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J. Biol. Chem., Vol. 276, Issue 20, 16690-16694, May 18, 2001

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Tyrosine Sulfation of Glycoprotein Ib

ROLE OF ELECTROSTATIC INTERACTIONS IN VON WILLEBRAND FACTOR BINDING^*

Jing-fei Dong, Pei Ye, Alicia J. Schade, Shan Gao, Gabriel M. Romo, Nancy T. Turner, Larry V. McIntire, and José A. López

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

	ABSTRACT

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Glycoprotein 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

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, 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 this window] [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.

	MATERIALS AND METHODS

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Site-directed Mutagenesis-- Polymerase chain reaction-based site-directed mutagenesis was performed using a commercial kit (QuickChange^TM, 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.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Effect of the Glu and Phe mutations on surface expression of GP Ib. Plasmids containing cDNAs encoding either the Glu or the Phe mutant of GP Ib were transiently transfected into CHO IX cells, and the surface expression of the mutant polypeptides was compared by flow cytometry to that of wild-type GP Ib (n = 6, paired t tests with p values of 0.09 for wild type versus 3Y-E and 0.18 for wild type versus 3Y-F).

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.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Effects of the Glu and Phe mutations on the conformation of the N-terminal region of GP Ib. The binding of five monoclonal GP Ib antibodies to both the Phe and Glu mutants was analyzed by flow cytometry and compared with the binding of WM23, which binds to the macroglycopeptide region, C-terminal to the ligand-binding region. Four of the five antibodies (AK2, AN51, C34, and TM60) were previously determined as conformation-dependent. The values are means of four independent experiments. *, SZ2 was defective in binding to both mutants; p < 0.05 for both analyses.

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).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Ristocetin- and botrocetin-induced 125I-vWF binding. 125I-vWF was incubated with cells expressing either wild-type GP Ib or one of the mutants in the presence of either 1.0 mg/ml ristocetin (upper) or 20 µg/ml botrocetin (lower) for 30 min. Membrane-bound radioactivity was then measured and specific binding was determined by subtracting the radioactivity associated with CHO (Chinese hamster ovary) IX cells. The experiment depicted is representative of six independent experiments.

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).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Adhesion of mutant-expressing cells to immobilized vWF. Cells expressing wild-type GP Ib or one of the mutants were incubated with immobilized vWF for 1 min. Buffer (PBS) was then perfused through the chamber at a flow rate that generated a wall shear stress of 10 dynes/cm2. The rolling velocities of the mutant-expressing cells were measured and compared with those of the wild-type cells (A). A portion of cells was taken from each cell suspension used for the flow experiments to determined surface receptor density by flow cytometry (B). Values are means ± S.E.; n = 129-230; *, p < 0.001.

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).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effects of the Glu and the Phe mutations on cell movement on the vWF matrix under fluid shear stress. Those cells that adhered to the vWF matrix at 10 dynes/cm2 shear stress were scored for their primary mode of translocation along that matrix. More than 70% of wild-type and Glu mutant-expressing cells rolled on the vWF matrix with the rest showing saltatory movement. In contrast, greater than 80% of the Phe mutant-expressing cells with high receptor density translocated in the direction of flow by saltation. Values are the mean ± S.E.; *, p < 0.001.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

	ACKNOWLEDGEMENTS

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.

	FOOTNOTES

^* 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

	ABBREVIATIONS

The abbreviations used are: vWF, von Willebrand factor; CHO, Chinese hamster ovary; -MEM, -minimal essential medium; PBS, phosphate-buffered saline; GP, glycoprotein.

	REFERENCES

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