A Receptor-like Protein-tyrosine Phosphatase PTPzeta /RPTPbeta Binds a Heparin-binding Growth Factor Midkine
INVOLVEMENT OF ARGININE 78 OF MIDKINE IN THE HIGH AFFINITY BINDING TO PTPzeta *

Nobuaki MaedaDagger , Keiko Ichihara-Tanaka§, Terutoshi Kimura, Kenji Kadomatsu§, Takashi Muramatsu§, and Masaharu NodaDagger parallel

From the Dagger  Division of Molecular Neurobiology, National Institute for Basic Biology, and Department of Molecular Biomechanics, Graduate University for Advanced Studies, Okazaki 444-8585, the § Department of Biochemistry, Nagoya University School of Medicine, Tsurumai-cho, Showa-ku, Nagoya 466-8550, and the  Peptide Institute Inc., 4-1-2, Ina, Minoh, Osaka 562-0015, Japan

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Midkine is a 13-kDa heparin-binding growth factor with 45% sequence identity to pleiotrophin. Pleiotrophin has been demonstrated to bind to protein-tyrosine phosphatase zeta  (PTPzeta ) with high affinity. In this study, we examined the binding of midkine to PTPzeta by solid-phase binding assay. Midkine and pleiotrophin binding to PTPzeta were equally inhibited by soluble pleiotrophin and also by some specific glycosaminoglycans. For both bindings, Scatchard analysis revealed low (3.0 nM) and high (0.58 nM) affinity binding sites. These results suggested that PTPzeta is a common receptor for midkine and pleiotrophin. Midkine is structurally divided into the N- and C-terminal halves, and the latter exhibited full activity for PTPzeta binding and neuronal migration induction. The C-terminal half contains two heparin-binding sites consisting of clusters of basic amino acids, Clusters I and II. A mutation at Arg78 in Cluster I resulted in loss of the high affinity binding and reduced neuronal migration-inducing activity, while mutations at Lys83 and Lys84 in Cluster II showed almost no effect on either activity. Chondroitinase ABC-treated PTPzeta exhibited similar low affinity binding both to the native midkine and midkine mutants at Arg78. These results suggested that Arg78 in midkine plays an essential role in high affinity binding to PTPzeta by interacting with the chondroitin sulfate portion of this receptor.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PTPzeta /RPTPbeta 1 is a receptor-like protein-tyrosine phosphatase, which is abundantly expressed in the central nervous system as a chondroitin sulfate proteoglycan (1-4). PTPzeta is composed of an N-terminal carbonic anhydrase-like domain, a fibronectin type III domain, a serine, glycine-rich domain that is thought to be chondroitin sulfate attachment region, a transmembrane segment, and two tyrosine phosphatase domains (1, 2). There are three splice variants of this molecule: (a) the full-length PTPzeta (PTPzeta -A); (b) the short form of PTPzeta , in which most of the serine, glycine-rich region is deleted (PTPzeta -B); and (c) the secreted form (PTPzeta -S), which corresponds to the extracellular region of PTPzeta -A and is also known as 6B4 proteoglycan/phosphacan (3, 5). All these splice variants are expressed as chondroitin sulfate proteoglycans in the brain (6), suggesting that chondroitin sulfate plays an essential role in receptor function.

Several proteins such as contactin, tenascin, L1, NCAM, and TAG1 have been reported to bind PTPzeta (7-9). Contactin is thought to be a neuronal receptor of PTPzeta expressed on glial cells (7). Recently, we found that PTPzeta binds with pleiotrophin/heparin-binding growth-associated molecule (10), in that a chondroitin sulfate portion of PTPzeta constitutes a part of the pleiotrophin binding site and regulates the affinity of PTPzeta -pleiotrophin binding (10). We further demonstrated that pleiotrophin-induced neurite outgrowth and neuronal migration were suppressed by chondroitin sulfate, polyclonal antibodies against the extracellular domain of PTPzeta , and sodium vanadate, a protein-tyrosine phosphatase inhibitor. These findings suggested that PTPzeta expressed on neurons is a signal transducing receptor for pleiotrophin (10, 11).

Pleiotrophin has 45% sequence identity to midkine, forming a new family of heparin-binding growth factors. These molecules share many biological activities (12, 13); both proteins promote neurite outgrowth (14-16), enhance plasminogen activator activity in aortic endothelial cells (17), and oncogenically transform NIH3T3 cells (18, 19). These findings suggest that they use a common or highly related receptors.

Midkine and pleiotrophin are structurally composed of two domains (the N- and C-terminal halves), each of which is tightly held through three or two disulfide bridges, respectively (20). The C-terminal half of midkine binds strongly to heparin and exhibits neurite outgrowth-promoting and plasminogen activator-enhancing activities (21, 22). On the other hand, the N-terminal half of midkine, which shows relatively weak heparin binding activity, does not promote neurite outgrowth or enhance plasminogen activator activity (21, 22). NMR spectroscopy revealed two clusters of basic amino acids in the C-terminal half of midkine, Clusters I and II, both of which interact with heparin oligosaccharides (23). Experiments using various midkine mutants indicated that Cluster II plays an essential role in its plasminogen activator-enhancing effect (22).

In this study, we examined the PTPzeta -midkine interaction using various midkine mutants. Native PTPzeta exhibited high affinity binding to midkine, and the binding properties were essentially the same as those of pleiotrophin. Moreover, PTPzeta -midkine binding was inhibited by the presence of pleiotrophin. These observations suggested that midkine and pleiotrophin share a common binding site on PTPzeta . PTPzeta bound to the C-terminal half of midkine, but not to the N-terminal half. A mutation R78Q in Cluster I reduced the binding affinity, while mutations K83Q, K84Q, and K83Q/K84Q in Cluster II did not affect binding. Furthermore, in these midkine mutants, the strength of binding affinities and the neuronal migration-inducing activities were highly correlated. These findings suggested that basic amino acids in Cluster I of midkine and pleiotrophin are crucial for high affinity binding to PTPzeta to transduce signals in neurons.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Chondroitin sulfate A from whale cartilage, chondroitin sulfate B from pig skin, chondroitin sulfates C and D from shark cartilage, chondroitin sulfate E from squid cartilage, heparan sulfate from bovine kidney, keratan sulfate from bovine cornea, and chondroitinase ABC were purchased from Seikagaku Corp. Heparin was obtained from Sigma. 125I-Bolton-Hunter reagent was purchased from DuPont NEN. Chroma Spin columns were obtained from CLONTECH. Maxisorp immunoplates were purchased from Nunc. Dulbecco's modified Eagle's medium, F-12 medium, and B-27 supplement were purchased from Life Technologies, Inc. TranswellsTM were obtained from Corning Coster Corp. Micro BCA kit was from Pierce. PTPzeta -S was purified as reported elsewhere (24). The N- and C-terminal half domains of human midkine (1-59 and 60-121, respectively) were synthesized as described previously (25). Mouse midkine mutants, R78Q, K83Q, K84Q, K83Q/K84Q and R78Q/K83Q/K84Q were prepared by site-directed mutagenesis (21, 22). Mutations are indicated by the amino acid residues (in one-letter code) in the wild-type and the mutant, preceding and following the numbers of the altered residues, respectively.

125I Labeling of PTPzeta -S-- PTPzeta -S was purified from rat brain and labeled as described previously (10, 24). Briefly, dried 125I-Bolton-Hunter reagent (100 µCi) was solubilized with samples (10 µg of protein in 100 µl of 100 mM sodium phosphate buffer, pH 8.0), followed by incubation for 3 h on ice and then mixed with 30 µl of 1 M glycine, pH 7.5. After a 2-h incubation at 4 °C, free 125I-Bolton-Hunter reagent was removed by passing through a Chroma Spin 30 column equilibrated with 0.05% Triton X-100, 0.5 mg/ml BSA, 0.15 M NaCl, 10 mM sodium phosphate, pH 7.2. The specific radioactivity of the sample thus prepared was 3.3 × 106 cpm/µg.

Binding Assay-- Wells of Nunc Maxisorp Immunoplates were coated with 35 µl of 1~5 µg/ml midkine or pleiotrophin in 5 mM Tris-HCl, pH 8.0, at 4 °C overnight. The wells were washed three times with phosphate-buffered saline and then blocked with 1% BSA/phosphate-buffered saline for 1 h at room temperature. 125I-PTPzeta -S diluted in 0.5% BSA, 2 mM CaCl2, 2 mM MgCl2, 0.1% CHAPS, 0.15 M NaCl, 10 mM sodium phosphate, pH 7.2, was added to the coated wells. When inhibition experiments were performed, inhibitors (pleiotrophin or glycosaminoglycans) were premixed with 125I-PTPzeta -S before addition to the wells. The plates were incubated for 5 h at room temperature and then the wells were washed three times with 1 mM CaCl2, 1 mM MgCl2, 0.15 M NaCl, 10 mM Tris-HCl, pH 7.2. The bound materials were released by adding 200 µl of 0.1 M NaOH, 0.2% SDS to the wells. The plates were shaken for 15 min at room temperature and then the eluted radioactivity was measured using a gamma  counter.

125I-Labeled PTPzeta -S was digested with chondroitinase ABC as described previously (10). Briefly, 125I-PTPzeta -S was diluted with 100 µl of 0.5% BSA, 2 mM MgCl2, 2 mM CaCl2, 0.15 M NaCl, 10 mM sodium acetate, 10 mM Tris-HCl, pH 7.5, to a final concentration of 2 µg/ml. Aliquots (5 milliunits) of protease-free chondroitinase ABC was added to the samples, and the solutions were incubated for 30 min at 30 °C for use in binding assays.

Other Methods-- Boyden chamber cell migration assays were performed using cortical neurons from embryonic day-17 Sprague-Dawley rats as described previously (11). Protein concentration was determined using a Micro BCA kit using BSA as a standard.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Binding of PTPzeta -S to Midkine-- Fig. 1 shows the binding profile of 125I-labeled PTPzeta -S to human midkine-coated ELISA plates. Scatchard analyses of the binding of PTPzeta -S to midkine showed low (Kd = 3.0 nM) and high (Kd = 0.58 nM) affinity binding sites (Fig. 1B), which were similar to those of pleiotrophin-PTPzeta binding (10). As shown in Fig. 1, the C-terminal half of midkine exhibited exactly the same binding properties to PTPzeta -S as native midkine. On the other hand, the N-terminal half of midkine showed no binding activity to PTPzeta -S (Fig. 1). Soluble pleiotrophin premixed with PTPzeta -S inhibited the binding of PTPzeta -S to pleiotrophin-coated ELISA plates (Fig. 2). In a similar dose-dependent manner, soluble pleiotrophin also inhibited the binding of PTPzeta -S to midkine on the plates (Fig. 2), suggesting that pleiotrophin and midkine bind to the same binding site on PTPzeta -S with a similar affinity. However, fairly high concentrations of pleiotrophin were required for inhibition (IC50 = ~600 nM) compared with the Kd values of midkine- or pleiotrophin-PTPzeta -S binding obtained by solid-phase binding assay. These observations suggested that substrate-bound forms of midkine and pleiotrophin exhibit orders of stronger affinity to PTPzeta -S than the soluble forms.


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Fig. 1.   Midkine binds to PTPzeta through the C-terminal half. A, wells of ELISA plates were coated with wild-type human midkine (), the C-terminal half (open circle ), or the N-terminal half of midkine (black-square), and the binding of 125I-PTPzeta -S was measured by solid-phase binding assay. B, 125I-PTPzeta -S binding to midkine (), C-terminal half (open circle ), or N-terminal half of midkine (black-square) was analyzed using Scatchard plots. Midkine exhibited high (Kd = 0.58 nM) and low (Kd = 3 nM) affinity binding sites. The C-terminal half of midkine also exhibited high (Kd = 0.55 nM) and low (Kd = 2.4 nM) affinity binding sites, but the N-terminal half of midkine showed no binding.


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Fig. 2.   Pleiotrophin inhibits the binding of midkine to PTPzeta -S. Binding of 125I-PTPzeta -S to midkine () or pleiotrophin (open circle ) on ELISA plates was measured by solid-phase binding assay in the presence of various concentrations of soluble pleiotrophin. Soluble pleiotrophin inhibited midkine-PTPzeta -S binding as well as pleiotrophin-PTPzeta -S binding.

Midkine has two clusters of basic amino acids (Clusters I and II) located at the surface on one side of the C-terminal half domain, which are considered to be heparin binding sites (23). Cluster I contains Lys76, Arg78, and Lys99, and Cluster II contains Lys83, Lys84, and Arg86; amino acids were numbered according to mouse midkine. Among these, Lys76, Arg78, Lys83, and Lys99 are conserved in midkine and pleiotrophin of all species examined to date. On the other hand, Lys84 is conserved only in midkine of various species but is changed to Arg in pleiotrophin, and Arg86 is changed to Leu in pleiotrophin of various species and midkine of some species (23).

Five mouse midkine mutants were prepared, in which some of the basic amino acids in the Cluster I and/or II were changed to glutamine: R78Q, K83Q, K84Q, K83Q/K84Q, and R78Q/K83Q/K84Q (21, 22). As shown in Fig. 3, K83Q, K84Q, and K83Q/K84Q exhibited essentially the same binding activities to PTPzeta -S as the native midkine, suggesting that Cluster II is not essential for midkine-PTPzeta binding. In contrast, R78Q and R78Q/K83Q/K84Q exhibited only low affinity binding to PTPzeta -S, suggesting that Cluster I plays an important role in the high affinity binding between PTPzeta and midkine (Fig. 3 and Table I).


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Fig. 3.   Loss of high affinity binding of midkine to PTPzeta by a mutation of Arg78 of midkine. A, the binding of 125I-PTPzeta -S to midkine mutants R78Q () or R78Q/K83Q/K84Q (open circle ) was measured by solid-phase binding assay and analyzed using Scatchard plots. B, the binding of 125I-PTPzeta -S to K83Q (open circle ), K84Q (), or K83Q/K84Q (black-square) was measured by solid-phase binding assay and analyzed using Scatchard plots. Dotted lines correspond to the binding of wild-type mouse midkine. Mutation at Arg78 resulted in the loss of high affinity binding. On the other hand, mutations at Lys83 and Lys84 showed no effect on PTPzeta -binding. The Kd values for the each midkine mutants are summarized in Table I.

                              
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Table I
Binding of PTPzeta to midkine mutants
NO, no binding; ND, not determined.

Effects of Chondroitinase ABC Digestion of PTPzeta -S on the PTPzeta -midkine Binding-- Chondroitin sulfate chains of PTPzeta play an essential role in its high affinity binding to pleiotrophin (10). Chondroitinase ABC digestion of PTPzeta -S reduced its affinity also to midkine (Fig. 4). In contrast to the intact PTPzeta -S showing high (Kd = ~0.5 nM) and low (Kd = ~3 nM) affinity binding sites, chondroitinase ABC-digested PTPzeta -S exhibited only a low affinity binding site (Kd = 8.8 nM) (Fig. 4, A and B). In addition, R78Q (Fig. 4, C and D) and R78Q/K83Q/K84Q (Table I), which have a mutation at Arg78, showed a single binding site to intact PTPzeta -S with a Kd value of 2.8 nM, in a similar affinity range to the chondroitinase ABC-digested PTPzeta -S (~8 nM). This suggested that Arg78 is involved in binding to chondroitin sulfate to make up the high affinity binding site.


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Fig. 4.   Involvement of chondroitin sulfate chains in the binding of PTPzeta to midkine. Binding of intact () or chondroitinase ABC-treated (open circle ) 125I-PTPzeta -S to native midkine (A, B) or R78Q (C, D) was measured by solid-phase binding assay. Scatchard analysis indicated that chondroitinase ABC-treated PTPzeta -S contained a single low affinity binding site for both R78Q (Kd = 8.0 nM) and native midkine (Kd = 8.8 nM).

Influence of Glycosaminoglycans on PTPzeta -midkine Binding-- Previously, we reported that pleiotrophin-PTPzeta -S binding is inhibited strongly by heparin, moderately by heparan sulfate and chondroitin sulfate C, and very weakly by chondroitin sulfate A (10). Glycosaminoglycans inhibited midkine-PTPzeta -S interactions similarly (Fig. 5). Heparin strongly inhibited binding of PTPzeta -S to midkine (IC50 = 10 ng/ml), heparan sulfate showed moderate inhibition (IC50 = 100 ng/ml), and keratan sulfate exerted almost no effect. On the other hand, various types of chondroitin sulfate exerted diverse influences on midkine-PTPzeta -S binding. Chondroitin sulfate D and chondroitin sulfate E strongly inhibited binding (IC50 = ~70 ng/ml for both types of chondroitin sulfate). Chondroitin sulfate B and chondroitin sulfate C showed moderate inhibitory effects (IC50 = 500 ng/ml and 1000 ng/ml, respectively), but chondroitin sulfate A exerted almost no effect (IC50 > 100 µg/ml). Similar sensitivities to the various chondroitin sulfates were observed for pleiotrophin-PTPzeta binding (data not shown; data partly shown in Ref. 10).


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Fig. 5.   Influence of various glycosaminoglycans on the binding of PTPzeta to midkine. Binding of 125I-PTPzeta -S to midkine was measured by a solid-phase binding assay in the presence of various concentrations of glycosaminoglycans. The effects of heparin (), heparan sulfate (black-square), chondroitin sulfate A (), chondroitin sulfate B (*), chondroitin sulfate C (open circle ), chondroitin sulfate D (+), chondroitin sulfate E (triangle ), and keratan sulfate (black-triangle) are shown.

Cell Migration-inducing Activity of Midkine-- We reported previously that pleiotrophin induced cell migration of cortical neurons (11). Midkine also induced neuronal migration in Boyden chamber cell migration assay with essentially the same dose dependence profile as that of pleiotrophin (data not shown; see Fig. 3A of Ref. 11). Boyden chamber cell migration assay indicated that the C-terminal half of midkine exhibited full cell migration-inducing activity but the N-terminal half was devoid of activity (Fig. 6A). Midkine mutants, K83Q, K84Q, and K83Q/K84Q, which have amino acid replacements in Cluster II, showed normal levels of activity. In contrast, R78Q and R78Q/K83Q/K84Q exhibited low cell migration-inducing activity (Fig. 6B). These results suggested that Cluster I is sufficient for the neuronal migration-inducing activity of midkine.


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Fig. 6.   Neuronal migration-inducing activity of midkine mutants. A, cortical neurons were analyzed by Boyden chamber cell migration assay using membranes coated with 70 µg/ml human midkine (wild), the N-terminal half (N-half), or C-terminal half (C-half) of midkine. The C-terminal half of midkine exhibited normal level of neuronal migration-inducing activity, whereas the N-terminal half was devoid of activity. B, cortical neurons were analyzed by Boyden chamber cell migration assay using membranes coated with 70 µg/ml mouse midkine (wild-type), or midkine mutants R78Q, K83Q, K84Q, K83Q/K84Q, or R78Q/K83Q/K84Q. R78Q and R78Q/K83Q/K84Q exhibited reduced neuronal migration-inducing activity.

Influence of Glycosaminoglycans on Midkine-induced Neuronal Migration-- Midkine-induced neuronal migration was inhibited strongly by heparin, moderately by heparan sulfate, but not by keratan sulfate (Fig. 7). As in the case of midkine-PTPzeta -S binding, various types of chondroitin sulfate exerted diverse effects on midkine-induced neuronal migration. Chondroitin sulfate A exhibited almost no effect (Fig. 7). On the other hand, midkine-induced neuronal migration was inhibited strongly by chondroitin sulfate E and moderately by chondroitin sulfates B, C, and D (Fig. 7). Similar inhibitory effects by chondroitin sulfates were observed for pleiotrophin-induced neuronal migration (data not shown; data partly shown in Ref. 11).


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Fig. 7.   Effects of various glycosaminoglycans on midkine-induced neuronal migration. Cortical neurons were analyzed by Boyden chamber cell migration assay using membranes coated with 33 µg/ml of midkine in the presence of various concentrations of glycosaminoglycans. Neurons were cultured in the presence of heparin (HR), heparan sulfate (HS), chondroitin sulfate A (CSA), chondroitin sulfate B (CSB), chondroitin sulfate C (CSC), chondroitin sulfate D (CSD), chondroitin sulfate E (CSE), and keratan sulfate (KS).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrated that midkine binds to PTPzeta . The characteristics of binding of midkine to PTPzeta were indistinguishable from those of pleiotrophin (10), suggesting that PTPzeta is a common receptor of midkine and pleiotrophin. Here, the C-terminal half of midkine was revealed to be sufficient for the binding. The C-terminal half domain of midkine exhibits various activities: strong heparin-binding activity, neurite promoting activity, and tissue plasminogen activator enhancing activity (21, 22). On the other hand, specific functions have not been found for the N-terminal half of midkine, although it weakly binds to heparin (21, 22, 26).

NMR spectroscopy indicated that there are two heparin-binding sites in the C-terminal half domain: Cluster I, which is composed of Lys76, Arg78, and Lys99, and Cluster II, which is composed of Lys83, Lys84, and Arg86 (23). On the other hand, in the N-terminal half domain, the basic amino acids do not form clusters which are expected to interact with the sulfate groups on heparin (23, 26). Our data showed that PTPzeta -midkine binding was significantly affected by the mutation of Arg78, but not by mutations of Lys83, Lys84, or Lys83 + Lys84. Here, mutation of Arg78 resulted in loss of high affinity binding between midkine and PTPzeta (Fig. 3), and the chondroitin sulfate portion of PTPzeta plays an essential role in formation of the high affinity binding site (Fig. 4). Therefore, it seems that Arg78 of midkine is involved in binding to chondroitin sulfate on PTPzeta . In support of this idea, various chondroitin sulfate preparations differentially affected midkine-PTPzeta binding (Fig. 5). Among various chondroitin sulfate species, there was a significant difference in the inhibiting activity. This finding suggested that there must be a specific structural motif of chondroitin sulfate that strongly inhibits midkine-PTPzeta binding. However, the nature of this structure is not known at present because commercially available chondroitin sulfate samples contain considerable heterogeneity. Nevertheless, it is possible to speculate that Arg78 of midkine recognizes a specific structure of chondroitin sulfate on the PTPzeta molecule, which is also present in chondroitin sulfates C, D, and E, but not in chondroitin sulfate A. An oversulfated structure is one of the candidates; however, the fine structure of chondroitin sulfate chains of PTPzeta must be determined to further clarify this point. A similar finding was reported for DSD-1-PG, a chondroitin sulfate proteoglycan expressed in the rodent central nervous system, that is recognized by a monoclonal antibody 473HD (27). DSD-1-PG exhibited neurite outgrowth-promoting activity, which was blocked by 473HD or by chondroitinase ABC digestion of this proteoglycan (27). The binding of 473HD to DSD-1-PG was inhibited by chondroitin sulfates C and D, but not by chondroitin sulfates A or B (27, 28), suggesting that a specific structural motif of chondroitin sulfate plays an important physiological function in the brain.

Chondroitinase ABC-treated PTPzeta showed markedly reduced binding affinity to midkine. Mutations of midkine at Arg78, Lys83, and Lys84 did not influence binding to the chondroitinase ABC-treated PTPzeta , suggesting that these amino acids do not play an essential role in binding to the core glycoprotein portion of PTPzeta . In summary, there seems to be a hierarchy with three steps in the binding between PTPzeta and midkine: 1) low affinity binding between midkine and core glycoprotein portion of PTPzeta (Kd = ~8 nM); 2) medium affinity binding between midkine and PTPzeta bearing general structure of chondroitin sulfate (Kd = ~3 nM); and 3) high affinity binding between midkine and PTPzeta bearing a specific structural motif of chondroitin sulfate (Kd = ~0.6 nM), which involves a specific contribution of Arg78 of midkine.

Boyden chamber cell migration assay indicated that the mutation of Arg78 of midkine significantly reduced the neuronal migration-inducing activity of this factor (Fig. 6). In contrast, mutations of Lys83 and Lys84 did not influence this activity. These observations suggested that the high affinity binding of midkine and PTPzeta is important for the neuronal migration-inducing activity. Here, heparin strongly inhibited midkine- and pleiotrophin-induced neuronal migration, and only the substrate-bound forms of these factors exhibit this activity (11, 17), which is consistent with the finding that PTPzeta exhibits very low affinity to soluble pleiotrophin (Fig. 2). In contrast, plasminogen activator-enhancing activity of midkine was markedly reduced by double mutation of Lys83 and Lys84, but not by the single mutation of Arg78, Lys83, or Lys84 (22). The soluble forms of midkine and pleiotrophin enhance plasminogen activator activity. However, it has been suggested that enzymatic dimerization of midkine and pleiotrophin induced by heparin-like oligosaccharides (presumably endogenous heparan sulfate) is required for plasminogen activator-enhancing activity (17). Here, exogenously added heparin could substitute for endogenous heparan sulfate (17). Taken together, these two activities of midkine and pleiotrophin are thought to be mediated by distinct receptors. Neurite-promoting activity of midkine was also markedly reduced by mutation of Arg78, while mutations of Lys83 and Lys84 were less effective (22). These observations suggested that the neurite-promoting and the neuronal migration-inducing activities of midkine are mediated at least partly by the same or similar receptor(s).

Midkine binds to a syndecan family heparan sulfate proteoglycan, ryudocan, with high affinity (29). Pleiotrophin/heparin-binding growth-associated molecule binds to N-syndecan, which is thought to be another pleiotrophin receptor involved in pleiotrophin-induced neurite extension (30). It would be helpful to examine the binding of syndecan family proteoglycans with midkine mutants to determine the physiological significance of these interactions.

    ACKNOWLEDGEMENT

We thank Akiko Kodama for secretarial assistance.

    FOOTNOTES

* This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan and from CREST of Japan Science and Technology Corporation.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.

parallel To whom correspondence should be addressed: Division of Molecular Neurobiology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji-cho, Okazaki 444-8585, Japan. Tel.: 81-564-55-7590; Fax: 81-564-55-7595; E-mail: madon{at}nibb.ac.jp.

    ABBREVIATIONS

The abbreviations used are: PTP, protein-tyrosine phosphatase; RPTP, receptor-like protein-tyrosine phosphatase; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ELISA, enzyme-linked immunosorbent assay.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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