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W2047731835 abstract "Laminins are a family of trimeric extracellular matrix proteins consisting of α, β, and γ chains. So far five different laminin α chains have been identified. The laminin α4 chain, which is present in laminin-8/9, is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes. Previously, we identified heparin-binding sites in the C-terminal globular domain (G domain) of the laminin α4 chain. Here we have focused on the biological functions of the laminin α4 chain G domain and screened active sites using a recombinant protein and synthetic peptides. The rec-α4G protein, comprising the entire G domain, promoted cell attachment activity. The cell attachment activity of rec-α4G was completely blocked by heparin and partially inhibited by EDTA. We synthesized 116 overlapping peptides covering the entire G domain and tested their cell attachment activity. Twenty peptides showed cell attachment activity, and 16 bound to heparin. We further tested the effect of the 20 active peptides in competition assays for cell attachment and heparin binding to rec-α4G protein. A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), A4G82 (TLFLAHGRLVFM), and A4G83 (LVFMFNVGHKKL), which promoted cell attachment and heparin binding, significantly inhibited both cell attachment and heparin binding to rec-α4G. These results suggest that the four active sites are involved in the biological functions of the laminin α4 chain G domain. Furthermore, rec-α4G, A4G6, and A4G20 were found to interact with syndecan-4. These active peptides may be useful for defining of the molecular mechanism laminin-receptor interactions and laminin-mediated cellular signaling pathways. Laminins are a family of trimeric extracellular matrix proteins consisting of α, β, and γ chains. So far five different laminin α chains have been identified. The laminin α4 chain, which is present in laminin-8/9, is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes. Previously, we identified heparin-binding sites in the C-terminal globular domain (G domain) of the laminin α4 chain. Here we have focused on the biological functions of the laminin α4 chain G domain and screened active sites using a recombinant protein and synthetic peptides. The rec-α4G protein, comprising the entire G domain, promoted cell attachment activity. The cell attachment activity of rec-α4G was completely blocked by heparin and partially inhibited by EDTA. We synthesized 116 overlapping peptides covering the entire G domain and tested their cell attachment activity. Twenty peptides showed cell attachment activity, and 16 bound to heparin. We further tested the effect of the 20 active peptides in competition assays for cell attachment and heparin binding to rec-α4G protein. A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), A4G82 (TLFLAHGRLVFM), and A4G83 (LVFMFNVGHKKL), which promoted cell attachment and heparin binding, significantly inhibited both cell attachment and heparin binding to rec-α4G. These results suggest that the four active sites are involved in the biological functions of the laminin α4 chain G domain. Furthermore, rec-α4G, A4G6, and A4G20 were found to interact with syndecan-4. These active peptides may be useful for defining of the molecular mechanism laminin-receptor interactions and laminin-mediated cellular signaling pathways. bovine serum albumin 9-fluorenylmethoxycarbonyl high performance liquid chromatography fetal bovine serum enzyme-linked immunosorbent assay Dulbecco's modified Eagle's medium Laminins, a family of extracellular matrix proteins, consist of three different subunits, α, β, and γ chains. So far, five α, three β, and three γ chains have been identified, and at least 15 isoforms (laminin 1–15) are formed by various combinations of each subunit (1Burgeson R.E. Chiquet M. Deutzmann R. Ekblom P. Engel J. Kleinman H.K. Martin G.R. Meneguzzi G. Paulson M. Sanes J. Timpl R. Tryggvason K. Yamada Y. Yurchenco P.D. Matrix Biol. 1994; 14: 209-211Crossref PubMed Scopus (694) Google Scholar, 2Miner J.H. Patton B.L. Lentz S.I. Gilbert D.J. Snider W.D. Jenkins N.A. Copeland N.G. Sanes J.R. J. Cell Biol. 1997; 137: 685-701Crossref PubMed Scopus (576) Google Scholar, 3Iivanainen A. Morita T. Tryggvason K. J. Biol. Chem. 1999; 274: 14107-14111Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 4Libby R.T. Champliaud M.F. Claudepierre T., Xu, Y. Gibbons E.P. Koch M. Burgeson R.E. Hunter D.D. Brunken W.J. J. Neurosci. 2000; 20: 6517-6528Crossref PubMed Google Scholar). Laminins have diverse biological activities including promotion of cell adhesion, migration, neurite outgrowth, angiogenesis, and tumor metastasis (5Colognato H. Yurchenco P.D. Dev. Dyn. 2000; 218: 213-234Crossref PubMed Scopus (1033) Google Scholar). More than 20 receptors have been reported for these laminin molecules (6Powell S.K. Kleinman H.K. Int. J. Biochem. Cell Biol. 1997; 29: 401-414Crossref PubMed Scopus (159) Google Scholar). Several active sites on laminin-1 have been identified using proteolytic fragments, recombinant proteins, and synthetic peptides (7Yamada Y. Kleinman H.K. Curr. Opin. Cell Biol. 1992; 4: 819-823Crossref PubMed Scopus (137) Google Scholar, 8Yamada K.M. J. Biol. Chem. 1991; 266: 12809-12912Abstract Full Text PDF PubMed Google Scholar). Previously, we screened for cell adhesive sequences on laminin-1 using 673 overlapping synthetic peptides covering the entire protein (9Nomizu M. Kim W.H. Yamamura K. Utani A. Song S.Y. Otaka A. Roller P.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1995; 270: 20583-20590Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 10Nomizu M. Kuratomi Y. Song S.Y. Ponce L.M. Hoffman M.P. Powell S.K. Miyoshi K. Otaka A. Kleinman H.K. Yamada Y. J. Biol. Chem. 1997; 272: 32198-32205Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 11Nomizu M. Kuratomi Y. Malinda M.K. Song S.Y. Miyoshi K. Otaka A. Powel S.K. Hoffman M.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1998; 273: 32491-32499Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 12Nomizu M. Kuratomi Y. Ponce L.M. Song S.Y. Miyoshi K. Otaka A. Powell S.K. Hoffman M.P. Kleinman H.K. Yamada Y. Arch. Biochem. Biophys. 2000; 378: 311-320Crossref PubMed Scopus (69) Google Scholar). Most of the active peptides were localized in the globular domains and found to play a critical role in binding to cell surface receptors in a peptide- and cell type-specific manner (13Makino M. Okazaki I. Nishi N. Nomizu M. Connect. Tissue. 1999; 31: 227-234Google Scholar, 14Hoffman M.P. Engbring J.A. Nielsen P.K. Vargas J. Steinberg Z. Karmand A.J. Nomizu M. Yamada Y. Kleinman H.K. J. Biol. Chem. 2001; 276: 22077-22085Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Several peptides were found to interact with integrins and syndecans (15Tashiro K. Sephel G.C. Greatorex D. Sasaki M. Shirashi N. Martin G.R. Kleinman H.K. Yamada Y. J. Cell. Physiol. 1991; 146: 451-459Crossref PubMed Scopus (97) Google Scholar, 16Hoffman M.P. Nomizu M. Roque E. Lee S. Jung D.W. Yamada Y. Kleinman H.K. J. Biol. Chem. 1998; 273: 28633-28641Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 17Ponce M.L. Nomizu M. Kleinman H.K. FASEB J. 2001; 15: 1389-1397Crossref PubMed Scopus (78) Google Scholar, 18Utani A. Nomizu M. Matsuura H. Kato K. Kobayashi T. Takeda U. Aota S. Nielsen P.K. Shinkai H. J. Biol. Chem. 2001; 276: 28779-28788Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Some of the peptides promoted neurite outgrowth, angiogenesis, and tumor metastasis (19Richard B.L. Nomizu M. Yamada Y. Kleinman H.K. Exp. Cell Res. 1996; 228: 98-105Crossref PubMed Scopus (74) Google Scholar, 20Kim W.H. Nomizu M. Song S.Y. Tanaka K. Kuratomi Y. Kleinman H.K. Yamada Y. Int. J. Cancer. 1998; 77: 632-639Crossref PubMed Scopus (48) Google Scholar, 21Malinda K.M. Nomizu M. Chung M. Delgado M. Kuratomi Y. Yamada Y. Kleinman H.K. Ponce M.L. FASEB J. 1999; 13: 53-62Crossref PubMed Scopus (164) Google Scholar, 22Ponce M.L. Nomizu M. Delgado M.C. Kuratomi Y. Hoffman M.P. Powell S. Yamada Y. Kleinman H.K. Malinda K.M. Circ. Res. 1999; 84: 688-694Crossref PubMed Scopus (90) Google Scholar).The laminin α chains are generally large (M r = 400,000) and contain a C-terminal globular domain consisting of five globular modules LG1–LG5. The laminin α4 chain lacks the N-terminal short arm and is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes (23Tokida Y. Aratani Y. Morita A. Kitagawa Y. J. Biol. Chem. 1990; 265: 18123-18129Abstract Full Text PDF PubMed Google Scholar, 24Sorokin L.M. Pausch F. Frieser M. Kröger S. Ohage E. Deutzmann R. Dev. Biol. 1997; 189: 285-300Crossref PubMed Scopus (215) Google Scholar, 25Niimi T. Kumagai C. Okano M. Kitagawa Y. Matrix Biol. 1997; 16: 223-230Crossref PubMed Scopus (43) Google Scholar). Laminin α4 chain expression is mainly localized to mesenchymal cells present in the lung and cardiac and skeletal muscles fibers (26Iivanainen A. Kortesmaa J. Sahlberg C. Morita T. Bergmann U. Thesleff I. Tryggvason K. J. Biol. Chem. 1997; 272: 27862-27868Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The α4 chain is also weakly expressed in other adult tissues, such as brain, spleen, liver, kidney, and testis (26Iivanainen A. Kortesmaa J. Sahlberg C. Morita T. Bergmann U. Thesleff I. Tryggvason K. J. Biol. Chem. 1997; 272: 27862-27868Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The laminin α4 chain may play a critical biological role in the tissues. Proteolytic processing of the laminin α4 chain G domain was confirmed with cultured endothelial and Schwannoma cells, where the C-terminal α4 LG4–5 module was released (27Talts J.F. Sasaki T. Miosge N. Göhring W. Mann K. Mayne R. Timpl R. J. Biol. Chem. 2000; 275: 35192-35199Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The LG4–5 fragment was detected in cell cultures but not in tissues by immunostaining. Laminin-8 containing the α4 chain bound to α6β1 and α3β1 integrins (28Kortesmaa J. Yurchenco P. Tryggvason K. J. Biol. Chem. 2000; 275: 14853-14859Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 29Fujiwara H. Kikkawa Y. Sanzen N. Sekiguchi K. J. Biol. Chem. 2001; 276: 17550-17558Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The α4 chain G domain bound to heparin, sulfatides, and fibulins but has relatively low affinity for α-dystroglycan receptors compared with other laminin α chains (27Talts J.F. Sasaki T. Miosge N. Göhring W. Mann K. Mayne R. Timpl R. J. Biol. Chem. 2000; 275: 35192-35199Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). By using recombinant proteins, we previously showed that the laminin α4 chain G domain bound to heparin, and this affinity was stronger than that of the α1 chain G domain (30Yamaguchi H. Yamashita H. Mori H. Okazaki I. Nomizu M. Beck K. Kitagawa Y. J. Biol. Chem. 2000; 275: 29458-29465Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Recently, we have identified the heparin-binding sites on the laminin α1, α3, and α5 chains LG4 modules (14Hoffman M.P. Engbring J.A. Nielsen P.K. Vargas J. Steinberg Z. Karmand A.J. Nomizu M. Yamada Y. Kleinman H.K. J. Biol. Chem. 2001; 276: 22077-22085Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 16Hoffman M.P. Nomizu M. Roque E. Lee S. Jung D.W. Yamada Y. Kleinman H.K. J. Biol. Chem. 1998; 273: 28633-28641Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 18Utani A. Nomizu M. Matsuura H. Kato K. Kobayashi T. Takeda U. Aota S. Nielsen P.K. Shinkai H. J. Biol. Chem. 2001; 276: 28779-28788Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 31Nielsen P.K. Gho Y.S. Hoffman M.P. Watanabe H. Makino M. Nomizu M. Yamada Y. J. Biol. Chem. 2000; 275: 14517-14523Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). These peptides interacted with syndecan-1 or -2, a membrane-associated proteoglycan, and promoted cell attachment. Heparin binding may be important for the biological activity of the laminin α4 chain.In this paper, we describe the systematic screening for biologically active sequences in the laminin α4 chain G domain (mouse laminin α4 chain 852–1816) using a recombinant protein and a large set of overlapping peptides. The laminin α4 chain G domain recombinant protein promoted cell attachment and heparin binding. For the initial stage of screening for identification of active sequences, we evaluated the cell attachment activities of 104 different peptides using peptide-conjugated Sepharose beads and peptide-coated plates. We also examined the effect of these peptides on heparin binding to the recombinant protein. Four sequences were identified that were active in all of the assays and were also evaluated for additional biological activities. Two were found to interact with syndecan-4.DISCUSSIONWe have found that the laminin α4 chain G domain recombinant protein promoted cell attachment and that was inhibited by heparin and partially inhibited by EDTA. These results suggest that heparin-like binding is involved in the cell attachment activity of the laminin α4 chain G domain. We identified cell binding and heparin-binding sites in the laminin α4 chain G domain by a systematic peptide screening. Twenty cell adhesive peptides were identified using peptide-coated plate and peptide-conjugated bead assays. Fourteen peptides promoted cell attachment activity in either the plate or bead assays, and six peptides were active in both assays. Four of the active peptides were not active when coated on the plates but were active in the bead assay, whereas 10 peptides were not active in the bead assay but were active in the plate assay. These results indicate that both assays should be employed when testing for active peptides. It is likely that the differential activities are due to conformational changes and/or poor coating efficiencies on the plates. For example, A4G59, A4G69, A4G79, and A4G84 showed strong cell attachment activity in the peptide-conjugated Sepharose bead assay, but these peptides were not active in the peptide-coated plate assay. We previously showed that a 12-mer peptide containing the RGD sequence (34Pierschbacher M.D. Ruoslahti E. Nature. 1984; 309: 30-33Crossref PubMed Scopus (2838) Google Scholar) was active in the peptide-conjugated Sepharose bead assay but not active in the peptide-coated plate assay (33Nomizu M. Song S.Y. Kuratomi Y. Tanaka M. Kim W.H. Kleinman H.K. Yamada Y. FEBS Lett. 1996; 396: 37-42Crossref PubMed Scopus (32) Google Scholar). The similarity of A4G59, A4G69, A4G79, and A4G84 and the RGD-containing peptide suggests that the cell binding of these peptides required an active conformation or higher coating efficiency to the plate. In contrast, A4G4, A4G10 A4G24, A4G25, A4G26, A4G31, A4G46, A4G78, A4G102, and A4G107 showed cell attachment activity in the peptide-coated plate assay, but these peptides were not active in the peptide-conjugated Sepharose bead assay. We showed previously (35Tashiro K. Sephel G.C. Weeks B. Sasaki M. Martin G.R. Kleinman H.K. Yamada Y. J. Biol. Chem. 1989; 264: 16174-16182Abstract Full Text PDF PubMed Google Scholar) that a 12-mer peptide containing the active IKVAV sequence from the laminin α1 chain was active in the peptide-coated plate assay but was not active in the peptide-conjugated Sepharose bead assay (11Nomizu M. Kuratomi Y. Malinda M.K. Song S.Y. Miyoshi K. Otaka A. Powel S.K. Hoffman M.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1998; 273: 32491-32499Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). These α4 chain G domain peptides may behave similarly to the IKVAV peptide and require a specific conformation for activity.We sought to partially characterize the cellular receptors for the most active peptides. Previously, we identified several cell binding sequences on the α1 chain that recognize integrins (9Nomizu M. Kim W.H. Yamamura K. Utani A. Song S.Y. Otaka A. Roller P.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1995; 270: 20583-20590Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 11Nomizu M. Kuratomi Y. Malinda M.K. Song S.Y. Miyoshi K. Otaka A. Powel S.K. Hoffman M.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1998; 273: 32491-32499Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17Ponce M.L. Nomizu M. Kleinman H.K. FASEB J. 2001; 15: 1389-1397Crossref PubMed Scopus (78) Google Scholar). EDTA partially inhibited cell attachment to A4G24 but did not affect the rest of the active peptides, suggesting that it has the potential to interact with integrins. Cell attachment to all of the active α4 chain G domain peptides was inhibited by heparin. Cell surface heparin-like molecules are likely important for cell attachment to these peptides. Furthermore, the heparin binding of rec-α4G is blocked by A4G6, A4G10, A4G20, A4G82, and A4G83 in a dose-dependent manner. In solid phase binding assays, biotinylated heparin also bound to these peptides. These data suggest that heparin-like cell surface molecules are important in α4 chain G domain-mediated cell attachment. Recently, we found that AG73, a laminin α1 chain LG4 module peptide, interacts with syndecan-1 (14Hoffman M.P. Engbring J.A. Nielsen P.K. Vargas J. Steinberg Z. Karmand A.J. Nomizu M. Yamada Y. Kleinman H.K. J. Biol. Chem. 2001; 276: 22077-22085Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,16Hoffman M.P. Nomizu M. Roque E. Lee S. Jung D.W. Yamada Y. Kleinman H.K. J. Biol. Chem. 1998; 273: 28633-28641Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), and A3G75, a laminin α3 chain LG4 module peptide, binds to syndecan-2 (18Utani A. Nomizu M. Matsuura H. Kato K. Kobayashi T. Takeda U. Aota S. Nielsen P.K. Shinkai H. J. Biol. Chem. 2001; 276: 28779-28788Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Here we showed the rec-α4G protein and the most active peptides A4G6 and A4G20 bound to syndecan-4. Taken together, the active peptides in the laminin α4 chain G domain have a potential to be involved in syndecan-mediated cell binding.Active sequences are mainly located on the LG1 and LG4 modules (Fig.10 A). Previously, we identified the AG73 and A3G75 peptides in the laminin α1 and α3 chains LG4 module. AG73 binds to syndecan-1 and promotes various biological activities including cell adhesion, neurite outgrowth, acinar cell differentiation, and liver metastasis (9Nomizu M. Kim W.H. Yamamura K. Utani A. Song S.Y. Otaka A. Roller P.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1995; 270: 20583-20590Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 11Nomizu M. Kuratomi Y. Malinda M.K. Song S.Y. Miyoshi K. Otaka A. Powel S.K. Hoffman M.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1998; 273: 32491-32499Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 16Hoffman M.P. Nomizu M. Roque E. Lee S. Jung D.W. Yamada Y. Kleinman H.K. J. Biol. Chem. 1998; 273: 28633-28641Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 19Richard B.L. Nomizu M. Yamada Y. Kleinman H.K. Exp. Cell Res. 1996; 228: 98-105Crossref PubMed Scopus (74) Google Scholar, 20Kim W.H. Nomizu M. Song S.Y. Tanaka K. Kuratomi Y. Kleinman H.K. Yamada Y. Int. J. Cancer. 1998; 77: 632-639Crossref PubMed Scopus (48) Google Scholar). A3G75 also promotes cell adhesion and binds to syndecan-2 (18Utani A. Nomizu M. Matsuura H. Kato K. Kobayashi T. Takeda U. Aota S. Nielsen P.K. Shinkai H. J. Biol. Chem. 2001; 276: 28779-28788Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). We have focused on homologous sites of the AG73 and A3G75 sequences in the α4 chain G domain (Fig. 10). Active sequences are mapped using a crystal structure-based sequential alignment of the LG modules reported previously (36Timpl R. Tisi D. Talts J.F. Andac Z. Sasaki T. Hohenester E. Matrix Biol. 2000; 19: 309-317Crossref PubMed Scopus (254) Google Scholar) (Fig. 10). The AG73 sequence is located on the β-strand C region, and the A3G75 sequence is located on the loop region between the β-strands E and F in the LG4 module. A4G6 and A4G82–83, which showed the strongest cell attachment and heparin binding activities, are located in the homologous region of A3G75 in the α4 chain LG1 and LG4 modules. A4G6 and A4G82–83 would be extruded at the opposite edge of the 14-stranded β-sheet sandwich structure where a calcium ion binds. These results suggest that the active sites in the α3 and α4 chains are conserved at the loop region between the β-strands E and F and that the region plays a critical role in the biological functions of the laminin α4 chain G domain. In contrast, A4G78 was found to locate on the homologous site of AG73 in the α4 chain LG4 module. The cell attachment activity of A4G78 was much lower than that of AG73. The AG73 site was previously found to be chain-specific and to promote cell type-specific activity (14Hoffman M.P. Engbring J.A. Nielsen P.K. Vargas J. Steinberg Z. Karmand A.J. Nomizu M. Yamada Y. Kleinman H.K. J. Biol. Chem. 2001; 276: 22077-22085Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). A4G20 located on the loop region between the β-strand L to N in the α4 chain LG1 module is not found on any other previously identified active sequences. These chain-specific active sites in the G domain may promote cell type-specific biological activities.Inhibition of cell attachment on the rec-α4G protein substrate by A4G6, A4G20, A4G24, A4G31, A4G78, A4G82, A4G83, A4G90, and A4G107 suggests that these active sites are available on the intact molecule. These results also confirm the importance of conformation for cell interactions. However, not all active sites function in the intact molecule. Recently, a proteolytic fragment of laminin-5 generated by matrix metalloproteinase-2 was found to induce cell migration (37Giannelli G. Falk-Marzillier J. Schiraldi O. Stetler-Stevenson W.G. Quaranta V. Science. 1997; 277: 225-228Crossref PubMed Scopus (1037) Google Scholar). Proteolytic fragments of other laminin chains may also contain activity that is cryptic in the intact molecule but is revealed after proteolysis. Fragments of plasminogen and collagen XVIII, designated angiostatin and endostatin, were found to have important functions in regulating angiogenesis and tumor growth (38O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 21: 315-328Abstract Full Text PDF Scopus (3150) Google Scholar, 39O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S. Flynn E. Birkhead J.R. Olsen B.R. Folkman J. Cell. 1997; 24: 277-285Abstract Full Text Full Text PDF Scopus (4211) Google Scholar). It is possible that the active peptide fragments of laminin play a critical role in its biological activity. Laminins, a family of extracellular matrix proteins, consist of three different subunits, α, β, and γ chains. So far, five α, three β, and three γ chains have been identified, and at least 15 isoforms (laminin 1–15) are formed by various combinations of each subunit (1Burgeson R.E. Chiquet M. Deutzmann R. Ekblom P. Engel J. Kleinman H.K. Martin G.R. Meneguzzi G. Paulson M. Sanes J. Timpl R. Tryggvason K. Yamada Y. Yurchenco P.D. Matrix Biol. 1994; 14: 209-211Crossref PubMed Scopus (694) Google Scholar, 2Miner J.H. Patton B.L. Lentz S.I. Gilbert D.J. Snider W.D. Jenkins N.A. Copeland N.G. Sanes J.R. J. Cell Biol. 1997; 137: 685-701Crossref PubMed Scopus (576) Google Scholar, 3Iivanainen A. Morita T. Tryggvason K. J. Biol. Chem. 1999; 274: 14107-14111Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 4Libby R.T. Champliaud M.F. Claudepierre T., Xu, Y. Gibbons E.P. Koch M. Burgeson R.E. Hunter D.D. Brunken W.J. J. Neurosci. 2000; 20: 6517-6528Crossref PubMed Google Scholar). Laminins have diverse biological activities including promotion of cell adhesion, migration, neurite outgrowth, angiogenesis, and tumor metastasis (5Colognato H. Yurchenco P.D. Dev. Dyn. 2000; 218: 213-234Crossref PubMed Scopus (1033) Google Scholar). More than 20 receptors have been reported for these laminin molecules (6Powell S.K. Kleinman H.K. Int. J. Biochem. Cell Biol. 1997; 29: 401-414Crossref PubMed Scopus (159) Google Scholar). Several active sites on laminin-1 have been identified using proteolytic fragments, recombinant proteins, and synthetic peptides (7Yamada Y. Kleinman H.K. Curr. Opin. Cell Biol. 1992; 4: 819-823Crossref PubMed Scopus (137) Google Scholar, 8Yamada K.M. J. Biol. Chem. 1991; 266: 12809-12912Abstract Full Text PDF PubMed Google Scholar). Previously, we screened for cell adhesive sequences on laminin-1 using 673 overlapping synthetic peptides covering the entire protein (9Nomizu M. Kim W.H. Yamamura K. Utani A. Song S.Y. Otaka A. Roller P.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1995; 270: 20583-20590Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 10Nomizu M. Kuratomi Y. Song S.Y. Ponce L.M. Hoffman M.P. Powell S.K. Miyoshi K. Otaka A. Kleinman H.K. Yamada Y. J. Biol. Chem. 1997; 272: 32198-32205Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 11Nomizu M. Kuratomi Y. Malinda M.K. Song S.Y. Miyoshi K. Otaka A. Powel S.K. Hoffman M.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1998; 273: 32491-32499Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 12Nomizu M. Kuratomi Y. Ponce L.M. Song S.Y. Miyoshi K. Otaka A. Powell S.K. Hoffman M.P. Kleinman H.K. Yamada Y. Arch. Biochem. Biophys. 2000; 378: 311-320Crossref PubMed Scopus (69) Google Scholar). Most of the active peptides were localized in the globular domains and found to play a critical role in binding to cell surface receptors in a peptide- and cell type-specific manner (13Makino M. Okazaki I. Nishi N. Nomizu M. Connect. Tissue. 1999; 31: 227-234Google Scholar, 14Hoffman M.P. Engbring J.A. Nielsen P.K. Vargas J. Steinberg Z. Karmand A.J. Nomizu M. Yamada Y. Kleinman H.K. J. Biol. Chem. 2001; 276: 22077-22085Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Several peptides were found to interact with integrins and syndecans (15Tashiro K. Sephel G.C. Greatorex D. Sasaki M. Shirashi N. Martin G.R. Kleinman H.K. Yamada Y. J. Cell. Physiol. 1991; 146: 451-459Crossref PubMed Scopus (97) Google Scholar, 16Hoffman M.P. Nomizu M. Roque E. Lee S. Jung D.W. Yamada Y. Kleinman H.K. J. Biol. Chem. 1998; 273: 28633-28641Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 17Ponce M.L. Nomizu M. Kleinman H.K. FASEB J. 2001; 15: 1389-1397Crossref PubMed Scopus (78) Google Scholar, 18Utani A. Nomizu M. Matsuura H. Kato K. Kobayashi T. Takeda U. Aota S. Nielsen P.K. Shinkai H. J. Biol. Chem. 2001; 276: 28779-28788Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Some of the peptides promoted neurite outgrowth, angiogenesis, and tumor metastasis (19Richard B.L. Nomizu M. Yamada Y. Kleinman H.K. Exp. Cell Res. 1996; 228: 98-105Crossref PubMed Scopus (74) Google Scholar, 20Kim W.H. Nomizu M. Song S.Y. Tanaka K. Kuratomi Y. Kleinman H.K. Yamada Y. Int. J. Cancer. 1998; 77: 632-639Crossref PubMed Scopus (48) Google Scholar, 21Malinda K.M. Nomizu M. Chung M. Delgado M. Kuratomi Y. Yamada Y. Kleinman H.K. Ponce M.L. FASEB J. 1999; 13: 53-62Crossref PubMed Scopus (164) Google Scholar, 22Ponce M.L. Nomizu M. Delgado M.C. Kuratomi Y. Hoffman M.P. Powell S. Yamada Y. Kleinman H.K. Malinda K.M. Circ. Res. 1999; 84: 688-694Crossref PubMed Scopus (90) Google Scholar). The laminin α chains are generally large (M r = 400,000) and contain a C-terminal globular domain consisting of five globular modules LG1–LG5. The laminin α4 chain lacks the N-terminal short arm and is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes (23Tokida Y. Aratani Y. Morita A. Kitagawa Y. J. Biol. Chem. 1990; 265: 18123-18129Abstract Full Text PDF PubMed Google Scholar, 24Sorokin L.M. Pausch F. Frieser M. Kröger S. Ohage E. Deutzmann R. Dev. Biol. 1997; 189: 285-300Crossref PubMed Scopus (215) Google Scholar, 25Niimi T. Kumagai C. Okano M. Kitagawa Y. Matrix Biol. 1997; 16: 223-230Crossref PubMed Scopus (43) Google Scholar). Laminin α4 chain expression is mainly localized to mesenchymal cells present in the lung and cardiac and skeletal muscles fibers (26Iivanainen A. Kortesmaa J. Sahlberg C. Morita T. Bergmann U. Thesleff I. Tryggvason K. J. Biol. Chem. 1997; 272: 27862-27868Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The α4 chain is also weakly expressed in other adult tissues, such as brain, spleen, liver, kidney, and testis (26Iivanainen A. Kortesmaa J. Sahlberg C. Morita T. Bergmann U. Thesleff I. Tryggvason K. J. Biol. Chem. 1997; 272: 27862-27868Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The laminin α4 chain may play a critical biological role in the tissues. Proteolytic processing of the laminin α4 chain G domain was confirmed with cultured endothelial and Schwannoma cells, where the C-terminal α4 LG4–5 module was released (27Talts J.F. Sasaki T. Miosge N. Göhring W. Mann K. Mayne R. Timpl R. J. Biol. Chem. 2000; 275: 35192-35199Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The LG4–5 fragment was detected in cell cultures but not in tissues by immunostaining. Laminin-8 containing the α4 chain bound to α6β1 and α3β1 integrins (28Kortesmaa J. Yurchenco P. Tryggvason K. J. Biol. Chem. 2000; 275: 14853-14859Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 29Fujiwara H. Kikkawa Y. Sanzen N. Sekiguchi K. J. Biol. Chem. 2001; 276: 17550-17558Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The α4 chain G domain bound to heparin, sulfatides, and fibulins but has relatively low affinity for α-dystroglycan receptors compared with other laminin α chains (27Talts J.F. Sasaki T. Miosge N. Göhring W. Mann K. Mayne R. Timpl R. J. Biol. Chem. 2000; 275: 35192-35199Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). By using recombinant proteins, we previously showed that the laminin α4 chain G domain bound to heparin, and this affinity was stronger than that of the α1 chain G domain (30Yamaguchi H. Yamashita H. Mori H. Okazaki I. Nomizu M. Beck K. Kitagawa Y. J. Biol. Chem. 2000; 275: 29458-29465Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Recently, we have identified the heparin-binding sites on the laminin α1, α3, and α5 chains LG4 modules (14Hoffman M.P. Engbring J.A. Nielsen P.K. Vargas J. Steinberg Z. Karmand A.J. Nomizu M. Yamada Y. Kleinman H.K. J. Biol. Chem. 2001; 276: 22077-22085Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 16Hoffman M.P. Nomizu M. Roque E. Lee S. Jung D.W. Yamada Y. Kleinman H.K. J. Biol. Chem. 1998; 273: 28633-28641Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 18Utani A. Nomizu M. Matsuura H. Kato K. Kobayashi T. Takeda U. Aota S. Nielsen P.K. Shinkai H. J. Biol. Chem. 2001; 276: 28779-28788Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 31Nielsen P.K. Gho Y.S. Hoffman M.P. Watanabe H. Makino M. Nomizu M. Yamada Y. J. Biol. Chem. 2000; 275: 14517-14523Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). These peptides interacted with syndecan-1 or -2, a membrane-associated proteoglycan, and promoted cell attachment. Heparin binding may be important for the biological activity of the laminin α4 chain. In this paper, we describe the systematic screening for biologically active sequences in the laminin α4 chain G domain (mouse laminin α4 chain 852–1816) using a recombinant protein and a large set of overlapping peptides. The laminin α4 chain G domain recombinant protein promoted cell attachment and heparin binding. For the initial stage of screening for identification of active sequences, we evaluated the cell attachment activities of 104 different peptides using peptide-conjugated Sepharose beads and peptide-coated plates. We also examined the effect of these peptides on heparin binding to the recombinant protein. Four sequences were identified that were active in all of the assays and were also evaluated for additional biological activities. Two were found to interact with syndecan-4. DISCUSSIONWe have found that the laminin α4 chain G domain recombinant protein promoted cell attachment and that was inhibited by heparin and partially inhibited by EDTA. These results suggest that heparin-like binding is involved in the cell attachment activity of the laminin α4 chain G domain. We identified cell binding and heparin-binding sites in the laminin α4 chain G domain by a systematic peptide screening. Twenty cell adhesive peptides were identified using peptide-coated plate and peptide-conjugated bead assays. Fourteen peptides promoted cell attachment activity in either the plate or bead assays, and six peptides were active in both assays. Four of the active peptides were not active when coated on the plates but were active in the bead assay, whereas 10 peptides were not active in the bead assay but were active in the plate assay. These results indicate that both assays should be employed when testing for active peptides. It is likely that the differential activities are due to conformational changes and/or poor coating efficiencies on the plates. For example, A4G59, A4G69, A4G79, and A4G84 showed strong cell attachment activity in the peptide-conjugated Sepharose bead assay, but these peptides were not active in the peptide-coated plate assay. We previously showed that a 12-mer peptide containing the RGD sequence (34Pierschbacher M.D. Ruoslahti E. Nature. 1984; 309: 30-33Crossref PubMed Scopus (2838) Google Scholar) was active in the peptide-conjugated Sepharose bead assay but not active in the peptide-coated plate assay (33Nomizu M. Song S.Y. Kuratomi Y. Tanaka M. Kim W.H. Kleinman H.K. Yamada Y. FEBS Lett. 1996; 396: 37-42Crossref PubMed Scopus (32) Google Scholar). The similarity of A4G59, A4G69, A4G79, and A4G84 and the RGD-containing peptide suggests that the cell binding of these peptides required an active conformation or higher coating efficiency to the plate. In contrast, A4G4, A4G10 A4G24, A4G25, A4G26, A4G31, A4G46, A4G78, A4G102, and A4G107 showed cell attachment activity in the peptide-coated plate assay, but these peptides were not active in the peptide-conjugated Sepharose bead assay. We showed previously (35Tashiro K. Sephel G.C. Weeks B. Sasaki M. Martin G.R. Kleinman H.K. Yamada Y. J. Biol. Chem. 1989; 264: 16174-16182Abstract Full Text PDF PubMed Google Scholar) that a 12-mer peptide containing the active IKVAV sequence from the laminin α1 chain was active in the peptide-coated plate assay but was not active in the peptide-conjugated Sepharose bead assay (11Nomizu M. Kuratomi Y. Malinda M.K. Song S.Y. Miyoshi K. Otaka A. Powel S.K. Hoffman M.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1998; 273: 32491-32499Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). These α4 chain G domain peptides may behave similarly to the IKVAV peptide and require a specific conformation for activity.We sought to partially characterize the cellular receptors for the most active peptides. Previously, we identified several cell binding sequences on the α1 chain that recognize integrins (9Nomizu M. Kim W.H. Yamamura K. Utani A. Song S.Y. Otaka A. Roller P.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1995; 270: 20583-20590Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 11Nomizu M. Kuratomi Y. Malinda M.K. Song S.Y. Miyoshi K. Otaka A. Powel S.K. Hoffman M.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1998; 273: 32491-32499Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17Ponce M.L. Nomizu M. Kleinman H.K. FASEB J. 2001; 15: 1389-1397Crossref PubMed Scopus (78) Google Scholar). EDTA partially inhibited cell attachment to A4G24 but did not affect the rest of the active peptides, suggesting that it has the potential to interact with integrins. Cell attachment to all of the active α4 chain G domain peptides was inhibited by heparin. Cell surface heparin-like molecules are likely important for cell attachment to these peptides. Furthermore, the heparin binding of rec-α4G is blocked by A4G6, A4G10, A4G20, A4G82, and A4G83 in a dose-dependent manner. In solid phase binding assays, biotinylated heparin also bound to these peptides. These data suggest that heparin-like cell surface molecules are important in α4 chain G domain-mediated cell attachment. Recently, we found that AG73, a laminin α1 chain LG4 module peptide, interacts with syndecan-1 (14Hoffman M.P. Engbring J.A. Nielsen P.K. Vargas J. Steinberg Z. Karmand A.J. Nomizu M. Yamada Y. Kleinman H.K. J. Biol. Chem. 2001; 276: 22077-22085Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,16Hoffman M.P. Nomizu M. Roque E. Lee S. Jung D.W. Yamada Y. Kleinman H.K. J. Biol. Chem. 1998; 273: 28633-28641Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), and A3G75, a laminin α3 chain LG4 module peptide, binds to syndecan-2 (18Utani A. Nomizu M. Matsuura H. Kato K. Kobayashi T. Takeda U. Aota S. Nielsen P.K. Shinkai H. J. Biol. Chem. 2001; 276: 28779-28788Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Here we showed the rec-α4G protein and the most active peptides A4G6 and A4G20 bound to syndecan-4. Taken together, the active peptides in the laminin α4 chain G domain have a potential to be involved in syndecan-mediated cell binding.Active sequences are mainly located on the LG1 and LG4 modules (Fig.10 A). Previously, we identified the AG73 and A3G75 peptides in the laminin α1 and α3 chains LG4 module. AG73 binds to syndecan-1 and promotes various biological activities including cell adhesion, neurite outgrowth, acinar cell differentiation, and liver metastasis (9Nomizu M. Kim W.H. Yamamura K. Utani A. Song S.Y. Otaka A. Roller P.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1995; 270: 20583-20590Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 11Nomizu M. Kuratomi Y. Malinda M.K. Song S.Y. Miyoshi K. Otaka A. Powel S.K. Hoffman M.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1998; 273: 32491-32499Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 16Hoffman M.P. Nomizu M. Roque E. Lee S. Jung D.W. Yamada Y. Kleinman H.K. J. Biol. Chem. 1998; 273: 28633-28641Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 19Richard B.L. Nomizu M. Yamada Y. Kleinman H.K. Exp. Cell Res. 1996; 228: 98-105Crossref PubMed Scopus (74) Google Scholar, 20Kim W.H. Nomizu M. Song S.Y. Tanaka K. Kuratomi Y. Kleinman H.K. Yamada Y. Int. J. Cancer. 1998; 77: 632-639Crossref PubMed Scopus (48) Google Scholar). A3G75 also promotes cell adhesion and binds to syndecan-2 (18Utani A. Nomizu M. Matsuura H. Kato K. Kobayashi T. Takeda U. Aota S. Nielsen P.K. Shinkai H. J. Biol. Chem. 2001; 276: 28779-28788Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). We have focused on homologous sites of the AG73 and A3G75 sequences in the α4 chain G domain (Fig. 10). Active sequences are mapped using a crystal structure-based sequential alignment of the LG modules reported previously (36Timpl R. Tisi D. Talts J.F. Andac Z. Sasaki T. Hohenester E. Matrix Biol. 2000; 19: 309-317Crossref PubMed Scopus (254) Google Scholar) (Fig. 10). The AG73 sequence is located on the β-strand C region, and the A3G75 sequence is located on the loop region between the β-strands E and F in the LG4 module. A4G6 and A4G82–83, which showed the strongest cell attachment and heparin binding activities, are located in the homologous region of A3G75 in the α4 chain LG1 and LG4 modules. A4G6 and A4G82–83 would be extruded at the opposite edge of the 14-stranded β-sheet sandwich structure where a calcium ion binds. These results suggest that the active sites in the α3 and α4 chains are conserved at the loop region between the β-strands E and F and that the region plays a critical role in the biological functions of the laminin α4 chain G domain. In contrast, A4G78 was found to locate on the homologous site of AG73 in the α4 chain LG4 module. The cell attachment activity of A4G78 was much lower than that of AG73. The AG73 site was previously found to be chain-specific and to promote cell type-specific activity (14Hoffman M.P. Engbring J.A. Nielsen P.K. Vargas J. Steinberg Z. Karmand A.J. Nomizu M. Yamada Y. Kleinman H.K. J. Biol. Chem. 2001; 276: 22077-22085Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). A4G20 located on the loop region between the β-strand L to N in the α4 chain LG1 module is not found on any other previously identified active sequences. These chain-specific active sites in the G domain may promote cell type-specific biological activities.Inhibition of cell attachment on the rec-α4G protein substrate by A4G6, A4G20, A4G24, A4G31, A4G78, A4G82, A4G83, A4G90, and A4G107 suggests that these active sites are available on the intact molecule. These results also confirm the importance of conformation for cell interactions. However, not all active sites function in the intact molecule. Recently, a proteolytic fragment of laminin-5 generated by matrix metalloproteinase-2 was found to induce cell migration (37Giannelli G. Falk-Marzillier J. Schiraldi O. Stetler-Stevenson W.G. Quaranta V. Science. 1997; 277: 225-228Crossref PubMed Scopus (1037) Google Scholar). Proteolytic fragments of other laminin chains may also contain activity that is cryptic in the intact molecule but is revealed after proteolysis. Fragments of plasminogen and collagen XVIII, designated angiostatin and endostatin, were found to have important functions in regulating angiogenesis and tumor growth (38O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 21: 315-328Abstract Full Text PDF Scopus (3150) Google Scholar, 39O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S. Flynn E. Birkhead J.R. Olsen B.R. Folkman J. Cell. 1997; 24: 277-285Abstract Full Text Full Text PDF Scopus (4211) Google Scholar). It is possible that the active peptide fragments of laminin play a critical role in its biological activity. We have found that the laminin α4 chain G domain recombinant protein promoted cell attachment and that was inhibited by heparin and partially inhibited by EDTA. These results suggest that heparin-like binding is involved in the cell attachment activity of the laminin α4 chain G domain. We identified cell binding and heparin-binding sites in the laminin α4 chain G domain by a systematic peptide screening. Twenty cell adhesive peptides were identified using peptide-coated plate and peptide-conjugated bead assays. Fourteen peptides promoted cell attachment activity in either the plate or bead assays, and six peptides were active in both assays. Four of the active peptides were not active when coated on the plates but were active in the bead assay, whereas 10 peptides were not active in the bead assay but were active in the plate assay. These results indicate that both assays should be employed when testing for active peptides. It is likely that the differential activities are due to conformational changes and/or poor coating efficiencies on the plates. For example, A4G59, A4G69, A4G79, and A4G84 showed strong cell attachment activity in the peptide-conjugated Sepharose bead assay, but these peptides were not active in the peptide-coated plate assay. We previously showed that a 12-mer peptide containing the RGD sequence (34Pierschbacher M.D. Ruoslahti E. Nature. 1984; 309: 30-33Crossref PubMed Scopus (2838) Google Scholar) was active in the peptide-conjugated Sepharose bead assay but not active in the peptide-coated plate assay (33Nomizu M. Song S.Y. Kuratomi Y. Tanaka M. Kim W.H. Kleinman H.K. Yamada Y. FEBS Lett. 1996; 396: 37-42Crossref PubMed Scopus (32) Google Scholar). The similarity of A4G59, A4G69, A4G79, and A4G84 and the RGD-containing peptide suggests that the cell binding of these peptides required an active conformation or higher coating efficiency to the plate. In contrast, A4G4, A4G10 A4G24, A4G25, A4G26, A4G31, A4G46, A4G78, A4G102, and A4G107 showed cell attachment activity in the peptide-coated plate assay, but these peptides were not active in the peptide-conjugated Sepharose bead assay. We showed previously (35Tashiro K. Sephel G.C. Weeks B. Sasaki M. Martin G.R. Kleinman H.K. Yamada Y. J. Biol. Chem. 1989; 264: 16174-16182Abstract Full Text PDF PubMed Google Scholar) that a 12-mer peptide containing the active IKVAV sequence from the laminin α1 chain was active in the peptide-coated plate assay but was not active in the peptide-conjugated Sepharose bead assay (11Nomizu M. Kuratomi Y. Malinda M.K. Song S.Y. Miyoshi K. Otaka A. Powel S.K. Hoffman M.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1998; 273: 32491-32499Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). These α4 chain G domain peptides may behave similarly to the IKVAV peptide and require a specific conformation for activity. We sought to partially characterize the cellular receptors for the most active peptides. Previously, we identified several cell binding sequences on the α1 chain that recognize integrins (9Nomizu M. Kim W.H. Yamamura K. Utani A. Song S.Y. Otaka A. Roller P.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1995; 270: 20583-20590Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 11Nomizu M. Kuratomi Y. Malinda M.K. Song S.Y. Miyoshi K. Otaka A. Powel S.K. Hoffman M.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1998; 273: 32491-32499Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 17Ponce M.L. Nomizu M. Kleinman H.K. FASEB J. 2001; 15: 1389-1397Crossref PubMed Scopus (78) Google Scholar). EDTA partially inhibited cell attachment to A4G24 but did not affect the rest of the active peptides, suggesting that it has the potential to interact with integrins. Cell attachment to all of the active α4 chain G domain peptides was inhibited by heparin. Cell surface heparin-like molecules are likely important for cell attachment to these peptides. Furthermore, the heparin binding of rec-α4G is blocked by A4G6, A4G10, A4G20, A4G82, and A4G83 in a dose-dependent manner. In solid phase binding assays, biotinylated heparin also bound to these peptides. These data suggest that heparin-like cell surface molecules are important in α4 chain G domain-mediated cell attachment. Recently, we found that AG73, a laminin α1 chain LG4 module peptide, interacts with syndecan-1 (14Hoffman M.P. Engbring J.A. Nielsen P.K. Vargas J. Steinberg Z. Karmand A.J. Nomizu M. Yamada Y. Kleinman H.K. J. Biol. Chem. 2001; 276: 22077-22085Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,16Hoffman M.P. Nomizu M. Roque E. Lee S. Jung D.W. Yamada Y. Kleinman H.K. J. Biol. Chem. 1998; 273: 28633-28641Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), and A3G75, a laminin α3 chain LG4 module peptide, binds to syndecan-2 (18Utani A. Nomizu M. Matsuura H. Kato K. Kobayashi T. Takeda U. Aota S. Nielsen P.K. Shinkai H. J. Biol. Chem. 2001; 276: 28779-28788Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Here we showed the rec-α4G protein and the most active peptides A4G6 and A4G20 bound to syndecan-4. Taken together, the active peptides in the laminin α4 chain G domain have a potential to be involved in syndecan-mediated cell binding. Active sequences are mainly located on the LG1 and LG4 modules (Fig.10 A). Previously, we identified the AG73 and A3G75 peptides in the laminin α1 and α3 chains LG4 module. AG73 binds to syndecan-1 and promotes various biological activities including cell adhesion, neurite outgrowth, acinar cell differentiation, and liver metastasis (9Nomizu M. Kim W.H. Yamamura K. Utani A. Song S.Y. Otaka A. Roller P.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1995; 270: 20583-20590Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 11Nomizu M. Kuratomi Y. Malinda M.K. Song S.Y. Miyoshi K. Otaka A. Powel S.K. Hoffman M.P. Kleinman H.K. Yamada Y. J. Biol. Chem. 1998; 273: 32491-32499Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 16Hoffman M.P. Nomizu M. Roque E. Lee S. Jung D.W. Yamada Y. Kleinman H.K. J. Biol. Chem. 1998; 273: 28633-28641Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 19Richard B.L. Nomizu M. Yamada Y. Kleinman H.K. Exp. Cell Res. 1996; 228: 98-105Crossref PubMed Scopus (74) Google Scholar, 20Kim W.H. Nomizu M. Song S.Y. Tanaka K. Kuratomi Y. Kleinman H.K. Yamada Y. Int. J. Cancer. 1998; 77: 632-639Crossref PubMed Scopus (48) Google Scholar). A3G75 also promotes cell adhesion and binds to syndecan-2 (18Utani A. Nomizu M. Matsuura H. Kato K. Kobayashi T. Takeda U. Aota S. Nielsen P.K. Shinkai H. J. Biol. Chem. 2001; 276: 28779-28788Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). We have focused on homologous sites of the AG73 and A3G75 sequences in the α4 chain G domain (Fig. 10). Active sequences are mapped using a crystal structure-based sequential alignment of the LG modules reported previously (36Timpl R. Tisi D. Talts J.F. Andac Z. Sasaki T. Hohenester E. Matrix Biol. 2000; 19: 309-317Crossref PubMed Scopus (254) Google Scholar) (Fig. 10). The AG73 sequence is located on the β-strand C region, and the A3G75 sequence is located on the loop region between the β-strands E and F in the LG4 module. A4G6 and A4G82–83, which showed the strongest cell attachment and heparin binding activities, are located in the homologous region of A3G75 in the α4 chain LG1 and LG4 modules. A4G6 and A4G82–83 would be extruded at the opposite edge of the 14-stranded β-sheet sandwich structure where a calcium ion binds. These results suggest that the active sites in the α3 and α4 chains are conserved at the loop region between the β-strands E and F and that the region plays a critical role in the biological functions of the laminin α4 chain G domain. In contrast, A4G78 was found to locate on the homologous site of AG73 in the α4 chain LG4 module. The cell attachment activity of A4G78 was much lower than that of AG73. The AG73 site was previously found to be chain-specific and to promote cell type-specific activity (14Hoffman M.P. Engbring J.A. Nielsen P.K. Vargas J. Steinberg Z. Karmand A.J. Nomizu M. Yamada Y. Kleinman H.K. J. Biol. Chem. 2001; 276: 22077-22085Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). A4G20 located on the loop region between the β-strand L to N in the α4 chain LG1 module is not found on any other previously identified active sequences. These chain-specific active sites in the G domain may promote cell type-specific biological activities. Inhibition of cell attachment on the rec-α4G protein substrate by A4G6, A4G20, A4G24, A4G31, A4G78, A4G82, A4G83, A4G90, and A4G107 suggests that these active sites are available on the intact molecule. These results also confirm the importance of conformation for cell interactions. However, not all active sites function in the intact molecule. Recently, a proteolytic fragment of laminin-5 generated by matrix metalloproteinase-2 was found to induce cell migration (37Giannelli G. Falk-Marzillier J. Schiraldi O. Stetler-Stevenson W.G. Quaranta V. Science. 1997; 277: 225-228Crossref PubMed Scopus (1037) Google Scholar). Proteolytic fragments of other laminin chains may also contain activity that is cryptic in the intact molecule but is revealed after proteolysis. Fragments of plasminogen and collagen XVIII, designated angiostatin and endostatin, were found to have important functions in regulating angiogenesis and tumor growth (38O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 21: 315-328Abstract Full Text PDF Scopus (3150) Google Scholar, 39O'Reilly M.S. Boehm T. Shing Y. Fukai N. Vasios G. Lane W.S. Flynn E. Birkhead J.R. Olsen B.R. Folkman J. Cell. 1997; 24: 277-285Abstract Full Text Full Text PDF Scopus (4211) Google Scholar). It is possible that the active peptide fragments of laminin play a critical role in its biological activity." @default.
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W2047731835 date "2002-10-01" @default.
W2047731835 modified "2023-10-18" @default.
W2047731835 title "Identification of Biologically Active Sequences in the Laminin α4 Chain G Domain" @default.
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W2047731835 doi "https://doi.org/10.1074/jbc.m201672200" @default.
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