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Dimerization of Midkine by Tissue Transglutaminase and Its Functional Implication

1997; Elsevier BV; Volume: 272; Issue: 14 Linguagem: Inglês

10.1074/jbc.272.14.9410

1083-351X

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

Proteoglycans and glycosaminoglycans research

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. 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. INTRODUCTIONTissue type II transglutaminase (R-glutaminylpeptide: amine γ-glutamyltransferase, EC 2.3.2.13) is a member of the transglutaminase family that catalyzes Ca2+-dependent acyl transfer reactions between γ-carboxamide groups of the Gln residues in peptides and either primary amines or ϵ-amino groups of the Lys residues in peptides, resulting in the formation of new γ-amides of glutamic acid or ϵ-(γ-glutamyl)lysine bonds and ammonia (1Lorand L. Conrad S.M. Mol. Cell. Biochem. 1984; 58: 9-35Google Scholar, 2Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Google Scholar). The molecular structure of tissue transglutaminase has been reported (3Gentile V. Saydak M. Chiocca E.A. Akande O. Birckbichler P.J. Lee K.N. Stein J.P. Davies P.J.A. J. Biol. Chem. 1991; 266: 478-483Google Scholar, 4Nakanishi K. Nara K. Hagiwara H. Aoyama Y. Ueno H. Hirose S. Eur. J. Biochem. 1991; 202: 15-21Google Scholar, 5Lu S. Saydak M. Gentile V. Stein J.P. Davies P.J.A. J. Biol. Chem. 1995; 270: 9748-9756Google Scholar). Although tissue transglutaminase is widely distributed in the body (6Thomázy V. Fésüs L. Cell Tissue Res. 1989; 255: 215-224Google Scholar), 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 (1Lorand L. Conrad S.M. Mol. Cell. Biochem. 1984; 58: 9-35Google Scholar, 2Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Google Scholar), and the formation of cross-linked envelopes during epidermal cell differentiation by tissue type I transglutaminase (7Floyd E.E. Jetten A.M. Mol. Cell. Biol. 1989; 9: 4846-4851Google Scholar, 8Marvin K.W. George M.D. Fujimoto W. Saunders N.A. Bernacki S.H. Jetten A.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11026-11030Google Scholar). Recently, tissue type II transglutaminase was implicated in the association of proteases and protease inhibitors with the cell surface (9Bendixen E. Borth W. Harpel P.C. J. Biol. Chem. 1993; 268: 21962-21967Google Scholar, 10Nara K. Ito S. Ito T. Suzuki Y. Ghoneim M.A. Tachibana S. Hirose S. J. Biochem. (Tokyo). 1994; 115: 441-448Google Scholar), in the activation of several cytokines (11Kojima S. Nara K. Rifkin D.B. J. Cell Biol. 1993; 121: 439-448Google Scholar, 12Eitan S. Schwartz M. Science. 1993; 261: 106-108Google Scholar), in signal transduction (13Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.-J. Graham R.M. Science. 1994; 264: 1593-1596Google Scholar), and in the process of apoptosis (14Zhang L.-X. Mills K.J. Dawson M.I. Collins S.J. Jetten A.M. J. Biol. Chem. 1995; 270: 6022-6029Google Scholar).Midkine (MK) 1The abbreviations used are: MKmidkinePTNpleiotrophinPAplasminogen activatorBAECsbovine aortic endothelial cellsN1/2N-terminal halfC1/2C-terminal halfTGF-βtransforming growth factor-β and pleiotrophin (PTN) constitute a new family of heparin-binding growth/differentiation factors (15Muramatsu T. Dev. Growth & Diff. 1994; 36: 1-8Google Scholar, 16Kurtz A. Schulte A.M. Wellstein A. Crit. Rev. Oncogen. 1995; 6: 151-177Google Scholar). MK has been found as a product of a retinoic acid-responsive gene (17Kadomatsu K. Tomomura M. Muramatsu T. Biochem. Biophys. Res. Commun. 1988; 151: 1312-1318Google Scholar), and exerts a variety of biological activities; it enhances neurite outgrowth and the survival of various embryonic neuron types (18Muramatsu H. Muramatsu T. Biochem. Biophys. Res. Commun. 1991; 177: 652-658Google Scholar, 19Nurcombe V. Fraser N. Herlaar E. Heath J.K. Development. 1992; 116: 1175-1183Google Scholar, 20Michikawa M. Kikuchi S. Muramatsu H. Muramatsu T. Kim S.U. J. Neurosci. Res. 1993; 35: 530-539Google Scholar, 21Satoh J. Muramatsu H. Moretto G. Muramatsu T. Chang H.J. Kim S.T. Cho J.M. Kim S.U. Dev. Brain Res. 1993; 75: 201-205Google Scholar), and is mitogenic for certain fibroblastic cell lines (18Muramatsu H. Muramatsu T. Biochem. Biophys. Res. Commun. 1991; 177: 652-658Google Scholar, 19Nurcombe V. Fraser N. Herlaar E. Heath J.K. Development. 1992; 116: 1175-1183Google Scholar). In addition, recently, we found that MK enhances the plasminogen activator (PA) activity in bovine aortic endothelial cells (BAECs; 22Kojima S. Muramatsu H. Amanuma H. Muramatsu T. J. Biol. Chem. 1995; 270: 9590-9596Google Scholar). PTN, also called heparin-binding growth-associated molecule (HB-GAM; Refs. 23Merenmies J. Rauvala H. J. Biol. Chem. 1990; 265: 16721-16724Google Scholar and 24Li Y.-S. Milner P.G. Chauhan A.K. Watson M.A. Hoffman R.M. Kodner C.M. Milbrandt J. Deuel T.F. Science. 1990; 250: 1690-1694Google Scholar), was found as another neurite-promoting factor (25Rauvala H. EMBO J. 1989; 8: 2933-2941Google Scholar). PTN has been shown to be mitogenic for endothelial cells (26Fang W. Hartmann N. Chow D.T. Riegel A.T. Wellstein A. J. Biol. Chem. 1992; 267: 25889-25897Google Scholar) and to enhance tube formation in vitro (27Laaroubi K. Delbé J. Vacherot F. Desgranges P. Tardieu M. Jaye M. Barritault D. Courty J. Growth Factors. 1994; 10: 89-98Google Scholar). Expression of these factors is strictly controlled during the processes of differentiation and development (28Kadomatsu K. Huang R.-P. Suganuma T. Murata F. Muramatsu T. J. Cell Biol. 1990; 110: 607-616Google Scholar, 29Mitsiadis T.A. Salmivirta M. Muramatsu T. Muramatsu H. Rauvala H. Lehtonen E. Jalkanen M. Thesleff I. Development. 1995; 121: 37-51Google Scholar, 30Mitsiadis T.A. Muramatsu T. Muramatsu H. Thesleff I. J. Cell Biol. 1995; 129: 267-281Google Scholar). MK is highly expressed in many human cancers (31Tsutsui J. Kadomatsu K. Matsubara S. Nakagawara A. Hamanoue M. Takao S. Shimazu H. Ohi Y. Muramatsu T. Cancer Res. 1993; 53: 1281-1285Google Scholar) and specifically localized in senile plaques of Alzheimer's disease (32Yasuhara O. Muramatsu H. Kim S.U. Muramatsu T. Maruta H. McGeer P.L. Biochem. Biophys. Res. Commun. 1993; 192: 246-251Google Scholar), and the overexpression of PTN in NIH3T3 cells results in transformation of the cells (33Chauhan A.K. Li Y.-S. Deuel T.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 679-682Google Scholar), suggesting the involvement of MK and PTN not only in normal development, but also in the pathogeneses of diseases (15Muramatsu T. Dev. Growth & Diff. 1994; 36: 1-8Google Scholar, 16Kurtz A. Schulte A.M. Wellstein A. Crit. Rev. Oncogen. 1995; 6: 151-177Google Scholar). MK is a 13-kDa heparin-binding polypeptide rich in basic amino acids and cysteine (17Kadomatsu K. Tomomura M. Muramatsu T. Biochem. Biophys. Res. Commun. 1988; 151: 1312-1318Google Scholar, 34Tomomura M. Kadomatsu K. Matsubara S. Muramatsu T. J. Biol. Chem. 1990; 265: 10765-10770Google Scholar), and exhibits 46% sequence identity with PTN (15Muramatsu T. Dev. Growth & Diff. 1994; 36: 1-8Google Scholar). 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 (35Fabri L. Nice E.C. Ward L.D. Maruta H. Burgess A.W. Simpson R.J. Biochem. Int. 1992; 28: 1-9Google Scholar, 36Fabri L. Maruta H. Muramatsu H. Muramatsu T. Simpson R.J. Burgess A.W. Nice E.C. J. Chromatogr. 1993; 646: 213-225Google Scholar). Of these two domains C1/2 is responsible for heparin-binding, neurite outgrowth-promoting, and PA-enhancing activities (37Muramatsu H. Inui T. Kimura T. Sakakibara S. Song X. Maruta H. Muramatsu T. Biochem. Biophys. Res. Commun. 1994; 203: 1131-1139Google Scholar, 38Kojima S. Inui T. Kimura T. Sakakibara S. Muramatsu H. Amanuma H. Maruta H. Muramatsu T. Biochem. Biophys. Res. Commun. 1995; 206: 468-473Google Scholar).During the course of studying the PA-enhancing properties of MK, we discovered that MK serves as a good substrate for tissue transglutaminase (22Kojima S. Muramatsu H. Amanuma H. Muramatsu T. J. Biol. Chem. 1995; 270: 9590-9596Google Scholar). 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 (39Muramatsu H. Shirahama H. Yonezawa S. Maruta H. Muramatsu T. Dev. Biol. 1993; 159: 392-402Google Scholar). Furthermore, Haynes and colleagues (40Perry M.J.M. Mahoney S.-A. Haynes L.W. Neuroscience. 1995; 65: 1063-1076Google Scholar, 41Mahoney S.-A. Perry M. Seddon A. Bohlen P. Haynes L. Biochem. Biophys. Res. Commun. 1996; 224: 147-152Google Scholar) 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 vivoIn the current study, we have investigated the mechanism underlying the transglutaminase-mediated cross-linking reaction and the relevance of dimer formation to MK activity.DISCUSSIONRetinol (vitamin A) and its derivatives (retinoids) have profound effects on the regulation of cell growth and differentiation (44Gudas L.J. Sporn M.B. Roberts A.B. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry, and Medicine. 2nd Ed. Raven Press, New York1994: 443-520Google Scholar). Using BAEC cultures, we found that retinoids induce the production of PA (45Krätzschmar J. Haendler B. Kojima S. Rifkin D.B. Schleuning W.-D. Gene (Amst.). 1993; 125: 177-183Google Scholar), transglutaminase (46Nara K. Nakanishi K. Hagiwara H. Wakita K. Kojima S. Hirose S. J. Biol. Chem. 1989; 264: 19308-19312Google Scholar), transforming growth factor-β (TGF-β; 47Kojima S. Rifkin D.B. J. Cell. Physiol. 1993; 155: 323-332Google Scholar), and MK (22Kojima S. Muramatsu H. Amanuma H. Muramatsu T. J. Biol. Chem. 1995; 270: 9590-9596Google Scholar), 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-β (11Kojima S. Nara K. Rifkin D.B. J. Cell Biol. 1993; 121: 439-448Google Scholar, 47Kojima S. Rifkin D.B. J. Cell. Physiol. 1993; 155: 323-332Google Scholar), whereas MK and TGF-β regulate PA activity, respectively, in the opposite way (11Kojima S. Nara K. Rifkin D.B. J. Cell Biol. 1993; 121: 439-448Google Scholar, 22Kojima S. Muramatsu H. Amanuma H. Muramatsu T. J. Biol. Chem. 1995; 270: 9590-9596Google Scholar). 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 Fig. 5, Fig. 6, Fig. 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 ϵ-(γ-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 (48Sekiguchi K. Yokota C. Asashima M. Kaname T. Fan Q.-W. Muramatsu T. Kadomatsu K. J. Biochem. (Tokyo). 1995; 118: 94-100Google Scholar). 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 spectroscopy 2F. Inagaki, personal communication. 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. 3N. Asai, unpublished result. 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 (43Kojima S. Inui T. Muramatsu H. Kimura T. Sakakibara S. Muramatsu T. Biochem. Biophys. Res. Commun. 1995; 216: 574-581Google Scholar). 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 (49Parameswaran K.N. Velasco P.T. Wilson J. Lorand L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8472-8475Google Scholar).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-β, which counteracts MK activity (22Kojima S. Muramatsu H. Amanuma H. Muramatsu T. J. Biol. Chem. 1995; 270: 9590-9596Google Scholar). Since the activation of latent TGF-β is required for transglutaminase to localize latent TGF-β on the surface, the inclusion of an anti-transglutaminase antibody in the culture medium prevents the formation of active TGF-β (11Kojima S. Nara K. Rifkin D.B. J. Cell Biol. 1993; 121: 439-448Google Scholar). 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 (37Muramatsu H. Inui T. Kimura T. Sakakibara S. Song X. Maruta H. Muramatsu T. Biochem. Biophys. Res. Commun. 1994; 203: 1131-1139Google Scholar, 38Kojima S. Inui T. Kimura T. Sakakibara S. Muramatsu H. Amanuma H. Maruta H. Muramatsu T. Biochem. Biophys. Res. Commun. 1995; 206: 468-473Google Scholar), 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; 10Nara K. Ito S. Ito T. Suzuki Y. Ghoneim M.A. Tachibana S. Hirose S. J. Biochem. (Tokyo). 1994; 115: 441-448Google Scholar). 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. Fig. 9, Fig. 10, Fig. 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 (46Nara K. Nakanishi K. Hagiwara H. Wakita K. Kojima S. Hirose S. J. Biol. Chem. 1989; 264: 19308-19312Google Scholar), 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 (11Kojima S. Nara K. Rifkin D.B. J. Cell Biol. 1993; 121: 439-448Google Scholar, 46Nara K. Nakanishi K. Hagiwara H. Wakita K. Kojima S. Hirose S. J. Biol. Chem. 1989; 264: 19308-19312Google Scholar), 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 (50Heldin C.-H. Cell. 1995; 80: 213-223Google Scholar, 51Wells J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1-6Google Scholar). 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-β (11Kojima S. Nara K. Rifkin D.B. J. Cell Biol. 1993; 121: 439-448Google Scholar), interleukin 2 (12Eitan S. Schwartz M. Science. 1993; 261: 106-108Google Scholar) and, now, MK. INTRODUCTIONTissue type II transglutaminase (R-glutaminylpeptide: amine γ-glutamyltransferase, EC 2.3.2.13) is a member of the transglutaminase family that catalyzes Ca2+-dependent acyl transfer reactions between γ-carboxamide groups of the Gln residues in peptides and either primary amines or ϵ-amino groups of the Lys residues in peptides, resulting in the formation of new γ-amides of glutamic acid or ϵ-(γ-glutamyl)lysine bonds and ammonia (1Lorand L. Conrad S.M. Mol. Cell. Biochem. 1984; 58: 9-35Google Scholar, 2Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Google Scholar). The molecular structure of tissue transglutaminase has been reported (3Gentile V. Saydak M. Chiocca E.A. Akande O. Birckbichler P.J. Lee K.N. Stein J.P. Davies P.J.A. J. Biol. Chem. 1991; 266: 478-483Google Scholar, 4Nakanishi K. Nara K. Hagiwara H. Aoyama Y. Ueno H. Hirose S. Eur. J. Biochem. 1991; 202: 15-21Google Scholar, 5Lu S. Saydak M. Gentile V. Stein J.P. Davies P.J.A. J. Biol. Chem. 1995; 270: 9748-9756Google Scholar). Although tissue transglutaminase is widely distributed in the body (6Thomázy V. Fésüs L. Cell Tissue Res. 1989; 255: 215-224Google Scholar), 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 (1Lorand L. Conrad S.M. Mol. Cell. Biochem. 1984; 58: 9-35Google Scholar, 2Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Google Scholar), and the formation of cross-linked envelopes during epidermal cell differentiation by tissue type I transglutaminase (7Floyd E.E. Jetten A.M. Mol. Cell. Biol. 1989; 9: 4846-4851Google Scholar, 8Marvin K.W. George M.D. Fujimoto W. Saunders N.A. Bernacki S.H. Jetten A.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11026-11030Google Scholar). Recently, tissue type II transglutaminase was implicated in the association of proteases and protease inhibitors with the cell surface (9Bendixen E. Borth W. Harpel P.C. J. Biol. Chem. 1993; 268: 21962-21967Google Scholar, 10Nara K. Ito S. Ito T. Suzuki Y. Ghoneim M.A. Tachibana S. Hirose S. J. Biochem. (Tokyo). 1994; 115: 441-448Google Scholar), in the activation of several cytokines (11Kojima S. Nara K. Rifkin D.B. J. Cell Biol. 1993; 121: 439-448Google Scholar, 12Eitan S. Schwartz M. Science. 1993; 261: 106-108Google Scholar), in signal transduction (13Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.-J. Graham R.M. Science. 1994; 264: 1593-1596Google Scholar), and in the process of apoptosis (14Zhang L.-X. Mills K.J. Dawson M.I. Collins S.J. Jetten A.M. J. Biol. Chem. 1995; 270: 6022-6029Google Scholar).Midkine (MK) 1The abbreviations used are: MKmidkinePTNpleiotrophinPAplasminogen activatorBAECsbovine aortic endothelial cellsN1/2N-terminal halfC1/2C-terminal halfTGF-βtransforming growth factor-β and pleiotrophin (PTN) constitute a new family of heparin-binding growth/differentiation factors (15Muramatsu T. Dev. Growth & Diff. 1994; 36: 1-8Google Scholar, 16Kurtz A. Schulte A.M. Wellstein A. Crit. Rev. Oncogen. 1995; 6: 151-177Google Scholar). MK has been found as a product of a retinoic acid-responsive gene (17Kadomatsu K. Tomomura M. Muramatsu T. Biochem. Biophys. Res. Commun. 1988; 151: 1312-1318Google Scholar), and exerts a variety of biological activities; it enhances neurite outgrowth and the survival of various embryonic neuron types (18Muramatsu H. Muramatsu T. Biochem. Biophys. Res. Commun. 1991; 177: 652-658Google Scholar, 19Nurcombe V. Fraser N. Herlaar E. Heath J.K. Development. 1992; 116: 1175-1183Google Scholar, 20Michikawa M. Kikuchi S. Muramatsu H. Muramatsu T. Kim S.U. J. Neurosci. Res. 1993; 35: 530-539Google Scholar, 21Satoh J. Muramatsu H. Moretto G. Muramatsu T. Chang H.J. Kim S.T. Cho J.M. Kim S.U. Dev. Brain Res. 1993; 75: 201-205Google Scholar), and is mitogenic for certain fibroblastic cell lines (18Muramatsu H. Muramatsu T. Biochem. Biophys. Res. Commun. 1991; 177: 652-658Google Scholar, 19Nurcombe V. Fraser N. Herlaar E. Heath J.K. Development. 1992; 116: 1175-1183Google Scholar). In addition, recently, we found that MK enhances the plasminogen activator (PA) activity in bovine aortic endothelial cells (BAECs; 22Kojima S. Muramatsu H. Amanuma H. Muramatsu T. J. Biol. Chem. 1995; 270: 9590-9596Google Scholar). PTN, also called heparin-binding growth-associated molecule (HB-GAM; Refs. 23Merenmies J. Rauvala H. J. Biol. Chem. 1990; 265: 16721-16724Google Scholar and 24Li Y.-S. Milner P.G. Chauhan A.K. Watson M.A. Hoffman R.M. Kodner C.M. Milbrandt J. Deuel T.F. Science. 1990; 250: 1690-1694Google Scholar), was found as another neurite-promoting factor (25Rauvala H. EMBO J. 1989; 8: 2933-2941Google Scholar). PTN has been shown to be mitogenic for endothelial cells (26Fang W. Hartmann N. Chow D.T. Riegel A.T. Wellstein A. J. Biol. Chem. 1992; 267: 25889-25897Google Scholar) and to enhance tube formation in vitro (27Laaroubi K. Delbé J. Vacherot F. Desgranges P. Tardieu M. Jaye M. Barritault D. Courty J. Growth Factors. 1994; 10: 89-98Google Scholar). Expression of these factors is strictly controlled during the processes of differentiation and development (28Kadomatsu K. Huang R.-P. Suganuma T. Murata F. Muramatsu T. J. Cell Biol. 1990; 110: 607-616Google Scholar, 29Mitsiadis T.A. Salmivirta M. Muramatsu T. Muramatsu H. Rauvala H. Lehtonen E. Jalkanen M. Thesleff I. Development. 1995; 121: 37-51Google Scholar, 30Mitsiadis T.A. Muramatsu T. Muramatsu H. Thesleff I. J. Cell Biol. 1995; 129: 267-281Google Scholar). MK is highly expressed in many human cancers (31Tsutsui J. Kadomatsu K. Matsubara S. Nakagawara A. Hamanoue M. Takao S. Shimazu H. Ohi Y. Muramatsu T. Cancer Res. 1993; 53: 1281-1285Google Scholar) and specifically localized in senile plaques of Alzheimer's disease (32Yasuhara O. Muramatsu H. Kim S.U. Muramatsu T. Maruta H. McGeer P.L. Biochem. Biophys. Res. Commun. 1993; 192: 246-251Google Scholar), and the overexpression of PTN in NIH3T3 cells results in transformation of the cells (33Chauhan A.K. Li Y.-S. Deuel T.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 679-682Google Scholar), suggesting the involvement of MK and PTN not only in normal development, but also in the pathogeneses of diseases (15Muramatsu T. Dev. Growth & Diff. 1994; 36: 1-8Google Scholar, 16Kurtz A. Schulte A.M. Wellstein A. Crit. Rev. Oncogen. 1995; 6: 151-177Google Scholar). MK is a 13-kDa heparin-binding polypeptide rich in basic amino acids and cysteine (17Kadomatsu K. Tomomura M. Muramatsu T. Biochem. Biophys. Res. Commun. 1988; 151: 1312-1318Google Scholar, 34Tomomura M. Kadomatsu K. Matsubara S. Muramatsu T. J. Biol. Chem. 1990; 265: 10765-10770Google Scholar), and exhibits 46% sequence identity with PTN (15Muramatsu T. Dev. Growth & Diff. 1994; 36: 1-8Google Scholar). 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 (35Fabri L. Nice E.C. Ward L.D. Maruta H. Burgess A.W. Simpson R.J. Biochem. Int. 1992; 28: 1-9Google Scholar, 36Fabri L. Maruta H. Muramatsu H. Muramatsu T. Simpson R.J. Burgess A.W. Nice E.C. J. Chromatogr. 1993; 646: 213-225Google Scholar). Of these two domains C1/2 is responsible for heparin-binding, neurite outgrowth-promoting, and PA-enhancing activities (37Muramatsu H. Inui T. Kimura T. Sakakibara S. Song X. Maruta H. Muramatsu T. Biochem. Biophys. Res. Commun. 1994; 203: 1131-1139Google Scholar, 38Kojima S. Inui T. Kimura T. Sakakibara S. Muramatsu H. Amanuma H. Maruta H. Muramatsu T. Biochem. Biophys. Res. Commun. 1995; 206: 468-473Google Scholar).During the course of studying the PA-enhancing properties of MK, we discovered that MK serves as a good substrate for tissue transglutaminase (22Kojima S. Muramatsu H. Amanuma H. Muramatsu T. J. Biol. Chem. 1995; 270: 9590-9596Google Scholar). 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 (39Muramatsu H. Shirahama H. Yonezawa S. Maruta H. Muramatsu T. Dev. Biol. 1993; 159: 392-402Google Scholar). Furthermore, Haynes and colleagues (40Perry M.J.M. Mahoney S.-A. Haynes L.W. Neuroscience. 1995; 65: 1063-1076Google Scholar, 41Mahoney S.-A. Perry M. Seddon A. Bohlen P. Haynes L. Biochem. Biophys. Res. Commun. 1996; 224: 147-152Google Scholar) 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 vivoIn the current study, we have investigated the mechanism underlying the transglutaminase-mediated cross-linking reaction and the relevance of dimer formation to MK activity.