Dimerization of Midkine by Tissue Transglutaminase and Its Functional Implication*

(Received for publication, September 25, 1996, and in revised form, December 23, 1996)

Soichi Kojima Dagger §, Tatsuya Inui , Hisako Muramatsu par , Yohko Suzuki **, Kenji Kadomatsu par , Misako Yoshizawa Dagger , Shigehisa Hirose **, Terutoshi Kimura , Shumpei Sakakibara and Takashi Muramatsu par

From the Dagger  Laboratory of Gene Technology and Safety, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Koyadai, Tsukuba, Ibaraki 305,  Peptide Institute Inc., 4-1-2 Ina, Minoh, Osaka 562, the par  Department of Biochemistry, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, and the ** Department of Biological Sciences, Tokyo Institute of Technology, Nagatsuda-machi, Midori-ku, Yokohama 226, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

Midkine (MK), a retinoic acid-inducible growth/differentiation factor, serves as a substrate for tissue transglutaminase (Kojima, S., Muramatsu, H., Amanuma, H., and Muramatsu, T. 1995. J. Biol. Chem. 270, 9590-9596). Upon incubation with transglutaminase MK forms multimers through cross-linkages. Here, we report the following results. 1) Heparin potentiated the multimer formation by MK. 2) The N- and C-terminal half domains each formed a dimer through the action of transglutaminase. 3) Gln42 or Gln44 in the N-terminal half and Gln95 in the C-terminal half served as amine acceptors in the cross-linking reaction, as judged from the incorporation of putrescine into whole MK or each half domain, and the competitive inhibition of the cross-linking by MK-derived peptides containing Gln residue(s). The strongest inhibition was obtained with Ala41-Pro51. 4) This peptide abolished the biological activity of MK to enhance the plasminogen activator activity in bovine aortic endothelial cells. The inhibition was limited against the MK monomer, and not seen against the MK dimer, separated by gel filtration chromatography. These results suggest that dimer formation through transglutaminase-mediated cross-linking is an important step as to the biological activity of MK.


INTRODUCTION

Tissue type II transglutaminase (R-glutaminylpeptide: amine gamma -glutamyltransferase, EC 2.3.2.13) is a member of the transglutaminase family that catalyzes Ca2+-dependent acyl transfer reactions between gamma -carboxamide groups of the Gln residues in peptides and either primary amines or epsilon -amino groups of the Lys residues in peptides, resulting in the formation of new gamma -amides of glutamic acid or epsilon -(gamma -glutamyl)lysine bonds and ammonia (1, 2). The molecular structure of tissue transglutaminase has been reported (3-5). Although tissue transglutaminase is widely distributed in the body (6), its physiological function is not well established compared those of other members of the transglutaminase family, e.g. the formation of cross-linkages between fibrin molecules by plasma Factor XIIIa (1, 2), and the formation of cross-linked envelopes during epidermal cell differentiation by tissue type I transglutaminase (7, 8). Recently, tissue type II transglutaminase was implicated in the association of proteases and protease inhibitors with the cell surface (9, 10), in the activation of several cytokines (11, 12), in signal transduction (13), and in the process of apoptosis (14).

Midkine (MK)1 and pleiotrophin (PTN) constitute a new family of heparin-binding growth/differentiation factors (15, 16). MK has been found as a product of a retinoic acid-responsive gene (17), and exerts a variety of biological activities; it enhances neurite outgrowth and the survival of various embryonic neuron types (18-21), and is mitogenic for certain fibroblastic cell lines (18, 19). In addition, recently, we found that MK enhances the plasminogen activator (PA) activity in bovine aortic endothelial cells (BAECs; Ref. 22). PTN, also called heparin-binding growth-associated molecule (HB-GAM; Refs. 23 and 24), was found as another neurite-promoting factor (25). PTN has been shown to be mitogenic for endothelial cells (26) and to enhance tube formation in vitro (27). Expression of these factors is strictly controlled during the processes of differentiation and development (28-30). MK is highly expressed in many human cancers (31) and specifically localized in senile plaques of Alzheimer's disease (32), and the overexpression of PTN in NIH3T3 cells results in transformation of the cells (33), suggesting the involvement of MK and PTN not only in normal development, but also in the pathogeneses of diseases (15, 16). MK is a 13-kDa heparin-binding polypeptide rich in basic amino acids and cysteine (17, 34), and exhibits 46% sequence identity with PTN (15). Both MK and PTN are largely composed of two domains, called the N-half (N1/2; Lys1-Gly59 in human MK) and the C-half (C1/2; Ala60-Asp121 in human MK), each of which contains a couple of intra-disulfide linkages (35, 36). Of these two domains C1/2 is responsible for heparin-binding, neurite outgrowth-promoting, and PA-enhancing activities (37, 38).

During the course of studying the PA-enhancing properties of MK, we discovered that MK serves as a good substrate for tissue transglutaminase (22). BAECs constitutively produce and secrete MK, which forms a transglutaminase-mediated complex in cultures. Prior to this discovery, a 29-kDa MK-related protein, which is now recognized as a dimer of MK, had been detected in a variety of tissues, such as the lymphonode, spleen, testis, small intestine, stomach, lung, kidney, and liver (39). Furthermore, Haynes and colleagues (40, 41) reported similar dimer formation by MK in the developing brain. These lines of accumulating evidence suggest that the cross-linking of MK by tissue transglutaminase may occur and play an important role in MK biology in vivo.

In the current study, we have investigated the mechanism underlying the transglutaminase-mediated cross-linking reaction and the relevance of dimer formation to MK activity.


EXPERIMENTAL PROCEDURES

Materials

The procedure for chemical synthesis of human MK and its fragments including N1/2 and C1/2 domains was described in a previous paper (42). Antibodies against the whole MK molecule, N1/2 domain, and C1/2 domain were produced in rabbits by injecting each antigen subcutaneously into animals after homogenization with Freund's complete adjuvant (Sigma). Immunization and bleeding were conducted biweekly. The IgG fraction was isolated from the sera using protein A-Sepharose (Pharmacia Biotech Inc.). The antibodies were shown to be immunospecific to each antigen by Western blotting as reported previously (37) or as shown in Fig. 1. The antibody against whole MK recognized the C-terminal tail (Thr105-Asp121) and thus recognized C1/2, but not N1/2 (37). The anti-N1/2 antibody did not cross-react with C1/2 (Fig. 1, panel A). The antibody to C1/2 recognized C-domain (Cys62-Cys104), which forms the compact structure maintained by two disulfide bonds in C1/2 (Fig. 1, panel B). This antibody showed strong, weak, and no cross-reactivity against C1/2, whole MK, and N1/2, respectively. Guinea pig liver transglutaminase and heparin sodium salt were purchased from Takara Biochemicals (Ohtsu, Japan) and Nacalai Tesque Inc. (Kyoto, Japan), respectively. Purified plasma factor XIII was a generous gift from Dr. Y. Saito (Tokyo Institute of Technology, Yokohama, Japan).


Fig. 1. Specificity of antibodies against N1/2 and C1/2 domains. Panel A, after synthetic human MK C1/2 domain (lane 1), N1/2 domain (lane 2), and intact molecule (lane 3; 300 ng each) were subjected onto SDS-PAGE on a 14% resolving gel, proteins were electrically transferred to Immobilon P (Millipore) polyvinylidene difluoride membrane and the membrane was blotted with 100 µg/ml anti-N1/2 antibody. The antibody-reacted protein bands were visualized by peroxidase-conjugated second antibody and ECL reagents. Panel B, after synthetic C1/2 (lane 1), N1/2 (lane 2), intact MK (lane 3), C-domain (lane 4), and C-terminal tail (lane 5; 300 ng each) were subjected onto SDS-PAGE on a 14% resolving gel, Western blotting was performed as in panel A using anti-C1/2 antibody.
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Putrescine Incorporation Assay

Intact (whole) MK, N1/2, or C1/2 was incubated with transglutaminase in Hepes-buffered saline (129 mM NaCl, 5 mM KCl, 0.3 mM Na2HPO4, 1 mM NaHCO3, 5 mM glucose, and 25 mM Hepes, pH 7.4) containing 10 mM CaCl2, 8 mM dithiothreitol, 0.3% (v/v) glycerol, and 3 mM [14C]putrescine (0.45 µCi, Amersham), in a final volume of 300 µl. The reaction mixture was incubated for the indicated time at 37 °C and then the reaction was stopped by the addition of 600 µl of 16.7% trichloroacetic acid with 100 µl of 2% bovine serum albumin. The precipitate was collected on Whatman GF/C glass filters and washed three times with 2 ml of 10% trichloroacetic acid, and then the radioactivity was measured by liquid scintillation counting. The amount of putrescine incorporated into each sample was expressed as moles of putrescine incorporated/mol of each sample.

Western Blotting

Western blotting was performed as described previously (22) using antibodies to intact MK (final 5 µg/ml), N1/2 (final 100 µg/ml), or C1/2 (final 100 µg/ml), and goat anti-rabbit IgG antibodies conjugated with peroxidase (Jackson ImmunoResearch Laboratories, Ltd., West Grove, PA). The signals were detected with an Amersham (Buckinghamshire, United Kingdom) ECL system.

Assaying of Cellular PA Activity

BAECs were isolated and grown in alpha -minimal essential medium containing 10% calf serum. The levels of cellular PA activity were measured using the chromogenic substrate, S-2403, as described previously (22), and expressed as urokinase units/mg of protein in each sample.

Gel Filtration Chromatography Using FPLC

Gel filtration chromatography was performed using a Superdex 75 HR 10/30 column and a FPLC System (Pharmacia). The column was equilibrated and eluted with Hepes-buffered saline buffer containing 1 M NaCl at the flow rate of 0.5 ml/min. The high salt was included to suppress the nonspecific adherence of MK to the apparatus (43). The absorbance at 280 nm was monitored, and fractions of 0.5 ml each collected.


RESULTS

Enhancement of the Cross-linking of MK by Heparin

Recently, we found that MK serves as a substrate for tissue transglutaminase (22). Incubation of recombinant murine MK with purified tissue transglutaminase readily yielded SDS- and beta -mercaptoethanol-resistant multimers, as detected by Western blotting (22). A similar result was obtained with synthetic human MK (Fig. 2, panel A). In BAECs, tissue transglutaminase functions in part on the cell surface (11), suggesting that cross-linking of MK might also occur in the cell surface milieu. On the other hand, MK is known to have strong heparin-binding activity (15). Therefore, we examined if the heparin-binding activity of MK affected its cross-linking by tissue transglutaminase. As shown in Fig. 2 (panel B), the inclusion of heparin in the reaction mixture significantly enhanced both the rate and the final degree of the formation of MK multimers by transglutaminase.


Fig. 2. Enhancement of transglutaminase-mediated cross-linking of MK by heparin. Synthetic human MK (final: 3 µM) was incubated with 1.25 µM tissue transglutaminase for the indicated time in the absence (panel A) or presence (panel B) of 30 µg/ml heparin, and then the formation of MK multimers was detected by Western blotting after SDS-PAGE on 14% resolving gels. Each similar experiment was repeated three times, and representative results are shown.
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Identification of Gln42, Gln44, and Gln95 as Possible Amine Acceptors

The formation of multimers indicates that both murine and human MK contain both Gln and Lys residues that participate in the transglutaminase-mediated bridge formation. Both murine and human MK contain 5 Gln residues/molecule (15, 17). To determine the number of acyl-donating Gln sites, a putrescine incorporation assay was performed using synthetic human MK. MK was incubated overnight with transglutaminase in the presence of [14C]putrescine, and then the number of putrescine molecules incorporated into one molecule of MK was calculated from the radioactivity. The result is shown in Fig. 3 (column 1). About 2 mol of putrescine was incorporated/mol of MK, suggesting the presence of at least two different Gln sites in a molecule. The incubation time and the concentration of putrescine employed gave the maximum amount of putrescine incorporated as a function of both the putrescine concentration and the incubation time (data not shown). MK is structurally composed of two domains, called N1/2 (Lys1-Gly59) and C1/2 (Ala60-Asp121; Ref. 15). To determine which domain contains the Gln sites, the incorporation of putrescine into synthetic N1/2 or C1/2 was measured. As can be seen in Fig. 3 (columns 2 and 3), both domains share one Gln site each. This also suggested that N1/2 and C1/2 alone each might be cross-linked by transglutaminase, as both domains contain many acyl-acceptable Lys sites (9 in N1/2 and 14 in C1/2). This was proved by the results of Western analyses performed following the incubation of either N1/2 or C1/2 with transglutaminase (Fig. 4). Although multimers larger than the tetramer were not observed much, a significant amount of the dimer was detected for both N1/2 and C1/2 (lanes 2 and 3). In contrast, comparable amounts of the dimer and tetramer were detected on the cross-linking of whole MK molecules (lane 1). These reactions did not occur in the absence of Ca2+ (data not shown). N1/2 and C1/2 contain two and three Gln residues, respectively (15). Therefore, it is suggested that either Gln42 or Gln44 in N1/2 and one of the three Gln residues (amino acids 82, 93, and 95) in C1/2 serve as acyl-donating sites. Plasma-derived FXIIIa catalyzed the incorporation of [14C]putrescine into MK, but did not catalyze the cross-linking of MK molecules, as assessed by Western blotting (data not shown).


Fig. 3. Incorporation of [14C]putrescine into intact MK, N1/2, and C1/2 by tissue transglutaminase. Synthetic human MK, N1/2, or C1/2 (final: 3 µM each) was incubated at 37 °C for 16 h with 3 mM [14C]putrescine in the presence of 1.25 µM tissue transglutaminase in Hepes buffer containing 10 mM Ca2+. After the incubation, proteins in the solution were precipitated by adding 10% trichloroacetic acid and spotted onto filter paper discs, which were repeatedly washed. Radioactivity precipitated on the discs was counted with a scintillation counter and expressed as moles of putrescine incorporated/mol of intact MK, N1/2, or C1/2. Column 1, intact MK; column 2, N1/2; column 3, C1/2. Data represent averages ± S.D. (n = 3).
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Fig. 4. Cross-linking of intact MK, N1/2, and C1/2 by transglutaminase. Synthetic human MK (final: 3 µM), N1/2 (final: 6 µM), or C1/2 (final: 6 µM) was incubated at 37 °C for 1 h with 1.25 µM tissue transglutaminase, in a total volume of 24 µl, in Hepes buffer containing 10 mM Ca2+. The reaction was terminated by adding 50 mM EDTA, samples were separated by SDS-PAGE on 14% resolving gels, and the formation of multimers was assessed by Western blotting with an anti-intact MK antibody, anti-N1/2 antibody, or anti-C1/2 antibody. The exposure time was greatly lengthened, compared with in Fig. 2. The 75-kDa band in lane 3 is a nonspecific band due to the first antibody. The numbers at the right of each band are the numbers of molecules cross-linked. Lane 1, intact MK; lane 2, N1/2; lane 3, C1/2. Each similar experiment was repeated three times, and representative results are shown.
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In parallel with putrescine incorporation assays, the effect of Gln-containing peptides on the cross-linking of intact MK, N1/2, and C1/2 was examined. In the process of chemical synthesis of human MK, we synthesized 13 segments that could be coupled to form N1/2 and C1/2, and finally intact MK (42). Of these 13 segments, segment V (Ala41-Pro51) contains both Gln42 and Gln44, and segments IX (Thr78-Gly83), X (Thr84-Gln93), and XI (Cys94-Pro103) contain Gln82, Gln93, and Gln95, respectively. The cross-linking reaction was performed in the presence of these peptides at the concentration of a 100-fold excess over moles of intact MK, N1/2, or C1/2, and the formation of each dimer was assessed by Western blotting. The results are shown in Fig. 5. Among the four peptides tested, Ala41-Pro51 (lane 2) and Cys94-Pro103 (lane 5) blocked the cross-linking reactions, whereas neither Thr78-Gly83 (lane 3) nor Thr84-Gln93 (lane 4) affected the reactions. The Ala41-Pro51 peptide showed a complete inhibition against intact MK (panel A), N1/2 (panel B), and C1/2 (panel C). The Cys94-Pro103 peptide also showed a complete inhibition toward the cross-linking of intact MK (panel A) and N1/2 (panel B), and partial inhibition against the cross-linking of C1/2 (panel C). The retardation and smear formation of each monomer observed in lanes 2 and 5 suggested that these peptides were preferentially cross-linked to molecules in question and, thereby, prevented cross-linking between monomer molecules. In this aspect, the results showed that Cys94-Pro103 was incorporated into MK or its fragment molecules more slowly than Ala41-Pro51. Next, to determine which Gln residue in Ala41-Pro51, namely either Gln42 or Gln44, participated in the cross-linking reaction, three mutant peptides were synthesized (Fig. 6); specifically, Gln42 was substituted with Ala, Gln44 was substituted with Ala, and both Gln42 and Gln44 were substituted with Ala, and then the competitive inhibition was tested. As shown in Fig. 7 (lane 2), the intact peptide was readily cross-linked to MK in a time-dependent manner and inhibited the cross-linking completely, consistent with the result in Fig. 5 (lane 2). Peptides in which one of the two Gln residues was substituted with Ala showed significant, but slower inhibition (lanes 3 and 4), suggesting that both Gln42 and Gln44 can serve as amine acceptor sites. In contrast, the double substituted peptide showed almost no inhibition (lane 5), suggesting that the inhibition by the peptides observed in lanes 2-4 was not due to the sequence structure other than Gln residues. Similar inhibition was observed with these peptides on the cross-linking of N1/2 or C1/2 (data not shown). In conclusion, Gln42 and Gln44 in the N1/2 domain as well as Gln95 in the C1/2 domain were suggested to function as potential amine-accepting sites in the transglutaminase-mediated cross-linking of MK.


Fig. 5. Effects of Gln-containing peptides on the cross-linking of intact MK, N1/2, and C1/2 by transglutaminase. Synthetic human MK, N1/2, or C1/2 (final: 3 µM each) was incubated for 30 min with 1.25 µM tissue transglutaminase and 30 µg/ml heparin in the absence or presence of 300 µM Gln-containing peptides, and then the formation of each dimer was detected by Western blotting after SDS-PAGE on 14% resolving gels. Panel A, intact MK; panel B, N1/2; panel C, C1/2. Lane 1, no competitor; lane 2, Ala41-Pro51; lane 3, Thr78-Gly83; lane 4, Thr84-Gln93; lane 5, Cys94-Pro103. Each similar experiment was repeated three times, and representative results are shown.
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Fig. 6. Sequences of three mutant peptides. The sequences of three mutant peptides derived from Ala41-Pro51 are shown. Lane 1, intact Ala41-Pro51, abbreviated as QQ; lane 2, Gln42 right-arrow Ala, abbreviated as AQ; lane 3, Gln44 right-arrow Ala, abbreviated as QA; lane 4, Gln42 right-arrow Ala and Gln44 right-arrow Ala, abbreviated as AA.
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Fig. 7. Effects of mutant peptides on the cross-linking of MK by tissue transglutaminase. Synthetic human MK (final: 3 µM) was incubated with 1.25 µM tissue transglutaminase and 30 µg/ml heparin in the absence or presence of 300 µM intact Ala41-Pro51 peptide or mutant peptides, whose sequences are presented in Fig. 6. After incubation for the indicated time, the formation of MK dimer was detected by Western blotting. Lane 1, no competitor; lane 2, intact Ala41-Pro51, abbreviated as QQ; lane 3, Gln42 right-arrow Ala, abbreviated as AQ; lane 4, Gln44 right-arrow Ala, abbreviated as QA; lane 5, Gln42 right-arrow Ala and Gln44 right-arrow Ala, abbreviated as AA. Each similar experiment was repeated three times, and representative results are shown.
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Requirement of the Cross-linking of MK for Enhancement of the PA Level

Next, we examined whether or not the cross-linking of MK participated in the MK action of enhancing the PA level, using a Gln-containing peptide, Ala41-Pro51, that showed the strongest inhibition against the cross-linking reaction (Figs. 5 and 7). BAECs were incubated with MK with increasing amounts of Ala41-Pro51, and then the PA levels were determined. Indeed, the peptide suppressed the enhancement by MK (Fig. 8, curve B), whereas a mutant peptide, with double substitution of two Gln residues with Ala, did not (curve A). This suggested that interference of the cross-linking by Ala41-Pro51 abolished the activity of MK added to the culture medium, namely that cross-linking of the MK monomer by cell surface transglutaminase might be required for the MK activity. If this speculation is correct and if the inhibition by the peptide only occurs at the step of the cross-linking, the once cross-linked dimer or tetramer should enhance the PA level without undergoing inhibition by the peptide. This was examined by means of an experiment in which the monomer, dimer, and other multimers were separated by gel filtration chromatography, followed by assaying for PA-enhancing activity in the absence and presence of the Ala41-Pro51 peptide (Fig. 9, panel A). The PA-enhancing activity (closed circles) was detected in fractions corresponding to the peak of absorbance at 280 nm (dotted line). From a comparison with the molecular standards used for the gel filtration chromatography and from the results of Western blotting of each fraction (panel B), three major peaks were determined as the monomer, the dimer, and a mixture of the tetramer and hexamer, from lower molecular weight fractions. As depicted by open circles, Ala41-Pro51 suppressed the PA-enhancing activity only in the monomer fraction, i.e. not in the dimer or tetramer/hexamer fraction. This supports the hypothesis that transglutaminase-mediated cross-linking of the MK monomer is required for its ability to enhance the PA level in BAECs.


Fig. 8. Suppression of MK activity by the A41-P51 peptide. Confluent BAEC cultures were incubated for 16 h in serum-free alpha -minimal essential medium containing 0.1% bovine serum albumin with 100 ng/ml (7.7 nM) synthetic human MK in the presence of increasing amounts of either the intact Ala41-Pro51 peptide or a mutant peptide, in which both the Gln42 and Gln44 residues were substituted with Ala. Cellular lysates were prepared in 0.5% Triton X-100 in 0.1 M Tris-HCl, pH 8.1, and the PA level of each cell lysate was measured using a chromogenic substrate and expressed as urokinase (UK) units/mg of protein in the sample. Curves A and B, MK-treated cells; curves C and D, unstimulated cells. Curves A and D, mutant peptide; curves B and C, Ala41-Pro51 peptide. Each value represents the average ± S.D. (n = 3). Each similar experiment was repeated twice, and representative results are shown.
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Fig. 9. PA-enhancing activity of the MK dimer. Synthetic human MK (final: 3 µM) was incubated at 37 °C for 1 h with 1.25 µM tissue transglutaminase (in a total volume of 1.25 ml), in Hepes buffer containing 10 mM Ca2+ and 30 µg/ml heparin, and the reaction was terminated by the addition of 50 mM EDTA. Samples were concentrated 12.5-fold using an Ultrafree C3LGC micro-concentrator (Millipore; molecular weight cut-off, 10,000) and then applied to a Superdex 75 HR 10/30 gel filtration column. The column was eluted with Hepes buffer containing M NaCl, using a FPLC system, at the flow rate of 0.5 ml/min. The absorbance at 280 nm was monitored and shown as a dotted line (panel A). 0.5-ml fractions were collected, concentrated to 24 µl, and then either examined for PA-enhancing activity in the absence (closed circles) or presence (open circles) of 24 µg/ml (15.2 µM) Ala41-Pro51 peptide, as described in Fig. 8 (panel A), or assessed the molecular species of MK by Western blotting (panel B). The PA activity levels plotted are averages (n = 3). Each similar experiment was repeated twice, representative results being shown.
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Non-covalent Association of MK

As can be seen in Fig. 9 (panel B), several bands were detected on Western blotting of the peak fractions. For example, the hexamer, tetramer, and dimer were seen in fraction 23, which was determined to be composed of the hexamer and tetramer, and the dimer and monomer were detected in fraction 26, which was determined to be the dimer. As a reason for this, we speculated that MK associated non-covalently in addition to the covalent cross-linking by transglutaminase. To determine if such a non-covalent association really occurs, we performed the cross-linking reaction in the presence or absence of free Ca2+, and then compared the chromatograms obtained on FPLC (Fig. 10). In the presence of free Ca2+, namely when transglutaminase functioned, almost the same amounts of the monomer, the dimer, and the tetramer/hexamer mixture were detected in fractions 36, 26, and 23, respectively (curve B, a solid line). However, even when transglutaminase was blocked by chelating Ca2+, almost the same peak was detected for fraction 26, and about a half amount of the peak was detected for fraction 23, in addition to a peak of the monomer for fraction 36 (curve A, a dashed line). Furthermore, the PA-enhancing activity was detected in accordance with the peak of multimers in addition to monomer (Fig. 11). Western blots of these fractions only showed the monomer (data not shown), indicating that the peaks detected for fractions 26 and 23 represented the non-covalently associated MK dimer, tetramer, or hexamer, which dissociated upon exposure to SDS. Therefore, under the current experimental conditions, MK forms multimers via two distinct mechanisms, transglutaminase-mediated covalent cross-linking and non-covalent association. We next examined the effect of Ala41-Pro51 on this non-covalent association. The cross-linking reaction was performed in the presence of an excess of the peptide, and multimer formation was assessed by gel filtration chromatography. As depicted by curve C (a double-dashed line) in Fig. 10, almost no peak was detected for fraction 26, although two small peaks were detected for fractions 22 and 23, suggesting that not only the cross-linking, but also the non-covalent association was suppressed by the Ala41-Pro51 peptide.


Fig. 10. Effect of the Ala41-Pro51 peptide on the non-covalent association of MK. Synthetic human MK was incubated with tissue transglutaminase as described in Fig. 9 in the absence (curves A and B) or presence (curve C) of 475 µg/ml (300 µM) Ala41-Pro51 peptide. Ca2+ was chelated by the addition of EDTA either before (curve A) or after (curves B and C) the incubation. Samples were concentrated and then subjected to the gel filtration chromatography, using a FPLC system. The absorbance at 280 nm was monitored and expressed. Each similar experiment was repeated twice, representative results being shown.
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Fig. 11. PA-enhancing activity of the non-covalently associated MK dimer. Synthetic human MK was incubated with tissue transglutaminase as described in Fig. 10 after the addition of EDTA. Samples were concentrated, fractionated through a Superdex 75 HR 10/30 gel filtration column, and eluted as described in Fig. 9. The absorbance at 280 nm was monitored and shown as a dashed line. 0.5-ml fractions were collected, concentrated to 24 µl, and then examined for PA-enhancing activity. The PA activity levels plotted are averages (n = 3). Each similar experiment was repeated twice, representative results being shown.
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DISCUSSION

Retinol (vitamin A) and its derivatives (retinoids) have profound effects on the regulation of cell growth and differentiation (44). Using BAEC cultures, we found that retinoids induce the production of PA (45), transglutaminase (46), transforming growth factor-beta (TGF-beta ; Ref. 47), and MK (22), and that these retinoic acid-inducible factors interact with each other. In retinoic acid-treated BAECs, PA and transglutaminase are required to promote the activation of latent TGF-beta (11, 47), whereas MK and TGF-beta regulate PA activity, respectively, in the opposite way (11, 22). The present paper describes an additional relationship between transglutaminase and MK.

MK was proved to be an excellent substrate for transglutaminase in that 1 mol of MK incorporated as much as 2 mol of [14C]putrescine. MK formed the dimer, tetramer, and hexamer on incubation with transglutaminase. Among multimers dimer seemed to be dominant. Whereas the results in Figs. 5 and 7 suggest that Gln42 and Gln44 in N1/2 as well as Gln95 in C1/2 may function as potential amine-accepting sites, the results in Fig. 3 suggest the existence of one Gln site in each of N1/2 and C1/2. The most likely explanation for this difference is that although both Gln42 and Gln44 are competent enough to serve as amine-accepting sites, only one of them is exposed to the surface of the MK molecule. The second likely explanation is that Gln42 and Gln44 serve heterogeneously as amine-accepting sites. Namely, when Gln42 is used to form an epsilon -(gamma -glutamyl) lysine bridge between a Lys residue in another MK molecule, or putrescine in the present experiment, neighboring Gln44 becomes inaccessible for further bridge formation due to stereohindrance by the MK molecule cross-linked to Gln42. Conversely, when Gln44 is cross-linked to another MK molecule, Gln42 becomes no longer accessible. Stereohindrance will also happen when Factor XIIIa is used as the enzyme. As Factor XIIIa is larger than tissue transglutaminase (~300 kDa versus 80 kDa), the cross-linking of MK molecules may not be performed by Factor XIIIa, even though it can stimulate putrescine incorporation into MK molecules. It is notable that these three acyl-donating Gln residues are conserved among the MK/PTN family, whereas the other two Gln residues are not (48). As can be seen in Fig. 4, the dimer and tetramer of MK C1/2 migrated slower than those of N1/2. This might be due to that C1/2 is much more highly charged being basic. The three-dimensional structure of MK C1/2 recently clarified by NMR spectroscopy2 is consistent with the results of the present investigation. In C1/2, basic amino acids, which are expected to form the heparin binding site (Arg81, Lys86, and Lys87), are clustered on one side. Site-directed mutagenesis of these amino acids resulted in decreased heparin-binding and neurite-promoting activities.3 Gln95, which was shown to be involved in the dimerization, is located on the opposite side. This distinct localization of the heparin binding site and the cross-linking site will permit the cross-linking between two MK molecules after binding to heparin. Thus, heparin may potentiate the cross-linking with transglutaminase by stabilizing the conformation of dimer. In addition, heparin increases the amount of products, too, by preventing the loss of MK molecules from the reaction mixture that happens due to static adherence of MK molecules to the vessel wall (43). Because of this reason and because we supposed the physiological reaction on the cell surface as discussed below, we analyzed the dimer formation in the presence of heparin. We are currently trying to determine the amine-donating Lys sites in the MK molecule. It is also of great importance to clarify the structural requirements for the substrate for transglutaminase, i.e. the role of flanking sequences in the Ala41-Pro51 peptide, and to compare it with the result obtained with fibronectin-derived sequence (49).

We have concluded that dimerization of MK by cell surface transglutaminase potentiates MK activity, based upon the fact that the Ala41-Pro51 peptide inhibits both the cross-linking and PA-enhancing activity of the MK monomer, with a double mutated peptide as an inactive control. We did not obtain additional proof utilizing an anti-transglutaminase antibody, because the antibody could inhibit the formation of TGF-beta , which counteracts MK activity (22). Since the activation of latent TGF-beta is required for transglutaminase to localize latent TGF-beta on the surface, the inclusion of an anti-transglutaminase antibody in the culture medium prevents the formation of active TGF-beta (11). Hence, the peptide was a strong tool to prove our hypothesis, although the specificity of the inhibition by the peptide was critical to obtain a conclusion. As a receptor interacting site(s) is located in C1/2 (37, 38), there is a low possibility that the Ala41-Pro51 peptide competes with the binding of MK to its receptor(s). Actually, the dimer exerted the PA-enhancing activity in the presence of this peptide. As Factor XIIIa functions only in putrescine incorporation, it is possible that Factor XIIIa might be used to cross-link peptide to MK molecule, serving as a control for this issue. It is true that when the PA-enhancing effect of the MK monomer is blocked by Ala41-Pro51, cross-linking of the MK monomer, especially formation of the dimer, is suppressed completely. However, it is possible that other transglutaminase-mediated cross-linking reactions were affected by the Ala41-Pro51 peptide, and that this caused the inhibition of MK activity by the peptide. In this context, we need to examine the specificity of the inhibition by this peptide. Although the peptide inhibited the cross-linking of intact MK, N1/2, and C1/2 by 80% at a concentration of as much as a 50-fold molar excess, a 200-fold molar excess of this peptide caused only weak (less than 5%) inhibition of the cross-linking of pro-SPAI (data not shown; Ref. 10). We are now investigating whether the peptide affects the cross-linking of other hitherto known substrates for transglutaminase. Nevertheless, we believe that the present conclusion is correct, because in the experiment in Fig. 9, whether an inhibitory effect of the peptide was observed or not depended only upon whether activity was derived from the monomer or dimer, and all other conditions were the same. Therefore, no matter what other cross-linking this peptide may interfere with, or if MK is cross-linked to other proteins such as matrix components and if the peptide blocks such cross-linking, we think that the results in Fig. 9 strongly suggest the relevance of the formation of the MK dimer.

The formation of the non-covalently associated dimer and other multimers explains why the MK monomer has always been detected on Western blotting, even on incubation with transglutaminase overnight. The Ala41-Pro51 peptide inhibited both the cross-linking and the non-covalent association through being incorporated into the Lys sites of MK molecules by transglutaminase. It appears that the peptide cannot dissociate the dimer once formed via either covalent cross-linking or even non-covalent association, as the peptide did not affect the PA-enhancing activity in the dimer fraction (Fig. 9). Once non-covalent association occurs between MK molecules, the cross-linking sites are supposed to be hidden, and thus the MK molecules might no longer act as a substrate for transglutaminase. Therefore, the non-covalently associated dimer co-existed with the cross-linked dimer, and the peptide was not cross-linked to the non-covalently associated dimer and, thus, did not affect its activity. We cannot explain why in the presence of Ala41-Pro51, the peak of the mixture of the tetramer and hexamer split into the hexamer and tetramer (Fig. 10); nor can we explain why the peptide did not inhibit non-covalent formation of the hexamer and tetramer completely, although the peptide inhibited the formation of the dimer completely. The non-covalent association may be apt to occur when the concentration of MK is unphysiologically high. At a physiological concentration, the chances of MK associating non-covalently will be low, whereas cross-linking may occur at sites where transglutaminase is exposed. Figs. 9 and 11 suggest that dimer/oligomer formation alone is sufficient to stimulate increases in PA-enhancing activity and that binding of MK to cells via cell surface transglutaminase is not necessary. However, since plasma Factor XIIIa is not able to cross-link MK molecules and tissue transglutaminase is not released from the cells (46), the surface reaction may be physiologically most important. The susceptibility of MK to transglutaminase suggests a mechanism whereby the interaction of MK with surface receptors and other surface-oriented structures could be enzymatically altered. Tissue transglutaminase is distributed mainly intracellularly and partially on the cell surface (11, 46), suggesting either that newly synthesized MK is cross-linked before secretion or that secreted MK is cross-linked on the cell surface as discussed above. The results in Fig. 2 suggest that the latter reaction may proceed very efficiently with the aid of heparan sulfate present on the cell surface. Recently, it was reported that hetero- or homodimerization of cytokine and hormone receptors is important for the emission of signals inside cells (50, 51). Together with the finding that the cross-linking is important for PA-enhancing activity, we imagine that the MK dimer, cross-linked on the cell surface, may activate its unidentified receptors by making them form the dimer. An experiment addressing this hypothesis is in progress.

The current study provided additional evidence for the hypothesis concerning the mechanism whereby transglutaminase plays a role in the regulation of cellular physiology through the cross-linking of cytokines such as TGF-beta (11), interleukin 2 (12) and, now, MK.


FOOTNOTES

*   This work was supported in part by a grant for the "Biodesign Research Program" from RIKEN, by Grants-in-aid 08780689 (to S. K.) and 08457035 (to T. M.) from the Japanese Ministry of Education, Science and Culture, and by grants from the Ito Foundation (to S. K.).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 all correspondence should be addressed. Tel.: 81-298-36-3522; Fax: 81-298-36-9050; E-mail: kojima{at}rtc.riken.go.jp.
1   The abbreviations used are: MK, midkine; PTN, pleiotrophin; PA, plasminogen activator; BAECs, bovine aortic endothelial cells; N1/2, N-terminal half; C1/2, C-terminal half; TGF-beta , transforming growth factor-beta .
2   F. Inagaki, personal communication.
3   N. Asai, unpublished result.

ACKNOWLEDGEMENT

We thank Dr. Y. Saito for supplying the Factor XIII as well as for comments on this study.


REFERENCES

  1. Lorand, L., and Conrad, S. M. (1984) Mol. Cell. Biochem. 58, 9-35 [Medline] [Order article via Infotrieve]
  2. Greenberg, C. S., Birckbichler, P. J., and Rice, R. H. (1991) FASEB J. 5, 3071-3077 [Abstract/Free Full Text]
  3. Gentile, V., Saydak, M., Chiocca, E. A., Akande, O., Birckbichler, P. J., Lee, K. N., Stein, J. P., and Davies, P. J. A. (1991) J. Biol. Chem. 266, 478-483 [Abstract/Free Full Text]
  4. Nakanishi, K., Nara, K., Hagiwara, H., Aoyama, Y., Ueno, H., and Hirose, S. (1991) Eur. J. Biochem. 202, 15-21 [Abstract]
  5. Lu, S., Saydak, M., Gentile, V., Stein, J. P., and Davies, P. J. A. (1995) J. Biol. Chem. 270, 9748-9756 [Abstract/Free Full Text]
  6. Thomázy, V., and Fésüs, L. (1989) Cell Tissue Res. 255, 215-224 [Medline] [Order article via Infotrieve]
  7. Floyd, E. E., and Jetten, A. M. (1989) Mol. Cell. Biol. 9, 4846-4851 [Medline] [Order article via Infotrieve]
  8. Marvin, K. W., George, M. D., Fujimoto, W., Saunders, N. A., Bernacki, S. H., and Jetten, A. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11026-11030 [Abstract]
  9. Bendixen, E., Borth, W., and Harpel, P. C. (1993) J. Biol. Chem. 268, 21962-21967 [Abstract/Free Full Text]
  10. Nara, K., Ito, S., Ito, T., Suzuki, Y., Ghoneim, M. A., Tachibana, S., and Hirose, S. (1994) J. Biochem. (Tokyo) 115, 441-448 [Abstract]
  11. Kojima, S., Nara, K., and Rifkin, D. B. (1993) J. Cell Biol. 121, 439-448 [Abstract]
  12. Eitan, S., and Schwartz, M. (1993) Science 261, 106-108 [Medline] [Order article via Infotrieve]
  13. Nakaoka, H., Perez, D. M., Baek, K. J., Das, T., Husain, A., Misono, K., Im, M.-J., and Graham, R. M. (1994) Science 264, 1593-1596 [Medline] [Order article via Infotrieve]
  14. Zhang, L.-X., Mills, K. J., Dawson, M. I., Collins, S. J., and Jetten, A. M. (1995) J. Biol. Chem. 270, 6022-6029 [Abstract/Free Full Text]
  15. Muramatsu, T. (1994) Dev. Growth & Diff. 36, 1-8
  16. Kurtz, A., Schulte, A. M., and Wellstein, A. (1995) Crit. Rev. Oncogen. 6, 151-177 [Medline] [Order article via Infotrieve]
  17. Kadomatsu, K., Tomomura, M., and Muramatsu, T. (1988) Biochem. Biophys. Res. Commun. 151, 1312-1318 [Medline] [Order article via Infotrieve]
  18. Muramatsu, H., and Muramatsu, T. (1991) Biochem. Biophys. Res. Commun. 177, 652-658 [Medline] [Order article via Infotrieve]
  19. Nurcombe, V., Fraser, N., Herlaar, E., and Heath, J. K. (1992) Development 116, 1175-1183 [Abstract/Free Full Text]
  20. Michikawa, M., Kikuchi, S., Muramatsu, H., Muramatsu, T., and Kim, S. U. (1993) J. Neurosci. Res. 35, 530-539 [Medline] [Order article via Infotrieve]
  21. Satoh, J., Muramatsu, H., Moretto, G., Muramatsu, T., Chang, H. J., Kim, S. T., Cho, J. M., and Kim, S. U. (1993) Dev. Brain Res. 75, 201-205 [Medline] [Order article via Infotrieve]
  22. Kojima, S., Muramatsu, H., Amanuma, H., and Muramatsu, T. (1995) J. Biol. Chem. 270, 9590-9596 [Abstract/Free Full Text]
  23. Merenmies, J., and Rauvala, H. (1990) J. Biol. Chem. 265, 16721-16724 [Abstract/Free Full Text]
  24. Li, Y.-S., Milner, P. G., Chauhan, A. K., Watson, M. A., Hoffman, R. M., Kodner, C. M., Milbrandt, J., and Deuel, T. F. (1990) Science 250, 1690-1694 [Medline] [Order article via Infotrieve]
  25. Rauvala, H. (1989) EMBO J. 8, 2933-2941 [Abstract]
  26. Fang, W., Hartmann, N., Chow, D. T., Riegel, A. T., and Wellstein, A. (1992) J. Biol. Chem. 267, 25889-25897 [Abstract/Free Full Text]
  27. Laaroubi, K., Delbé, J., Vacherot, F., Desgranges, P., Tardieu, M., Jaye, M., Barritault, D., and Courty, J. (1994) Growth Factors 10, 89-98 [Medline] [Order article via Infotrieve]
  28. Kadomatsu, K., Huang, R.-P., Suganuma, T., Murata, F., and Muramatsu, T. (1990) J. Cell Biol. 110, 607-616 [Abstract]
  29. Mitsiadis, T. A., Salmivirta, M., Muramatsu, T., Muramatsu, H., Rauvala, H., Lehtonen, E., Jalkanen, M., and Thesleff, I. (1995) Development 121, 37-51 [Abstract/Free Full Text]
  30. Mitsiadis, T. A., Muramatsu, T., Muramatsu, H., and Thesleff, I. (1995) J. Cell Biol. 129, 267-281 [Abstract]
  31. Tsutsui, J., Kadomatsu, K., Matsubara, S., Nakagawara, A., Hamanoue, M., Takao, S., Shimazu, H., Ohi, Y., and Muramatsu, T. (1993) Cancer Res. 53, 1281-1285 [Abstract]
  32. Yasuhara, O., Muramatsu, H., Kim, S. U., Muramatsu, T., Maruta, H., and McGeer, P. L. (1993) Biochem. Biophys. Res. Commun. 192, 246-251 [CrossRef][Medline] [Order article via Infotrieve]
  33. Chauhan, A. K., Li, Y.-S., and Deuel, T. F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 679-682 [Abstract]
  34. Tomomura, M., Kadomatsu, K., Matsubara, S., and Muramatsu, T. (1990) J. Biol. Chem. 265, 10765-10770 [Abstract/Free Full Text]
  35. Fabri, L., Nice, E. C., Ward, L. D., Maruta, H., Burgess, A. W., and Simpson, R. J. (1992) Biochem. Int. 28, 1-9 [Medline] [Order article via Infotrieve]
  36. Fabri, L., Maruta, H., Muramatsu, H., Muramatsu, T., Simpson, R. J., Burgess, A. W., and Nice, E. C. (1993) J. Chromatogr. 646, 213-225 [CrossRef][Medline] [Order article via Infotrieve]
  37. Muramatsu, H., Inui, T., Kimura, T., Sakakibara, S., Song, X., Maruta, H., and Muramatsu, T. (1994) Biochem. Biophys. Res. Commun. 203, 1131-1139 [CrossRef][Medline] [Order article via Infotrieve]
  38. Kojima, S., Inui, T., Kimura, T., Sakakibara, S., Muramatsu, H., Amanuma, H., Maruta, H., and Muramatsu, T. (1995) Biochem. Biophys. Res. Commun. 206, 468-473 [CrossRef][Medline] [Order article via Infotrieve]
  39. Muramatsu, H., Shirahama, H., Yonezawa, S., Maruta, H., and Muramatsu, T. (1993) Dev. Biol. 159, 392-402 [CrossRef][Medline] [Order article via Infotrieve]
  40. Perry, M. J. M., Mahoney, S.-A., and Haynes, L. W. (1995) Neuroscience 65, 1063-1076 [CrossRef][Medline] [Order article via Infotrieve]
  41. Mahoney, S.-A., Perry, M., Seddon, A., Bohlen, P., and Haynes, L. (1996) Biochem. Biophys. Res. Commun. 224, 147-152 [CrossRef][Medline] [Order article via Infotrieve]
  42. Inui, T., Bódi, J., Kubo, S., Nishio, H., Kimura, T., Kojima, S., Maruta, H., Muramatsu, T., and Sakakibara, S. (1996) J. Peptide Sci. 2, 28-39 [CrossRef][Medline] [Order article via Infotrieve]
  43. Kojima, S., Inui, T., Muramatsu, H., Kimura, T., Sakakibara, S., and Muramatsu, T. (1995) Biochem. Biophys. Res. Commun. 216, 574-581 [CrossRef][Medline] [Order article via Infotrieve]
  44. Gudas, L. J., Sporn, M. B., and Roberts, A. B. (1994) in The Retinoids: Biology, Chemistry, and Medicine (Sporn, M. B., Roberts, A. B., and Goodman, D. S., eds), 2nd Ed., pp. 443-520, Raven Press, New York
  45. Krätzschmar, J., Haendler, B., Kojima, S., Rifkin, D. B., and Schleuning, W.-D. (1993) Gene (Amst.) 125, 177-183 [CrossRef][Medline] [Order article via Infotrieve]
  46. Nara, K., Nakanishi, K., Hagiwara, H., Wakita, K., Kojima, S., and Hirose, S. (1989) J. Biol. Chem. 264, 19308-19312 [Abstract/Free Full Text]
  47. Kojima, S., and Rifkin, D. B. (1993) J. Cell. Physiol. 155, 323-332 [Medline] [Order article via Infotrieve]
  48. Sekiguchi, K., Yokota, C., Asashima, M., Kaname, T., Fan, Q.-W., Muramatsu, T., and Kadomatsu, K. (1995) J. Biochem. (Tokyo) 118, 94-100 [Abstract]
  49. Parameswaran, K. N., Velasco, P. T., Wilson, J., and Lorand, L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 8472-8475 [Abstract]
  50. Heldin, C.-H. (1995) Cell 80, 213-223 [Medline] [Order article via Infotrieve]
  51. Wells, J. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1-6 [Abstract/Free Full Text]

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