Mutational Analysis of Thrombopoietin for Identification of Receptor and Neutralizing Antibody Sites*

(Received for publication, February 11, 1997, and in revised form, April 30, 1997)

Kenneth H. Pearce Jr. Dagger §, Beverly J. Potts , Leonard G. Presta par , Laura N. Bald , Brian M. Fendly and James A. Wells Dagger **

From the Departments of Dagger  Protein Engineering,  Antibody and Bioassay Technology, and par  Immunology, Genentech, Inc., South San Francisco, California 94080

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Thrombopoietin (TPO) is a hematopoietin important for megakaryocyte proliferation and production of blood platelets. We sought to characterize how TPO binds and activates its receptor, myeloproliferative leukemia virus receptor. The erythropoietin-like domain of TPO (TPO1-153) has been fused to the gIII coat protein of M13 bacteriophage. Forty residues were chosen for mutation to alanine using the criteria that they were charged residues or predicted to be solvent-exposed, based on a homology model. Phage enzyme-linked immunosorbent assay was used to determine affinities for binding to both the TPO receptor and five anti-TPO1-153 monoclonal antibodies. Mutations at mostly positively charged residues (Asp8, Lys14, Lys52, Lys59, Lys136, Lys138, Arg140) caused the greatest reduction in receptor-binding affinity. Most of these residues mapped to helices-1 and -4 and a loop region between helix-1 and helix-2. Two of the monoclonal antibodies that blocked TPO binding and bioactivity had determinants in helix-4. In contrast, the other three monoclonal antibodies, which were effective at blocking TPO activity but did not block initial binding of TPO to its receptor, had epitopes predominantly on helix or 3. These results suggest that TPO has two distinct receptor-binding sites that function to dimerize TPO receptors in a sequential fashion.


INTRODUCTION

Thrombopoietin (TPO)1 is the hematopoietic cytokine responsible for stimulation of megakaryocyte formation and regulation of platelet release (1-3). The gene for TPO encodes a 38-kDa protein (332 amino acids) that can be divided into two distinct domains (1, 4). The N-terminal region (153 amino acids) is predicted to be a four-helix bundle and has considerable sequence similarity to erythropoietin (EPO; 23% amino acid identity), whereas the C-terminal domain has no homology to known proteins and contains several asparagine-linked glycosylation sites. A number of studies have indicated that the N-terminal domain, TPO1-153, is responsible for binding and activation of the TPO receptor (MPL) (1, 5). The extracellular domain of the receptor for TPO has considerable sequence similarity to the other receptors in the class I hematopoietin receptor superfamily (6).

The four-helix bundle cytokines typically activate their receptors by homo- or hetero-oligomerization in which two distinct binding sites on a single hormone sequentially bind two of the same receptor molecules or two or more different receptor molecules, respectively (for recent review, see Ref. 7). The receptor binding sites for a number of cytokines (hGH, human prolactin, IL-4, IL-6, and EPO) map to similar regions of the hormone: one of these sites (site 1) has primary determinants in helix-4 and the loop connecting helix-1 and helix-2, whereas the other site (site 2) has primary determinants in helix-1 and helix-3. In the case of hGH, site 1 binds first followed by site 2 (8); for IL-4, site 2 appears to bind first followed by site 1 (9). We wondered if TPO contains two distinct receptor binding sites, and if so, do these map to regions seen for the other members of this class.

Alanine-scanning mutagenesis has been used to probe binding determinants in protein-protein complexes (10) (for review, see Ref. 11). Here, the sequence of TPO1-153 was aligned to that of IL-4 and mapped upon the known structure of IL-4 (12). Forty residues predicted to be solvent-exposed (mostly charged residues) were mutated to alanine and displayed as a single copy on M13 phagemid particles (13). The alanine mutants were analyzed by phage ELISA (14) for binding to the TPO receptor and to five monoclonal antibodies (mAbs) that block TPO bioactivity. Interestingly, mAbs having binding determinants in helix-4 blocked receptor binding and bioactivity, whereas those having determinants in helix-1 or helix-3 blocked bioactivity but not receptor binding. These data plus the receptor epitope mapping suggest that TPO1-153 has two receptor-binding sites that bind and activate its receptor by a sequential dimerization-type mechanism similar to that for hGH.


MATERIALS AND METHODS

Phagemid Construction, Alanine-scanning Mutagenesis, and Phage Preparation

The vector, pMP11 containing the TPO1-155 gene (gift of Dan Yansura, Genentech, Inc.), was tailored by site-directed mutagenesis (15) to create EcoRI and AvrII restriction sites that facilitated excision of the TPO gene. This was inserted into an EcoRI/XbaI-digested derivative of phGHam-g3 (16). This construct places the TPO gene (codons 1-155) at the N-terminal end of two Gly-Gly-Gly-Ser repeats that is followed by the C-terminal domain (codons 249-406) of M13 gene III. Deletion mutagenesis (15) was used to remove codons 154, 155, and the amber stop codon (TAG) to produce the phagemid vector pML0433 to display TPO1-153. Alanine mutations in the TPO1-153 gene were made by site-directed mutagenesis (15). Monovalent phage particles were prepared as described previously (13, 16) with the exception that phagemid and M13K07-infected XL-1-Blue cells (Strategene, La Jolla, CA) were cultured at 30 °C. Additionally, monovalent phage particles were generated using the VCS helper phage (Strategene). Typically, phage particles obtained from 30 ml of cell culture were resuspended into 0.5 ml of 10 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.2 (PBS) for preparation of the final phage stock.

Preparation of TPO Receptor (MPL-IgG)

Recombinant MPL-IgG was prepared essentially as described by de Sauvage et al. (1). Cell culture supernatant from calcium phosphate transfected 293 cells was passed over a protein A-agarose (Repligen, Cambridge, MA) column at 1 ml/min then washed extensively with 10 mM tris-HCl, 150 mM NaCl, pH 7.5 (TBS). Material was obtained from the column with a gradient elution using ImmunoPure Gentle Ag/Ab Elution Buffer (Pierce, Rockford, IL). Fractions containing MPL-IgG were immediately desalted using PD-10 columns (Pharmacia Biotech, Uppsala, Sweden), concentrated, and dialyzed against TBS. Concentration of MPL-IgG was estimated using epsilon 280 = 1.9 ml mg-1 cm-1.

Generation of Anti-TPO1-153 mAbs

mAb 1831

Balb/c mice were immunized with approximately 10 µg per dose of TPO expressed from 293 cells. TPO was formulated in QS21/alum and RIBI adjuvant (RIBI ImmunoChem Research Inc., Hamilton, MT) and injected intraperitoneally on weeks 1, 2, 5, 6, 9, 10, 13, 23, and 26. The immunized mice were tested for antibody response by ELISA. The mouse with the highest titer was given an additional boost of 10 µg of TPO in RIBI during week 27, and 3 days later the spleen was fused with the mouse myeloma line X63-Ag8.653 (17) using 50% polyethylene glycol 4000 (Boehringer Mannheim) by the procedure of Kohler and Milstein (18).

mAbs 1643, 1645, 1646, and 1653

Balb/c mice were immunized with approximately 20 µg of TPO in RIBI adjuvant on days 0, 3, and 21. The mice were given an additional boost of 10 µg of TPO in RIBI on day 31. Three days later the popliteal nodes were fused with the mouse myeloma line X63-Ag8.653. The fused cells were plated in 96 well tissue culture plates at a density of 200,000 cells per well. Hybridoma selection, using HAT media supplement (Sigma) was begun 1 day postfusion. Beginning on day 10, the hybridoma supernatants were screened for the presence of TPO1-153-specific antibodies using a solid phase ELISA. Stable antibody producing clones were obtained by limiting dilution and large quantities of specific mAbs were produced in ascites fluid. The antibodies were purified on protein A-Sepharose columns (Fementech, Inc., Edinburgh, Scotland) and stored sterile in PBS at 4 °C. Isotyping of the monoclonal antibodies was done using a Mouse MonoAb ID/SP isotyping kit from Zymed (Zymed, South San Francisco, CA) according to the manufacturer's instructions.

TPO-phage ELISA for Determination of TPO Receptor and mAb EC50 Values

Typically, 96-well Maxisorp immunoplates (NUNC, Roskilde, Denmark) were coated with either the respective anti-TPO1-153 mAb or anti-hFc F(ab')2 (Jackson ImmunoResearch, West Grove, PA) (100 µl at 1 and 2 µg/ml, respectively, in 50 mM carbonate buffer, pH 9.6) overnight. Plates were washed using PBS, 0.05% Tween 20 (Sigma); for TPO receptor experiments, MPL-IgG was added to anti-hFc F(ab')2-coated wells (100 µl at 1 µg/mL in PBS) and incubated at room temperature for 2 h. Plates were again washed using PBS, 0.05% Tween 20. Phagemid particles were diluted serially using either PBS, 0.05% BSA, 0.05% Tween 20 (BSA blocking buffer) or 25 mM Na2HPO4/200 mM NaCl, 1% Hammarsten grade casein (BDH Lab Supplies, Poole, UK) (casein blocking buffer) and then transferred (100 µl) to coated wells. After 2 h, plates were washed extensively with PBS, 0.05% Tween 20, incubated with 100 µl of 1:5000 HRP/anti-M13 conjugate (Pharmacia Biotech) in BSA blocking buffer for 15 min then washed with PBS, 0.05% Tween 20, and PBS. Plates were developed using an o-phenylenediamine dihydrochloride/H2O2 (Sigma) solution, stopped with H2SO4, and read spectrophotometrically at 492 nm. Additionally, phage binding to polyclonal anti-TPO antisera was performed as described above to confirm TPO and TPO mutant expression. For the competition phase of the assay, each phage stock was diluted into either BSA or casein blocking buffer to give a subsaturating phage concentration in the final assay volume (100 µl). Typically, an amount of phage giving approximately 0.3-0.4 OD units after development was chosen. The diluted phage (50 µl) was combined with serially diluted MPL-IgG or mAb (50 µl) and then rapidly transferred to either MPL-IgG- or mAb-coated wells, respectively. Samples were incubated for 2 h before development as described above.

BIAcore Determination of anti-TPO1-153 mAb Affinity

Apparent equilibrium dissociation constants (Kd) for anti-TPO1-153 mAb binding to TPO were determined using surface plasmon resonance (19) on a Pharmacia BIAcore instrument. Typically, full-length TPO (Genentech, Inc.) was reacted with the activated sensor chip by random lysines to yield either 500 or 1500 resonance units. Dissociation rates were measured by passing 35 µl of either 1.3 µM, 0.65 µM, or 0.33 µM mAb in PBS with 0.05% Tween 20 at a flow rate of 20 µl/min over the sensor chip; after the injection, the decrease in resonance units was monitored with time. Binding profiles were analyzed by nonlinear regression using a two-component, bivalent binding model (BIAevaluation version 2.0; Pharmacia Kinetics). Average dissociation rates were determined using f(1)koff(1) + f(2)koff(2), where f is the fractional response value derived from the nonlinear curve fits. Association rates were determined from the concentration dependence of the binding profiles for various mAb solutions. Typically, 325 nM solutions of ligand were serially diluted (2-fold) four times, and 35 µl were injected with flow rates of 20 µl/min. Sensor chips were regenerated with injection of 5 µl of 10 mM HCl at a flow rate of 5 µl/min. Equilibrium dissociation constants were calculated by Kd = koff/kon.

TPO Receptor Binding Inhibition Assay

MPL-IgG was captured on 96-well immunoplates (NUNC) as described above. Wells were blocked with 150 µl of PBS, 0.5% Tween and washed. Each mAb stock was serially diluted 2-fold in PBS, 0.5% BSA, 0.05% Tween 20, pH 7.4, then combined with a solution of biotinylated full-length TPO to give a final TPO concentration of 35 pM. Mixtures were incubated for 1 h before transferring 100 µl to TPO receptor-coated wells. Samples were incubated for 1 h at room temperature with gentle agitation. Plates were washed, incubated with 1:5000 streptavidin-HRP (Sigma; 100 µl), then washed again. Wells were developed using TMB substrate (Kirkegaard and Perry Labs, Inc., Gaithersburg, MD), quenched with H3PO4, and read by spectrophotometer at 450 nm.

MPL/Rse KIRA

A vector containing the chimeric receptor, the extracellular domain of MPL epitope-tagged with glycoprotein D (gD-MPL) and the transmembrane/intracellular domain of the receptor-type tyrosine kinase Rse (20), was transfected into CHO cells (21).2 The cells expressing this chimeric receptor have been shown to be responsive to TPO, as measured by intracellular tyrosine phosphorylation. Intracellular tyrosine phosphorylation was detected by incubation of the wells with biotinylated 4G10 antibody (Upstate Biotechnology Inc., Lake Placid, NY; 0.05 µg/ml), streptavidin-HRP (Zymed; 1:40,000), and then developed using TMB substrate (Kirkegaard and Perry). Reactions were stopped and samples were read by spectrophotometer as described above.


RESULTS

Structure-based Sequence Alignment and Model of TPO

Members of the four-helix bundle cytokine family fall into two general classes depending on their length (for review, see Ref. 22). The long-chain class that includes hGH have lengths that range from 175 to 200 residues, whereas the short-chain class that includes IL-4 have lengths that range from 105 to 160 residues. Despite differences in length and sequence, alignments from known structures show that the helical cores of both classes of molecules are very similar (12, 23-25). In fact, predictions using structural pattern-matching methods (26) have been successful for predicting the basic four-alpha -helical cores (12, 25, 27), and these can be useful for guiding mutational analysis.

To identify the helical core in TPO1-153 we chose to align its sequence with that of IL-4, because the structure of IL-4 is known to high resolution (12) and the four cysteines in TPO1-153 match most closely to four of the six cysteines in IL-4 (Fig. 1A). Hydrophobic residues at the i to i + 3 and/or i to i + 4 positions in the predicted helical segments of TPO1-153 align with buried residues on the IL-4 helices consistent with their having similar helical cores. Although two hydrophilic residues of TPO1-153 are predicted to be partially buried in the core (Asp18 and Lys138), IL-4 also contains hydrophilic residues at these analogous positions (Thr13 and Lys123). The segment connecting alpha -helices 1 and 2 is much longer in TPO1-153 than in IL-4, and in this regard TPO resembles members of the long-chain alpha -helical family. The alignment shows that alpha -helices-2, -3, and -4 in TPO have one to three glycine residues per helix, but glycines also occur within helices of other cytokines (28). The alignment was used as a guide for alanine-scanning (Fig. 1B).


Fig. 1. A, sequence alignment of human TPO with human IL-4. The sequences were aligned to best match the cysteines and regions predicted to be helical using the crystal structure of IL-4 as a guide (12). Those residues in IL-4 that are classified as consensus inner core side chains by comparison with other four-alpha -helical cytokines (24) are shown by asterisks. Residues in TPO that when mutated to alanine caused 2-fold or greater disruption in receptor binding are shown as bold characters with dots under them. B, residues in shaded boxes are those that were chosen for mutagenesis to alanine. Dotted lines indicate the location of the predicted helical regions in TPO1-153.
[View Larger Version of this Image (32K GIF file)]

Expression of TPO1-153 on Phage

TPO1-153 was displayed monovalently on the surface of bacteriophage as a gene III fusion (13, 16). Expression of active protein was increased by growing infected Escherichia coli at 30 °C instead of 37 °C (data not shown). The TPO1-153 displayed on phage bound tightly to five anti-TPO1-153 mAbs with EC50 values in the phage ELISA that ranged between 1 and 10 nM (data not shown). Phage particles displaying TPO1-153 bound specifically to immobilized TPO receptor (MPL-IgG) and the interaction was competed by soluble TPO receptor (Fig. 2). Because the amount of phage added in the competition phase of the ELISA is subsaturating, analysis of the competition curve yields an EC50 value that is related to the dissociation constant (Kd) for the soluble receptor. The EC50 value for TPO1-153-phage (44 ± 15 nM; Fig. 2) was almost 100-fold higher than the apparent Kd (approx 0.5 nM; data not shown) for the soluble ligand-receptor interaction. This apparent reduction in affinity is likely due to linkage to the gene III protein and others have reported similar reductions in affinity when proteins were displayed on phage. Nonetheless, the relative changes in affinity caused by alanine mutations when measured on the phage have been shown to parallel those measured on soluble proteins (14).3 Thus, it seems reasonable that the TPO phage ELISA can be used to compare relative affinities for various site-directed mutants of TPO.


Fig. 2. Typical phage ELISA for TPO1-153-phage versus TPO receptor. A, a representative phage titration. TPO1-153-phage was added to MPL-IgG coated (open circle ) and noncoated (square ) wells as described under "Materials and Methods." Data represent the average from five independent phage preparations. B, competition of TPO1-153 phage by TPO receptor. A subsaturating concentration of TPO phage was combined with TPO receptor dilutions then rapidly transferred to TPO receptor-coated wells. Typically, the amount of phage chosen for competition yielded between 0.3 and 0.4 A492 units. Each point represents the average of five independent experiments, and errors are standard error of the mean. The solid line shows the curve fit for a four parameter analysis and yields an EC50 = 44 (±15) nM.
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Alanine-scanning Mutagenesis for Location of Receptor Binding Determinants

Using the alignment of TPO1-153 on to the structure of IL-4, 40 charged and surface residues were mutated to alanine (Fig. 1B). As a control for functional display, all the alanine mutants were tested for binding to anti-TPO polyclonal antisera (data not shown) and to a panel of five anti-TPO1-153 mAbs (see below). All the variants bound the polyclonal antisera, except H23A which did not bind to either the polyclonal or to the five monoclonal antibodies suggesting it was either not expressed on the phage or grossly misfolded. The R78A variant bound the polyclonal antibody but did not bind with high affinity to three of the five mAbs tested. Thus, of the forty alanine mutants, we suspect that only two variants (H23A and R78A) are grossly disruptive to the structure or are not displayed on the phage.

A phage ELISA was used to test binding of the remaining thirty-eight alanine mutants to an immobilized and dimeric form of the TPO receptor (MPL-IgG). All but two variants (D8A and K138A) retained sufficient affinity for the TPO receptor to allow for competition in the assay (Table I; Fig. 3A). Very little signal was detected for D8A and K138A in the phage titration, and we estimate that their EC50 values were at least 20-fold greater than the wild-type TPO1-153. The second class of residues, including K14A, K52A, R136A, and R140A, had EC50 values that ranged from 5-10-fold greater than TPO1-153. Nine other mutations (R17A, S24A, K59A, R98A, S106A, L129A, Q132A, H133A, and L144A) were found to have 2-5-fold higher EC50 values than TPO1-153, and these decreases in affinity were significant (p < 0.01). Interestingly, two of the alanine mutants (D18A and H20A) showed EC50 values that were two to three times lower TPO1-153. Overall, the most functionally important receptor-binding residues are located in regions predicted to be helix-1, a loop region preceding helix-2, and along helix-3 and helix-4 (Fig. 3B).

Table I. Relative EC50 values for binding of alanine mutants of TPO1-153 to the TPO receptor

The phage ELISA was performed as described under "Materials and Methods." Relative EC50 values were determined using EC50 = 44 ± 15 nM for wild-type TPO1-153.

TPO1-153 mutant EC50 mutant/ EC50 wild type Standard deviationa p valueb

D8A >20c <<0.0001
R10A 1.72 1.14 0.116
S13A 0.77 0.11 0.360
K14A 6.05 1.20 <<0.0001
R17A 2.54 0.56 0.0002
D18A 0.27 0.03 0.0085
H20A 0.51 0.01 0.057
H23A NDd
S24A 2.07 0.06 0.00058
R25A 0.85 0.07 0.564
S27A 1.02 0.06 0.927
Q28A 0.89 0.11 0.667
E31A 1.72 0.55 0.0257
H33A 0.93 0.34 0.808
D45A 2.02  ---e
E50A 1.95 1.02 0.032
K52A 9.69 6.02 0.00061
K59A 4.04 1.27 <0.0001
Q61A 1.34 0.72 0.304
D62A 1.91 1.50 0.107
E72A 1.57 1.03 0.171
R78A NDd
S87A 1.13 0.03 0.567
S88A 1.23 0.19 0.342
Q92A 1.46 0.13 0.071
S94A 1.32 0.11 0.18
R98A 3.61 0.62 <<0.0001
Q105A 1.74 0.46 0.018
S106A 2.24 0.59 0.0012
R117A 1.14 0.12 0.53
K122A 1.08 0.01 0.699
N125A 0.85 0.18 0.559
L129A 2.63 0.25 <<0.0001
S130A 1.11 0.09 0.63
Q132A 1.91 0.04 0.0019
H133A 4.36 0.08 <<0.0001
R136A 5.33 0.82 <<0.0001
K138A >20c <<0.0001
R140A 9.86 5.00 0.00011
L144A 3.54 0.67 <<0.0001

a Errors are derived from two independent trials.
b Values of p were calculated using a students t-test for unpaired values (29).
c Competition of phage with soluble TPO receptor was not performed due to poor signal in the phage titration phase of the assay.
d H23A did not bind to polyclonal or monoclonal anti-TPO1-153 antibodies. R78A did not bind with high affinity to three of the five mAbs. We could not detect binding to the TPO receptor but these mutants may have been poorly expressed so we denote them as ND, not determined.
e Only a single trial was performed.


Fig. 3. Alanine scan of TPO displayed on phage for binding to the TPO receptor. A, the EC50 value for each alanine mutant was determined and plotted as an EC50 ratio to wild type TPO1-153. A value above unity indicates the alanine mutation disrupted affinity by the value shown. H23A did not bind to anti-TPO1-153 mAbs or to polyclonal antibodies suggesting that this mutant does not express (DNE). Additionally, R78A is a mutation that disrupted binding to three mAbs and therefore may play a structural role in TPO. Errors represent standard deviations from two trials using independent TPO phage and TPO receptor preparations. For all mutants with EC50 ratios greater than two, values of p obtained using a two-tailed t test (29) were less than 0.01. For some mutants (R10A, E31A, E50A, D62A, E72A, and Q105A) with EC50 ratios between 1.5 and 2.0, the t test yields 0.02 < p < 0.17; therefore, these differences from wild-type are not statistically significant. B, TPO residues that had a significant effect on TPO receptor-binding are shown plotted on the structure of IL-4 (12). Red indicates a >20-fold disruption in affinity and these residues are labeled; dark blue indicates a 5-10-fold drop in affinity and light blue indicates a 2-5-fold loss in affinity. Because of gaps in the sequence alignment (Fig. 1A), only approximate locations are shown for some residues (Asp45, Glu50, Lys52, Lys59, and Leu144). Because TPO is larger than IL-4 residues 1-6, 47-59, and 145-153 are not included in the model. Figs. were made using Insight II (Biosym).
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Alanine-scanning Mutagenesis for Epitope Mapping Anti-TPO1-153 mAbs

Five monoclonal antibodies that bind TPO1-153 (mAbs 1643, 1645, 1646, 1653, and 1831) were tested for binding to phage expressing the alanine mutations (Fig. 4A). As mentioned above, only H23A did not bind any of the mAbs, and R78A bound poorly to mAbs 1643, 1645, and 1653. The remaining 38 mutants bound to at least three of the five mAbs with near wild-type affinity suggesting that any disruptive binding effects were not the result of gross misfolding or poor expression. The dynamic range in the phage ELISA for the mAbs allows us to measure increases in EC50 (decreases in affinity) of up to 20-fold. In general, only a small set of alanine mutants (one to eight) caused a >20-fold increase in EC50 to any particular mAb. That only a few critical residues are important for governing mAb binding has been previously reported for a number of antibodies against several other proteins (30-33).


Fig. 4. Alanine scan for TPO1-153-phage versus five monoclonal antibodies. A, each alanine mutant was tested for binding to five anti-TPO1-153 mAbs using the TPO phage ELISA. The effects of the alanine mutations are expressed as EC50 ratios relative to TPO1-153. For mutants with bars denoted as >20-fold, the ELISA signal was too weak for the competition phase of the experiment. Data represent the average of two trials with the exception of mAb 1645. B, residues that were determined to be important for mAb binding are shown labeled on the IL-4 structure. Color coding for the residues is described in Fig. 3B.
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Mapping the disruptive alanine mutations on to the structural model of TPO shows that the epitopes for the five mAbs fall into three groups: mAbs 1643 and 1645 bind similarly, mAbs 1653 and 1831 bind similarly, and mAb 1646 is rather distinct from either of these two groups (Fig. 4B). Excluding R78A as a binding determinant, binding of mAbs 1643 and 1645 to TPO is dominated by residues on helix-3. The mutations, S87A and S94A were most disruptive to binding of mAb 1643, and S88A and Q92A and were most disruptive to binding of mAb 1645. mAbs 1653 and 1831 had primary determinants in helix-4 as well as a short segment at the N-terminal region of helix-2 (Fig. 4B). Binding of mAb 1653 was significantly effected by the mutations K52A, D62A, N125A, L129A, Q132A, H133A, R136A, K138A, and R140A. For mAb 1831, residues K59A, Q61A, D62A, and K138A (on helix-4) were found to be important for binding TPO.

Effects of alanine mutations on binding mAb 1646 were somewhat less disruptive (between 2- and 3-fold) and generally larger in number (Fig. 4A). Mutations that caused a greater than two-fold increase in the EC50 for mAb 1646 were R10A, R17A, D18A, S24A, Q92A, and R98A. Generally, these mutations map to predicted helix-1 and helix-3 (Fig. 4B). Binding of phage expressing R78A could not be detected for mAb 1646. Because this residue is predicted to be located distal to the other binding determinants, it is presumed that this mutation is important for maintaining global structural integrity.

Receptor Binding and Neutralizing Activities for Anti-TPO1-153 mAbs

Using surface plasmon resonance, the affinities and kinetics were measured for TPO binding to the five anti-TPO1-153 mAbs (Table II). All the mAbs bound TPO with similar Kd values (ranging from 1.2 to 8.3 nM) and similar kinetic parameters. Each mAb was then tested for its ability to block either direct receptor binding or activity of full-length TPO. mAbs 1653 and 1831 effectively blocked TPO binding to immobilized TPO receptor with IC50 values of 1.7 and 1.3 nM, respectively, (Fig. 5A). These values were reasonably close to their respective Kd values for binding to TPO. In contrast, the IC50 value for mAb 1646 inhibiting binding of the TPO to its receptor was approximately 10-fold higher (13.5 nM) than its Kd for binding to TPO (13.5 nM versus 1.1 nM). mAbs 1643 and 1645 only weakly inhibited TPO receptor binding and showed only approx 30% inhibition of TPO receptor binding even at a concentration of 500 nM (Fig. 5A), yet they bind TPO with Kd values of 4.0 and 4.8 nM, respectively (Table II). Thus, while all five the mAbs bound with similar affinities only two effectively blocked initial binding to receptor.

Table II. Kinetic and equilibrium binding constants for binding of mAbs to TPO as determined from BIAcore

Full-length TPO was randomly immobilized to the sensor chip via lysine residues at densities ranging from 1500 and 500 resonance units. Dilutions of each mAb were passed over the immobilized TPO and kinetic constants were obtained by nonlinear regression of association/dissociation phases. Association rates were determined by linear regression of rates for various mAb concentrations. Errors represent the standard error of the linear regression analysis. Dissociation rates were determined using a two-component fit analysis and average koff was calculated using koff(ave) = f(1)koff(1) f(2)koff(2), where f is the fractional response attributed each rate. Errors are given as the standard deviation using at least three determinations. Apparent affinities were determined by Kd koff(ave)/kon.

 alpha -TPO1-153 mAb f(1) koff(1) × 10-2 f(2) koff(2) × 10-4 koff(ave) × 10-4 kon × 105 Kd

s-1 s-1 s-1 M-1 s-1 nM
1643 0.03 1.9  ± 0.2 0.97 2.6  ± 0.3 8.2  ± 0.2 1.7  ± 0.3 4.8  ± 0.8
1645 0.03 2.3  ± 0.2 0.97 1.6  ± 0.3 8.4  ± 0.3 2.1  ± 0.4 4.0  ± 0.7
1646 0.01 2.9  ± 1.3 0.99 0.9  ± 0.4 3.7  ± 0.1 1.4  ± 0.4 2.6  ± 0.5
1653 0.02 2.8  ± 0.3 0.98 2.8  ± 0.1 8.3  ± 0.1 1.0  ± 0.1 8.3  ± 1.0
1831 0.01 1.8  ± 0.6 0.99 0.8  ± 0.2 2.6  ± 0.1 2.2  ± 0.2 1.2  ± 0.1


Fig. 5. Competition of TPO receptor binding and activity. A, TPO receptor binding inhibition assay. The TPO receptor (MPL-IgG) was captured on ELISA plates using an anti-hFc antibody. Each anti-TPO1-153 mAb was serially diluted and then incubated with biotinylated full-length TPO (35 pM). Mixtures were then transferred to TPO receptor coated wells and amount of TPO bound was measured spectrophotometrically. Data was normalized to maximum signal (A450) and is expressed as percent inhibition. Each data point represents the average between two experiments and the solid line is the best fit using a four parameter analysis. IC50 values determined for mAb 1643 (bullet ), 1645 (black-triangle), 1646 (square ), 1653 (black-diamond ), and 1831 (open circle ) are 86.7, 51.2, 13.5, 1.7, and 1.3 nM, respectively. B, MPL/Rse KIRA assay for measurement of receptor intracellular domain phosphorylation. CHO cells were transfected with a gD-MPL/Rse chimeric receptor. Treatment of these cells with TPO results in activation of the fusion receptor via phosphorylation of the tyrosines in the Rse intracellular domain. Activity is measured spectrophotometrically after treatment and cell lysis using a gD-capture ELISA format with an anti-phosphotyrosine mAb. Each mAb was tested for the ability to block TPO activity (150 pM). Shown as controls are three mAbs 1664 (black-triangle]), 1682 (diamond ), and 1908 (black-down-triangle ) that recognize TPO within the COOH domain. Data represents the average of three trials. Symbols for the neutralizing mAbs are as denoted above.
[View Larger Version of this Image (22K GIF file)]

A MPL/Rse KIRA assay was used to monitor TPO-induced intracellular domain phosphorylation. A receptor fusion, consisting of the extracellular domain of the TPO receptor linked to the transmembrane and intracellular domains of the Rse kinase, was transfected into CHO cells; TPO-specific tyrosine phosphorylation was measured in these cells using an ELISA-based format. All five of the mAbs were able to neutralize nearly all of the TPO-induced phosphorylation with similar IC50 values (Fig. 5B). As a control, mAbs that recognize the C-terminal domain of full-length TPO have no antagonistic effect (Fig. 5B). To confirm that the mAbs could also neutralize TPO activity for the full-length TPO receptor, a cell proliferation assay was performed using the cell line HU-03 which normally expresses the TPO receptor. Using 70 nM of each mAb, inhibition of TPO activity (at 1 pM TPO) was found to be 83.6, 80.1, 84.8, 90.6, and 87.3% for mAb 1643, 1645, 1646, 1653, and 1831, respectively (data not shown). Furthermore, all antibodies were fully capable of neutralizing TPO activity for BAF-3 cells that were transfected with the full-length TPO receptor (1) (data not shown). Thus, all the mAbs, including the three mAbs that do not block initial binding of TPO (1643, 1645, and 1646), effectively neutralize bioactivity of TPO in three cell-based assays.


DISCUSSION

The glycoprotein, TPO, is a key regulator of megakaryocyte and platelet production (1, 2). This molecule has tremendous therapeutic potential for patients who have undergone chemotherapy and consequently suffer from thrombocytopenia. The N-terminal domain (amino acids 1-153) is sufficient for receptor-binding and megakaryocyte proliferation (1, 5). The N-terminal domain of TPO is predicted to be a four-helix bundle like its most closely related homolog EPO (34) and more distantly related ones for which structures are known like hGH, granulocyte colony-stimulating factor, and interleukin-4. In the absence of the three-dimensional structure for TPO, a structural alignment was made based on the sequence and the crystal structure of IL-4; this model was used as a guide for site-directed mutagenesis to begin to elucidate the functional features of TPO1-153·

The TPO residues that were found to affect receptor binding could be divided into three classes. The first class consists of residues that caused greater than a 20-fold increase in the EC50 (D8A and K138A). A second class of TPO mutants increased the EC50 ranging from between five- and fifteen-fold (K14A, K52A, R136A, and R140). A third class of mutants reduced affinity between 2- and 5-fold (R17A, S24A, K59A, R98A, S106A, L129A, Q132A, H133A, and L144A). It is likely that other residues near these also play roles in receptor binding because only charged and polar residues were targeted for mutagenesis. Without structural information, we cannot rule out the possibility that some of the alanine mutations effect receptor binding by affecting the structure of the molecule. However, we believe the vast majority of these mutations cause only minor changes in structure because thirty-eight of the forty mutants tested bound to at least three of five anti-TPO mAbs.

Generally, the side chains important receptor-binding map to one side of the predicted four helix bundle (Fig. 3B). Most of the 15 functional residues reside on helix-1 and helix-4. Seven of the 15 identified residues map onto helix-4 and four of these are positively charged side chains (His133, Arg136, Lys138, and Arg140). Most of the residues in EPO that align with the functionally important residues on helix-4 of TPO are predicted to be solvent-exposed in a model of EPO (1, 34). Lys138 in helix-4 of TPO plays a very significant role in receptor binding, yet may be involved in inner core packing of the four-alpha -helix bundle as predicted by our sequence alignment with IL-4 (Fig. 1A) and model (Fig. 3B). Tyr145 in EPO aligns with Lys138 of TPO (1), and a structural model of EPO (34) predicts that Tyr145 is buried in the core of the globular structure. Thus, even though the K138A mutant in TPO bound to three of the five anti-TPO1-153 mAbs, without a structure we cannot exclude the possibility that this causes local structural perturbations.

Most of the residues found to be important for receptor binding are completely conserved among humans, pigs, and mice (4). It is likely that the residues identified here for human TPO binding are also important for murine and pig TPO binding because TPO reacts broadly across these species (4). Slight variation does occur at minor binding determinants. For example, S24A which causes a 2.1-fold reduction in affinity is glycine in pig; S106 which causes a 2.2-fold reduction in affinity is aspartate in pig and glycine in mouse; His133 which causes a 4.4-fold reduction in affinity is glutamine in both pig and mouse.

There are striking similarities between those regions found to be functional for TPO receptor binding and two other well characterized hematopoietic cytokine systems, hGH and EPO. For the hGH-receptor interaction, most of the binding determinants for initial receptor binding (site 1) are found on helix-4 (10, 35). Most of the residues dominating receptor binding by EPO reside in regions predicted to be helix-1 and helix-4 (36). Overall, the alanine-scanning results for EPO corroborate very well with those presented here for TPO in that a number of positively charged side chains along helix-4 are important for modulating receptor binding.

Two of the alanine mutations, D18A and H20A, were found to improve binding to the TPO receptor by 4- and 2-fold, respectively. Similarly, it was found that alanine mutations at several contact residues in helix-1 and helix-4 in hGH gave affinity improvements for hGH receptor binding (10, 28). Interestingly, these steric hindrance residues in hGH (His18, Phe25, Gln29, Glu65, and Glu174) are known to be critical for hGH binding to the prolactin receptor (8) or for forming a dimeric complex that chelates Zn2+ (37). Thus, it is likely that Asp18 and His2O are buried at the TPO-receptor interface, and it is possible they play additional roles as yet to be determined.

Although a number of alanine mutations reduce the affinity of TPO for its receptor, most were found to have no effect (Fig. 3B). These "nonfunctional" residues are found in loops and throughout helices-1, -2, and -3. A number of these residues are involved in binding one or more of the neutralizing mAbs. Two of the antibodies (mAbs 1653 and 1831) that map to the N-terminal end of helix-2 and helix-4 are neutralizing for both receptor binding and TPO receptor activation (Fig. 5). These results suggest that this face of TPO is required for TPO receptor binding and activation, analogous to site 1 in hGH. mAbs 1643 and 1645 map to regions on helix-3 and do not block receptor binding but do inhibit receptor activation. Correlating this result to the receptor alanine-scanning data strongly suggests that residues near Ser87, Ser88, Gln92, and Ser94 are involved in binding of possibly a second TPO receptor. In addition, mAb 1646 blocked TPO activity but was only moderately effective at competing for receptor binding. This antibody maps to helix-1 and helix-3 further indicating that residues on this side of the molecule (opposite that of site 1) are required for receptor activation.

A report by Alexander et al. (38) suggests that TPO activates its receptor by homodimerization. By engineering cysteine residues into a conserved receptor dimer interface region proximal to the membrane-spanning helix in the TPO receptor, receptor mutants were found that were constitutively active in a cell-based assay. Similar findings were first reported for the EPO receptor (39). Furthermore, chimeric receptors consisting of the extracellular domains of the granulocyte colony-stimulating factor receptor (6, 40) and the hGH receptor (41) fused to the intracellular region of the TPO receptor have demonstrated that TPO receptor homodimerization precedes signal transduction.


CONCLUSIONS

Using a structural alignment and alanine-scanning mutagenesis we have begun to dissect the interactions between TPO and its receptor. As for a number of other ligand-receptor systems within the hematopoietic family (hGH, EPO, and granulocyte colony-stimulating factor receptor), our data suggest that activation of the TPO receptor occurs by a sequential homodimerization mechanism. These results provide a foundation for further analysis of TPO-receptor interactions, and suggest yet another example of how a single hormone molecule can have multiple receptor binding sites to facilitate receptor oligomerization and consequent activation.


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.
§   Present address: Molecular Microbiology, SmithKline Beecham Pharmaceuticals, 1250 South Collegeville Rd., Collegeville, PA 19426.
**   To whom correspondence should be addressed: Dept. of Protein Engineering, Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080. Tel.: 415-225-1000; Fax: 415-225-6000.
1   The abbreviations used are: TPO, thrombopoietin; TPO1-153, the N-terminal domain of thrombopoietin; MPL, myeloproliferative leukemia virus receptor for TPO; EPO, erythropoietin; mAb, monoclonal antibody; HRP, horse radish peroxidase; ELISA, enzyme-linked immunosorbent assay; KIRA assay, kinase receptor activation assay; gD, glycoprotein D; PBS, phosphate-buffered saline; TBS, tris-buffered saline; CHO, Chinese hamster ovary; BSA, bovine serum albumin; hGH, human growth hormone; IL, interleukin.
2   M. Sadick, A. Galloway, and M. R. Mark, personal communication.
3   J. T. Jones, M. D. Ballinger, J. A. Lofgren, V. D. Fitzpatrick, J. A., Wells, and M. X. Sliwkowski, manuscript in preparation.

ACKNOWLEDGEMENTS

We thank Paul Sims for conducting the TPO receptor binding assays, Amy Galloway for MPL/Rse KIRA assays, Chris Jung for performing the HU-03 and BaF-3 assays, Rob Shawley and Kurt Schroeder for providing MPL-IgG cell culture supernatant and for purification advice, Raymond Kwan for DNA sequencing help, the DNA oligonucleotide group for oligo synthesis, Dan Yansura for providing the TPO clone, David Wood for help with graphics, and Drs. Fred de Sauvage and Richard Vandlen for many helpful discussions and advice.


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