell was defined as a cell moving in the direction of fluid flow while maintaining constant contact with the vWF matrix. Saltatory movement describes a cell moving along the flow direction by "skipping" through discontinuous contact with the vWF matrix. The rolling velocity was the distance that a single cell rolled during a defined time period (26, 28, 29).
Statistics--
The data were analyzed using either Student's
t test (paired samples) or the analysis of variance
F-test (multiple sample comparison). The results are expressed
as means ±S.E.
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RESULTS |
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The Mutations Do Not Affect the Surface Expression of the GP Ib-IX
Complex--
We first examined whether the mutations affected surface
expression of the GP Ib-IX complex by expressing the mutant transiently in CHO IX cells. By this strategy, the only variable affecting expression was the mutation itself, as the same plasmid expression vector was used for all transfections and the GP Ib
inserts differed only at the mutated nucleotides. Expression of GP Ib
in the cells was determined by flow cytometry 72 h after transfection. As shown in Fig. 2, the expression of the mutants
on the cell surface was similar to that of wild-type GP Ib
,
indicating that the mutations had no effect on the synthesis, assembly,
or transport of the GP Ib-IX complex.
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The Mutations Did Not Change the Conformation of the GP Ib
Ligand-binding Region--
The potential effects of the mutations on
the global conformation of the GP Ib
ligand-binding region were
examined with five GP Ib
monoclonal antibodies known to bind
different epitopes within this region: AK2, AN51, C34, TM60, and SZ2
(30). Of these, all but SZ2 recognize conformation-sensitive epitopes
(Fig. 1). SZ2 binds the SDS-denatured polypeptide on immunoblots and
has been mapped to the anionic region mutated in this study (13). The
five antibodies are each also capable of blocking modulator-induced vWF
binding to the GP Ib-IX-V complex, albeit to different extents (13, 23,
31-34). Each of the antibodies except SZ2 bound to both the Phe and
Glu mutants in a manner similar to their binding to wild-type GP Ib
(Fig. 3). SZ2 recognized neither mutant,
as expected from the known location of its epitope within the anionic tyrosine-sulfated region of GP Ib
(13, 30). Thus, the mutations preserved the overall conformation of the GP Ib
ligand-binding region.
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Modulator-induced vWF Binding--
The binding of
125I-vWF to cells expressing the mutants was measured at
several vWF concentrations in the presence of either 1.0 mg/ml
ristocetin or 20 µg/ml botrocetin. Ristocetin-induced vWF binding to
cells expressing the Phe mutants was markedly decreased compared with
the binding to cells expressing wild-type GP Ib, whereas binding to
cells expressing the Glu mutant was similar to the binding to wild-type
cells (Fig. 4 upper). In
contrast, both mutations abolished botrocetin-induced vWF binding (Fig. 4 lower).
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Adhesion of Mutant-expressing Cells to Immobilized vWF under Fluid
Shear Stress--
The behavior of cells expressing the mutants was
examined under flow conditions in a parallel-plate flow chamber. Cells
were allowed to settle on the vWF matrix and were then subjected to 10 dynes/cm2 fluid shear stress. Under these
conditions, cells expressing wild-type GP Ib adhered to the matrix
and rolled at a mean velocity of 54.20 ± 1.44 µm/s (Fig.
5A). At a comparable receptor
density, cells expressing the Glu mutant rolled at a similar velocity
(56.75 ± 1.39 µm/s, Fig. 5, A and B). In
contrast, cells expressing the Phe mutant did not adhere to the
vWF-coated surface at these shear rates. However, when the surface
levels of this mutant were increased 2-fold (by repeated cell sorting,
mean fluorescence of 46.25 ± 11.46 for wild-type GP Ib
and
115.74 ± 28.36 for the Phe mutant (Fig. 5B)), almost
20% of the cells adhered to and rolled on the vWF matrix (Fig.
5A). Nevertheless, they still rolled significantly faster
than wild-type cells (69.20 ± 2.47 versus 52.20 ± 1.44 µm/s, Student's t test, n = 271-419, p < 0.005, Fig. 5A).
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In addition to rolling after initial adhesion, some cells seemed to
skip over the surface, a process we termed "saltation." The
percentage of cells translocating by this method was similar between
wild-type cells and cells expressing the Glu mutant (26.70 ± 6.81 for wild type cells versus 30.22 ± 4.49 of the Glu
mutant, Student's t test, n = 79-143,
p = 0.09), whereas greater than 80% of adherent Phe
mutant-expressing cells demonstrated saltatory movement (26.70 ± 6.81 versus 82.32 ± 9.44, Student's t
test, n = 89-143, p < 0.001) (Fig.
6).
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DISCUSSION |
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Tyrosine sulfation is a widespread posttranslational protein
modification that occurs in the trans-Golgi compartment where it is
catalyzed by the membrane-bound enzyme, aryl sulfotransferase (15,
35-37). A large number of proteins have been found to contain this
modification, but its functional role has only been demonstrated in a
few cases (19, 38, 39), GP Ib being one of them (11-13). Even with
this, the mechanism of how sulfation affects protein function has not
been addressed for GP Ib
and has only been addressed in one other
case to our knowledge (40). The two most obvious means by which
tyrosine sulfation may participate in protein function is by either
providing or contributing to a specific structure (e.g.
through folding) or by contributing negative charges. An alternative
has been suggested by the work of Somers et al. (40) who
recently showed that the sulfated tyrosines of P-selection glycoprotein
ligand-1 participate in calcium ion coordination when this
protein binds its counter-receptor, P-selectin. In the current study we
examined whether sulfate is specifically required for GP Ib
function
or whether substituting another acidic residue for the sulfate will
maintain vWF binding function. We addressed this issue by constructing
cell lines expressing either wild-type GP Ib
or mutant receptors
substituting the three tyrosines with either phenylalanine or glutamate
residues. The Phe substitutions maintained the phenyl groups of the
tyrosines but eliminated the p-hydroxy group, thereby
preventing sulfation. The Glu substitutions, on the other hand, changed
the side chains radically but preserved their net charge.
Both mutants were expressed on the cell surface normally and retained
the normal conformation of the GP Ib N terminus, as determined by
the binding of several conformation-sensitive antibodies. The
monoclonal antibody SZ2 failed to bind both mutants because the anionic
sulfated region constitutes its binding site (13).
Whether the mutations affected vWF binding depended on how that binding
was induced. When vWF binding was induced by botrocetin, a snake venom
protein known to bind vWF and alter its conformation, both mutants were
found to be markedly defective (Fig. 4 lower). This is
consistent with extensive previous work showing that the anionic
sulfated site of GP Ib is important in botrocetin-induced vWF
binding and that even minor perturbations of this site can alter
binding drastically (7, 12, 30). In contrast, only the Phe mutant was
defective when vWF binding was induced by ristocetin (Fig. 4
upper), a modulator that we have recently demonstrated to
more closely mimic physiological vWF binding, i.e. binding induced by high shear stress or by vWF immobilization onto a surface (41). This defect in ristocetin-induced vWF binding was indeed mimicked
by a defect in attachment of the cells to immobilized vWF in a flow
chamber. At surface densities of the Phe mutant similar to those on
wild-type cells that bound and rolled on the surface, cells expressing
the mutant failed to attach to the surface. At roughly double those
surface densities, the cells attached and a few rolled, but most of
them translocated along the surface by saltation (Fig. 6). This is in
contrast to the Glu mutants, which attached and rolled on the surface
in a manner indistinguishable from the wild-type cells. Thus, with
regard to the more physiological measures of GP Ib
's vWF binding
function, substitution of the sulfated tyrosines with glutamic acid
residues seems to fully maintain function.
These results have several implications. First, they demonstrate that
tyrosine sulfation influences the interaction between GP Ib and vWF
either by an electrostatic mechanism or by metal ion coordination, both
of which can be replaced by the carboxylic acid-containing side chain
of glutamic acid. Second, the correspondence between the effects of the
mutations on shear-dependent vWF binding function and on
ristocetin-induced vWF binding is consistent with our previous findings
that ristocetin is a better mimic of the physiologic interaction than
is botrocetin (41). The specific sequence is not as critical for
ristocetin-induced binding as for botrocetin-induced binding, which is
markedly perturbed by both the Phe and the Glu substitutions. The
latter indicates that the anionic sulfated region of GP Ib
may
constitute, at least partially, a direct site for botrocetin-induced
vWF binding, with the sulfates being absolutely required for the
interaction. Third, the results suggest that the three negative charges
contributed by the sulfated tyrosines are important in maintaining the
affinity of the GP Ib
-vWF interaction. The Phe mutants, lacking
these negative charges, interact abnormally with immobilized vWF under conditions of flow, demonstrated by the presence of fewer rolling cells
and by the fact that those cells that did adhere to the surface
translocated by saltation rather than by continuous rolling (Figs. 5
and 6). This defect was partially compensated by increasing the surface
levels of the mutant receptor.
In summary, we present evidence that sulfation of tyrosine residues
within the anionic sulfated region of platelet GP Ib contributes
negative charges that are critical for the interaction of GP Ib
with
vWF under flow and in the presence of ristocetin. Although charge
rather than a specific sequence is important for these interactions,
the same is not true for the interaction induced by botrocetin. Our
results strongly support the notion that there is more than one binding
site on GP Ib
for vWF and that the choice of that site depends on
the means by which the interaction is induced.
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ACKNOWLEDGEMENTS |
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We thank Leticia H. Nolasco for assistance in purifying human von Willebrand factor. We also thank Dr. Michael C. Berndt for providing reagents and for helpful discussion of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL02463, HL46416, HL18673, and NS23327, Robert A. Welch Foundation Grant C938, a grant-in-aid from the American Heart Association-Texas Affiliate, and by the National Institutes of Health Medical Scientist Training Program at Baylor College of Medicine.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: Thrombosis Research
Section, Dept. of Medicine, BCM286, N1319, Baylor College of Medicine,
One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-3470; Fax:
713-798-3415; E-mail: josel@bcm.tmc.edu.
Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M101035200
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ABBREVIATIONS |
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The abbreviations used are:
vWF, von Willebrand
factor;
CHO, Chinese hamster ovary;
-MEM,
-minimal essential
medium;
PBS, phosphate-buffered saline;
GP, glycoprotein.
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