Dimerization of Midkine by Tissue Transglutaminase and Its Functional Implication

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. 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; Scholar) reported dimer formation by MK in the These lines of suggest that the cross-linking of MK by tissue transglutaminase and an important in MK in the we the the transglutaminase-mediated cross-linking and the of dimer formation to MK and its the of cell growth and differentiation The and Scholar). we found that the of J. S. Rifkin D.B. 1993; Scholar), transglutaminase K. K. Hagiwara H. K. S. Hirose S. J. Biol. Chem. 1989; 264: Scholar), growth factor-β S. Rifkin D.B. J. Cell. 1993; 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 with each In retinoic and transglutaminase are to the activation of (11Kojima S. Nara K. Rifkin D.B. J. Cell Biol. 1993; 121: 439-448Google Scholar, S. Rifkin D.B. J. Cell. 1993; Scholar), MK and in the (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 an between transglutaminase and was to be an substrate for transglutaminase in that of MK as as of MK formed the dimer, and incubation with transglutaminase. multimers dimer to be the results in suggest that Gln42 and Gln44 in as well as Gln95 in C1/2 function as the results in suggest the of Gln in each of and The for this is that Gln42 and Gln44 are to as only of is to the surface of the MK The is that Gln42 and Gln44 as Gln42 is used to an between a Lys in another MK or putrescine in the Gln44 for formation to by the MK molecule cross-linked to Gln44 is cross-linked to another MK Gln42 also Factor XIIIa is used as the Factor XIIIa is tissue transglutaminase the cross-linking of MK molecules not be by Factor it putrescine incorporation into MK is that these Gln residues are the family, the other two Gln residues are not K. M. T. Muramatsu T. Kadomatsu K. J. Biochem. (Tokyo). 1995; Scholar). be seen in the dimer and of MK C1/2 those of This be to that C1/2 is highly The structure of MK C1/2 by is with the results of the In basic amino which are to the and are of these amino acids in heparin-binding and neurite-promoting which was shown to be in the is the This of the and the cross-linking the cross-linking between two MK molecules to the cross-linking with transglutaminase by the of In addition, the of by the of MK molecules from the that to of MK molecules to the S. Inui T. Muramatsu H. Kimura T. Sakakibara S. Muramatsu T. Biochem. Biophys. Res. Commun. 1995; Scholar). of this and we the physiological the cell surface as we the dimer formation in the of are to the Lys in the MK is also of to the for the substrate for the of in the and to it with the obtained with sequence K.N. J. L. Proc. Natl. Acad. Sci. U. S. A. 1990; that of MK by cell surface transglutaminase MK the that the peptide the cross-linking and PA-enhancing activity of the MK monomer, with a peptide as an not an the the formation of which MK activity (22Kojima S. Muramatsu H. Amanuma H. Muramatsu T. J. Biol. Chem. 1995; 270: 9590-9596Google Scholar). the activation of is for transglutaminase to the the of an in the the formation of (11Kojima S. Nara K. Rifkin D.B. J. Cell Biol. 1993; 121: 439-448Google Scholar). the peptide was a to the of the inhibition by the peptide was to a a is 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), is a that the peptide with the of MK to its the dimer the PA-enhancing activity in the of this Factor XIIIa only in putrescine it is that Factor XIIIa be used to peptide to MK as a for this is that the PA-enhancing of the MK is by cross-linking of the MK monomer, formation of the dimer, is it is that other transglutaminase-mediated cross-linking reactions by the and that this the inhibition of MK activity by the In this we to the of the inhibition by this Although the peptide the cross-linking of MK, and C1/2 by a of as as a a of this peptide only inhibition of the cross-linking of not 10Nara K. Ito S. Ito T. Suzuki Y. Ghoneim M.A. Tachibana S. Hirose S. J. Biochem. (Tokyo). 1994; 115: 441-448Google Scholar). are now the peptide the cross-linking of other for transglutaminase. we that the is in the in an of the peptide was or not only activity was from the or dimer, and other the other cross-linking this peptide or MK is cross-linked to other such as and the peptide such we that the results in suggest the of the formation of the MK formation of the dimer and other multimers the MK has been detected incubation with transglutaminase The peptide the cross-linking and the association through into the Lys of MK molecules by transglutaminase. that the peptide the dimer formed either cross-linking or as the peptide not the PA-enhancing activity in the dimer association between MK the cross-linking are to be and the MK molecules as a substrate for transglutaminase. the dimer with the cross-linked dimer, and the peptide was not cross-linked to the dimer not its in the of the of the of the and into the and we the peptide not formation of the and the peptide the formation of the dimer The association be to the of MK is a physiological the of MK be cross-linking transglutaminase is suggest that formation is to in PA-enhancing activity and that of MK to cells cell surface transglutaminase is not plasma Factor XIIIa is not to MK molecules and tissue transglutaminase is not from the cells K. K. Hagiwara H. K. S. Hirose S. J. Biol. Chem. 1989; 264: Scholar), the surface be The of MK to transglutaminase a the of MK with surface and other be Tissue transglutaminase is distributed and the cell surface (11Kojima S. Nara K. Rifkin D.B. J. Cell Biol. 1993; 121: 439-448Google Scholar, K. K. Hagiwara H. K. S. Hirose S. J. Biol. Chem. 1989; 264: Scholar), suggesting either that MK is cross-linked or that MK is cross-linked the cell surface as The results in suggest that the with the of the cell Recently, it was reported that or of and is important for the of cells Cell. 1995; Scholar, Proc. Natl. Acad. Sci. U. S. A. 1996; Scholar). with the that the cross-linking is important for PA-enhancing we that the MK dimer, cross-linked the cell its by the this is in for the the transglutaminase a in the of through the cross-linking of cytokines such as (11Kojima S. Nara K. Rifkin D.B. J. Cell Biol. 1993; 121: 439-448Google Scholar), S. Schwartz M. Science. 1993; 261: 106-108Google Scholar) 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; Scholar) reported dimer formation by MK in the These lines of suggest that the cross-linking of MK by tissue transglutaminase and an important in MK in the we the the transglutaminase-mediated cross-linking and the of dimer formation to MK