Recombinant Human Laminin-5 Domains

Human laminin-5 fragments, comprising the heterotrimeric C-terminal part of the coiled-coil (CC) domain and the globular (G) domain with defined numbers of LG subdomains, were produced recombinantly. The α3′ chain with all five LG subdomains was processed proteolytically in a manner similar to the wild-type α3 chain. Conditions were established under which the proteolytic cleavage was either inhibited in cell culture or was brought to completion in vitro. The shorter chains of the laminin-5CCG molecule, β3′and γ2′, produced in a bacterial expression system associated into heterodimers, which then combined spontaneously with the α3′ chains in vitro to form heterotrimeric laminin-5CCG molecules. Only heterotrimeric laminin-5CCG with at least subdomains LG1–3, but not the single chains, supported binding of soluble α3β1 integrin, proving the coiled-coil domain of laminin-5 to be essential for its interaction with α3β1 integrin. The N-glycosylation sites in wild-type α3 chain were mapped by mass spectrometry. Their location in a structural model of the LG domain suggested that large regions on both faces of the LG1 and LG2 domains are inaccessible by other proteins. However, neither heterotrimerization nor α3β1 integrin binding was affected by the loss of N-linked glycoconjugates. After the proteolytic cleavage between the subdomains LG3 and LG4, the LG4–5 tandem domain dissociated from the rest of the G domain. Further, the laminin-5CCG molecule with the α3′LG1–3 chain showed an increased binding affinity for α3β1 integrin, indicating that proteolytic processing of laminin-5 influences its interaction with α3β1 integrin. Human laminin-5 fragments, comprising the heterotrimeric C-terminal part of the coiled-coil (CC) domain and the globular (G) domain with defined numbers of LG subdomains, were produced recombinantly. The α3′ chain with all five LG subdomains was processed proteolytically in a manner similar to the wild-type α3 chain. Conditions were established under which the proteolytic cleavage was either inhibited in cell culture or was brought to completion in vitro. The shorter chains of the laminin-5CCG molecule, β3′and γ2′, produced in a bacterial expression system associated into heterodimers, which then combined spontaneously with the α3′ chains in vitro to form heterotrimeric laminin-5CCG molecules. Only heterotrimeric laminin-5CCG with at least subdomains LG1–3, but not the single chains, supported binding of soluble α3β1 integrin, proving the coiled-coil domain of laminin-5 to be essential for its interaction with α3β1 integrin. The N-glycosylation sites in wild-type α3 chain were mapped by mass spectrometry. Their location in a structural model of the LG domain suggested that large regions on both faces of the LG1 and LG2 domains are inaccessible by other proteins. However, neither heterotrimerization nor α3β1 integrin binding was affected by the loss of N-linked glycoconjugates. After the proteolytic cleavage between the subdomains LG3 and LG4, the LG4–5 tandem domain dissociated from the rest of the G domain. Further, the laminin-5CCG molecule with the α3′LG1–3 chain showed an increased binding affinity for α3β1 integrin, indicating that proteolytic processing of laminin-5 influences its interaction with α3β1 integrin. As a member of the laminin superfamily (for review, see Refs. 1Tunggal P. Smyth N. Paulsson M. Ott M.-C. Microsc. Res. Tech. 2000; 51: 214-227Crossref PubMed Scopus (168) Google Scholar and 2Colognato H. Yurchenco P.D. Dev. Dyn. 2000; 218: 213-234Crossref PubMed Scopus (1049) Google Scholar), laminin-5 is characterized by the rod-like α-helical coiled-coil (CC) 1The abbreviations used are: CC domaincoiled-coil domainBMP-1bone morphogenetic protein-1G domainglobular domainGSTglutathione S-transferaseHAhemagglutininHEKhuman embryonic kidneymAbmonoclonal antibodyMALDImatrix-assisted laser desorption ionizationpAbpolyclonal antibodyPBSphosphate-buffered salinePNGase Fpeptidyl-N-glycosidase FYFPyellow fluorescent protein. domain (3Beck K. Hunter I. Engel J. FASEB J. 1990; 4: 148-160Crossref PubMed Scopus (660) Google Scholar) of all three laminin-5 chains, α3, β3, and γ2, and by the C-terminal globular or G domain of the α3 chain, which consists of five homologous LG subdomains (4Timpl R. Tisi D. Talts J.F. Andac Z. Sasaki T. Hohenester E. Matrix Biol. 2000; 19: 309-317Crossref PubMed Scopus (258) Google Scholar). LG domains consist of two sheets of six and seven antiparallel β-strands, which form the convex and concave face of the domain (5Hohenester E. Tisi D. Talts J.F. Timpl R. Mol. Cell. 1999; 4: 783-792Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 6Tisi D. Talts J.F. Timpl R. Hohenester E. EMBO J. 2000; 19: 1432-1440Crossref PubMed Google Scholar). Within the G domain, the LG1–3 subdomains form a three-bladed propeller, from which the LG4–5 tandem domain protrudes (4Timpl R. Tisi D. Talts J.F. Andac Z. Sasaki T. Hohenester E. Matrix Biol. 2000; 19: 309-317Crossref PubMed Scopus (258) Google Scholar). In the elongated spacer sequence connecting LG3 and LG4, a proteolytic cleavage occurs. Although plasmin, bone morphogenetic protein-1 (BMP-1) and its homolog mammalian tolloid (7Tsubota Y. Mizushima H. Hirosaki T. Higashi S. Yasumitsu H. Miyazaki K. Biochem. Biophys. Res. Commun. 2000; 278: 614-620Crossref PubMed Scopus (62) Google Scholar, 8Amano S. Scott I.C. Takahara K. Koch M. Champliaud M.-F. Gerecke D.R. Keene D.R. Hudson D.L. Nishiyama T. Lee S. Greenspan D.S. Burgeson R.E. J. Biol. Chem. 2000; 275: 22728-22735Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 9Goldfinger L.E. Stack M.S. Jones J.C.R. J. Biol. PubMed Scopus Google Scholar, P. R. D.R. Greenspan D.S. J. Biol. Chem. 278: Full Text Full Text PDF PubMed Scopus Google Scholar) are the in processing not proteolytic of laminin-5 to a in cell L.E. S. Jones J.C.R. J. 1999; PubMed Google Scholar) in and J. J. PubMed Scopus Google Scholar, S. Stack M.S. Microsc. Res. Tech. 2000; 51: PubMed Scopus Google Scholar). coiled-coil domain bone morphogenetic protein-1 globular domain embryonic laser desorption fluorescent protein. from the other of the laminin superfamily not in its chains, α3, β3, and γ2, and its but in its its and its is the of which are an essential part of the R.E. Biol. PubMed Scopus Google Scholar). to the the consists of a a other laminin-5 and which are by R. Biol. PubMed Scopus Google Scholar). an part in the of the H. Yurchenco P.D. Dev. Dyn. 2000; 218: 213-234Crossref PubMed Scopus (1049) Google Scholar, M. E. S. Burgeson R.E. Nishiyama T. Matrix Biol. PubMed Scopus Google Scholar). or in laminin-5 in the which be P. Smyth N. Paulsson M. Ott M.-C. Microsc. Res. Tech. 2000; 51: 214-227Crossref PubMed Scopus (168) Google Scholar, 2Colognato H. Yurchenco P.D. Dev. Dyn. 2000; 218: 213-234Crossref PubMed Scopus (1049) Google Scholar, J. PubMed Scopus Google Scholar). cell with laminin-5 (for review, see Refs. 2Colognato H. Yurchenco P.D. Dev. Dyn. 2000; 218: 213-234Crossref PubMed Scopus (1049) Google Scholar and Microsc. Res. Tech. 2000; 51: PubMed Scopus Google Scholar), and the and (for review, see Refs. Microsc. Res. Tech. 2000; 51: PubMed Scopus Google Scholar, M. P. P. Timpl R. The Scholar, J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and chains M. H. K. T. S. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The interaction sites for integrin and to the G domain of laminin-5 M. H. K. T. S. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Mizushima H. Y. K. Miyazaki K. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, M. N. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Y. Hirosaki T. Mizushima H. Y. Miyazaki K. J. Cell. Biochem. PubMed Scopus Google Scholar). Further, a of the to laminin-5 J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). its laminin-5 with other laminin M.-F. P. Nishiyama T. Keene D.R. Burgeson R. J. Biol. PubMed Scopus Google Scholar) and P. Keene D.R. Champliaud M.-F. M. Burgeson R.E. J. Biol. PubMed Scopus Google Scholar). laminin-5 both and In a the of a soluble α3β1 integrin, a for laminin-5 P. E. PubMed Scopus Google Scholar). the of laminin-5CCG which of the CC domain and the G domain with numbers of LG laminin-5 fragments, the interaction of α3β1 integrin with its affinity was for the location of the α3β1 integrin binding and its the of proteolytic processing of the laminin-5 G domain on α3β1 integrin and were from the The a sequence was from The was from The the was from and the the LG4–5 tandem domain of laminin-5 was from P. The the LG1 domain of laminin-5 M.-F. P. Nishiyama T. Keene D.R. Burgeson R. J. Biol. PubMed Scopus Google Scholar) and the integrin D. of were from of was with a of for at with to of the α3′ by and which the sequence of the laminin-5 α3 chain, an a spacer the C-terminal of CC domain, and the of LG1 of laminin-5 α3 chain sequence R. D.R. J. Biol. Chem. Full Text PDF PubMed Google was by the by J. PubMed Scopus Google Scholar) and the two of with the and and G with the The the rest of the G domain of the laminin-5 α3 chain was by The and were into the and into from to the expression the chain. fragments, were by at the of the the LG The with the and in were used to the and and and The sites were used to the sequence the and for the shorter of the and the and the were a expression two bacterial expression were which for of the two laminin chains to an The in was in the the a and the C-terminal of the laminin-5 chain D.R. Champliaud M.-F. Burgeson R.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar) was by the by and two of with the and and with the an the the a and the C-terminal of the chain J. P. Champliaud M.-F. Gerecke D. D. Burgeson R. E. J. Biochem. PubMed Scopus Google Scholar) was by by and two of with the and and with the The and chains were with a sequence into the expression to the The the and chains with an domain were by the and the sites that were to the into the bacterial expression The for the were with the and with the The for the were with the and with the were by α3′ were in and with and The the α3′ were into the to the were in the of were established by for of α3′ chains in a of α3′ cell the α3′ chains were in for processing of the chain, and were were and with and and α3′ chains were with and a After the with the α3′ chains were with an of and in the After was by the of the α3′ chains was in and of and with the expression or were at and for with were then in and and by by with for and by three for The or were and in and and After the laminin chains were were by affinity on to the and were by and of the chain was processed proteolytically with at an of in at of the α3′ chains was with at an of at the the chain was with for at at an of The was by of The chain was in the of a the of the α3 wild-type laminin-5 was from of P. E. PubMed Scopus Google Scholar). After of the three laminin-5 chains, the α3 chain was from the and with and in Hunter Biochem. PubMed Scopus Google The and were from the and by mass spectrometry. was a mass in was used with the to and a at were in of and and were to The was to a of and the was and were in the and in the cell at and of and of were with the was used mass for and mass of all was mass and by mass were mass and to α3′ and α3′ cell and to the heterotrimeric of the laminin-5CCG were The α3′ chains or the were with the and the which were at to at After with in of cell or of heterotrimeric laminin-5CCG at were to the After the α3′ chains and heterotrimeric laminin-5CCG were by by of by was at in an of α3β1 to were with the or in at After two with the and with in laminin-5CCG or its chains in and were with α3β1 integrin was in in with and a of the or in the of for at After two with α3β1 integrin was with in the and with a the integrin and an P. E. PubMed Scopus Google Scholar). of α3′ were laminin-5 α3′ chains, which the an and the C-terminal of the CC domain but in the numbers of LG the the subdomains the subdomains LG1–3, the subdomains or LG1 with the shorter to the wild-type α3 chain α3′ chains were produced and by which not and The of α3′ chains in the culture with numbers of LG subdomains for the and were to the culture of with α3′ the of in the increased indicating that were to proteolytic in the of a in and of the and chains were not in for the of the α3′ chains, and was to the wild-type α3 chain, the G domain of the was not in the sequence connecting the LG3 and proteolytic processing of the chain was by a of the and the which is a for a of and the mammalian of both the of the chains, to with an mass of in the other the processing of chain in the cell culture system in be brought to completion by in vitro of the chain by plasmin, in the processed chain, with an mass of in After proteolytic cleavage in the spacer sequence between LG3 and LG4, the LG4–5 tandem domain dissociated from the rest of the α3′ chain and After its the tandem domain be in the of the chain by with a LG4–5 domain in to be under cell culture in that other are for the of the dissociated LG4–5 tandem in the cell culture the chain in a similar to the of that are for chain processing in The α3′LG1–3 chain the LG4–5 tandem domain a was the to be the cleavage of is the spacer sequence between the LG3 and subdomains (7Tsubota Y. Mizushima H. Hirosaki T. Higashi S. Yasumitsu H. Miyazaki K. Biochem. Biophys. Res. Commun. 2000; 278: 614-620Crossref PubMed Scopus (62) Google Scholar) α3′ chains by and by the LG1 domain and the LG4–5 domain α3′ and α3′LG1–3 were by in a under and with of or of the and processed of the α3′ chains After to the were with the LG1 or with the LG4–5 tandem The was with and a of are to the of processing of the G domain in the of the LG4–5 domain. the processed of the chains or the α3′LG1–3 chain were with the LG4–5 domain was with the LG4–5 tandem domain and of the α3′ α3′ chains produced by N-linked which were by with not of the chain to the N-glycosylation the α3 chain of wild-type laminin-5 was by The was with and The and were by mass spectrometry. the the a and a N-glycosylation showed a mass of the sites for and and be for the two N-glycosylation sites at and sequence between LG3 and the LG3 mass that the of the α3 chain were to both the and the of N-glycosylation sites the wild-type α3 chain sequence of laminin-5 R. D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar), by mass spectrometry. After and of the wild-type α3 chain, its were either by mass mass with or both sites to be are by the sequence of the α3 chain R. D.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). α3′ chains of laminin-5CCG at of and LG3 are of and of and Their both and chains of laminin-5CCG were produced in a bacterial expression system proteins. The was a spacer sequence to a sequence and the C-terminal of the laminin and As the sequence and the in the and chains, In the the chain was in a of the chain was in the cell chains were from under and by affinity on a of the two chains a single not and chains with of and but and with of and of a the chain was in the chain of chain were to the by Although the chain at the between the domain and the of the chain in of the chain not the chain was and the was used sequence for the chain. of the and chains, the and were under and in an a of the two chains, a of for be However, of and the of the and chains and was to The was by an by its mass of in under was into the single and chains by The was with both the and the proving the of both chains in the of the and of the α3′ chains were under heterotrimeric laminin-5CCG The of all three laminin-5 chains the molecule was by in which a was all three chains were associated in In the of the α3′ chains were with a the and the or chains of the laminin-5CCG were with the and with a the for the chain were for the chain, a of a was In the heterotrimerization was by the with the As in and the α3′ chains in the heterotrimeric laminin-5CCG were by the a sequence the LG1 domain M.-F. P. Nishiyama T. Keene D.R. Burgeson R. J. Biol. PubMed Scopus Google Scholar). was an α3′ chain associated with a to the the nor the α3′ chains not However, the for the laminin-5CCG α3′ chains is by of the of the in the α3′ After the chain associated with the to the chain that the of the α3′ chains not the of heterotrimeric in which laminin-5CCG with α3′ chains were and with the LG4–5 domain, showed that the chain cleavage is not by the LG4–5 domain similar to the α3′LG1–3 chain, proving that proteolytic cleavage between the LG3 and subdomains of laminin-5 in the of the LG4–5 tandem domain from the rest of the laminin-5 G domain α3β1 to the with soluble α3β1 integrin were laminin-5CCG or single chains with the laminin-5CCG chains by G domain in a the soluble α3β1 integrin. binding of soluble α3β1 integrin was the single α3′ chains or the was used In all three chains in the heterotrimeric laminin-5CCG binding with the wild-type laminin-5 The integrin binding was and on the binding to The was in the binding to the affinity of α3β1 integrin the laminin-5CCG In the of α3β1 integrin showed binding not The laminin-5CCG molecule with the three subdomains LG1–3 was for α3β1 integrin binding showed a binding with that of the laminin-5CCG molecule with the chain, indicating that the α3β1 integrin binding be the three LG subdomains of the heterotrimerization of the α3′ chain with the β3′and chains is for α3β1 integrin binding to the laminin-5CCG the of proteolytic processing of the laminin-5 G domain on α3β1 integrin the laminin-5CCG with soluble α3β1 integrin of laminin-5CCG either or chains that the cleavage between the LG3 and subdomains and the of LG4–5 tandem domain from the G domain not α3β1 integrin However, the laminin-5CCG molecule the α3′LG1–3 chain with the (7Tsubota Y. Mizushima H. Hirosaki T. Higashi S. Yasumitsu H. Miyazaki K. Biochem. Biophys. Res. Commun. 2000; 278: 614-620Crossref PubMed Scopus (62) Google Scholar) showed a of affinity α3β1 integrin. of α3β1 integrin affinity laminin-5CCG with the α3′LG1–3 chain was the were in the of M. H. P. D. S. PubMed Scopus Google Scholar) The binding of α3β1 integrin to the heterotrimeric laminin-5CCG not were in the or the which for α3β1 integrin interaction with wild-type laminin-5 P. E. PubMed Scopus Google Scholar). of in α3β1 the of chains of laminin-5 on integrin the binding of soluble α3β1 integrin to the laminin-5CCG a α3′LG1–3 chain. The laminin-5CCG was by the soluble α3β1 integrin to the the form that its chains neither to nor the α3β1 integrin binding laminin-5 with α3β1 integrin by a the interaction of laminin-5 with integrin on the other cell and a system in which the binding of soluble α3β1 integrin to laminin-5 with defined G domains was to laminin-5 and its M. N. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. H. Y. Y. Yasumitsu H. K. Miyazaki K. Google Scholar) with both LG subdomains of or laminin-5 α3 chain in a bacterial expression Although a cell the LG2 or LG3 was in both which the LG domain, were used the of the LG of the heterotrimeric CC domain, which essential for α3β1 integrin binding to expression of laminin-5 was in a expression either in which α3 chain with and chains T. Mizushima H. Y. K. Miyazaki K. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), or in which were with of all three laminin-5 chains Y. K. Y. Y. Hirosaki T. T. Miyazaki K. J. Biochem. PubMed Scopus Google Scholar). However, with the laminin-5 molecule are to with cell which or other cell molecules. the were with which its G domain, the and H. Yurchenco P.D. Dev. Dyn. 2000; 218: 213-234Crossref PubMed Scopus (1049) Google Scholar, Microsc. Res. Tech. 2000; 51: PubMed Scopus Google Scholar). are to with the R. E. J. 2000; PubMed Google Scholar) in part of cell between the integrin and the of the laminin-5 chain P. J. 2000; Scholar), or and with the binding in the laminin-5 M. H. K. T. S. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of cell with laminin-5 is is processed by L.E. Stack M.S. Jones J.C.R. J. Biol. PubMed Scopus Google Scholar, S. Stack M.S. Microsc. Res. Tech. 2000; 51: PubMed Scopus Google Scholar), the and of which on the cell to laminin-5 Y. Z. Mol. Biol. Cell. PubMed Scopus Google Scholar). However, the proteolytic processing of the G domain the interaction of α3β1 integrin and other with and cell the chains of the laminin-5CCG were in and and in vitro. The α3′ chains were in and and chains, the single α3′ chain was by the into the culture of the α3′ chains was not and with other for laminin I. T. Engel J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the α3′ chain not to with the and chains to be However, the of α3′ chains in which not laminin-5 or of its chains is with its expression in which the molecule J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the α3′ chains comprising at least the three subdomains LG1–3 were at in part be of a proteolytic be by the structural model of the laminin G domain (4Timpl R. Tisi D. Talts J.F. Andac Z. Sasaki T. Hohenester E. Matrix Biol. 2000; 19: 309-317Crossref PubMed Scopus (258) Google Scholar, E. Tisi D. Talts J.F. Timpl R. Mol. Cell. 1999; 4: 783-792Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 6Tisi D. Talts J.F. Timpl R. Hohenester E. EMBO J. 2000; 19: 1432-1440Crossref PubMed Google Scholar), is that the three subdomains LG1–3 form a three-bladed propeller, from which the LG4–5 tandem domain Although the spacer sequence connecting the subdomains LG3 and is to proteolytic the three-bladed of LG1–3 to be a Further, loss of of the three LG domains the three-bladed and in and to and were and the proteolytic cleavage of the G domain a for processing of the laminin-5 G domain in S. Scott I.C. Takahara K. Koch M. Champliaud M.-F. Gerecke D.R. Keene D.R. Hudson D.L. Nishiyama T. Lee S. Greenspan D.S. Burgeson R.E. J. Biol. Chem. 2000; 275: 22728-22735Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 9Goldfinger L.E. Stack M.S. Jones J.C.R. J. Biol. PubMed Scopus Google Scholar, P. R. D.R. Greenspan D.S. J. Biol. Chem. 278: Full Text Full Text PDF PubMed Scopus Google Scholar). that processing of the chain in the cell culture was inhibited by a of to both and but not by either of the that the cleavage of the α3′ chain the G domain at least and that the chain is by plasmin, of the for G domain processing L.E. Stack M.S. Jones J.C.R. J. Biol. PubMed Scopus Google Scholar). The and chains were produced in a bacterial expression to the and chain of P. T. Engel J. J. Mol. Biol. PubMed Scopus Google Scholar), the and chains of laminin-5 showed a in with in J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the is spontaneously by the α3′ chains under to form the heterotrimeric laminin-5CCG laminin-5 is a molecule, similar to R. M. H. Timpl R. D. J. Biochem. 1990; PubMed Scopus Google Scholar, Yurchenco P.D. J. Biol. PubMed Scopus Google Scholar, P. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and R. M. H. Timpl R. D. J. Biochem. 1990; PubMed Scopus Google Scholar) and Yurchenco P.D. J. Biol. PubMed Scopus Google Scholar) that the C-terminal of all three chains are to form a heterotrimeric comprising the CC and G Further, all three chains of were for cell by integrin. that the C-terminal from the CC domain are to the of the coiled-coil domain is a for α3β1 integrin an that the loss of cell of laminin-5 P. R. M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the CC domain the the LG1–3 domain, the α3β1 integrin binding the of the CC domain for α3β1 integrin binding be by a interaction of domain with the integrin, that α3β1 integrin a binding comprising regions of both the G domain and the CC domain. with laminin-5 shorter regions of the CC domains be to between heterotrimerization nor α3β1 integrin binding was affected by the N-glycosylation of the α3 chain in of the subdomains LG1 and LG2 two of the of the laminin α3 and chains and of the to the of domains (5Hohenester E. Tisi D. Talts J.F. Timpl R. Mol. Cell. 1999; 4: 783-792Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 6Tisi D. Talts J.F. Timpl R. Hohenester E. EMBO J. 2000; 19: 1432-1440Crossref PubMed Google Scholar) that the two N-linked chains are on face of the two α3 the of N-linked chains, large of the two subdomains LG1 and LG2 not be for the α3β1 integrin. be for the α3β1 integrin to both faces of the LG3 domain, which N-glycosylation In to other cell with laminin-5 M. H. K. T. S. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that the interaction of α3β1 integrin with laminin-5 is by Although not in laminin-5 to be in its chain or in its α3β1 integrin the chains laminin-5 from proteolytic not and its proteolytic processing in its α3β1 integrin binding that the binding affinity of α3β1 integrin to laminin-5 is by the proteolytic processing of the G domain. of the α3′ chain by plasmin, a for the in processing of to a of LG4–5 domain from the rest of the α3′ chain. However, is to the dissociated domain, other be for in and loss of the LG4–5 tandem domain not α3β1 integrin binding to In the laminin-5CCG molecule the α3′LG1–3 chain with the (7Tsubota Y. Mizushima H. Hirosaki T. Higashi S. Yasumitsu H. Miyazaki K. Biochem. Biophys. Res. Commun. 2000; 278: 614-620Crossref PubMed Scopus (62) Google Scholar) was by α3β1 integrin with a in laminin-5 not for the of laminin-5 processing in the of the other and its (7Tsubota Y. Mizushima H. Hirosaki T. Higashi S. Yasumitsu H. Miyazaki K. Biochem. Biophys. Res. Commun. 2000; 278: 614-620Crossref PubMed Scopus (62) Google Scholar, 8Amano S. Scott I.C. Takahara K. Koch M. Champliaud M.-F. Gerecke D.R. Keene D.R. Hudson D.L. Nishiyama T. Lee S. Greenspan D.S. Burgeson R.E. J. Biol. Chem. 2000; 275: 22728-22735Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 9Goldfinger L.E. Stack M.S. Jones J.C.R. J. Biol. PubMed Scopus Google Scholar, P. R. D.R. Greenspan D.S. J. Biol. Chem. 278: Full Text Full Text PDF PubMed Scopus Google Scholar) in the in the α3β1 integrin affinity for into the of After at the and laminin-5 with a G domain Biol. 2000; PubMed Scopus Google Scholar). proteolytic processing and the loss of the LG4–5 tandem domain Biol. 2000; PubMed Scopus Google Scholar) to an in binding affinity of α3β1 integrin, integrin and into the L.E. Stack M.S. Jones J.C.R. J. Biol. PubMed Scopus Google Scholar, L.E. S. Jones J.C.R. J. 1999; PubMed Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In by characterized laminin-5CCG with defined numbers of LG subdomains, that heterotrimerization of the three laminin-5 chains is a for α3β1 integrin the subdomains LG1–3 of the α3 chain the α3β1 integrin binding that of the G domain not to α3β1 integrin and location α3β1 integrin to LG1 and but not to and that the proteolytic processing the G domain α3β1 integrin binding The of interaction on the in α3β1 cell and which are by laminin-5 and a in and P. for the the LG4–5 domain, for the laminin-5 chain and M. for the