Self-assembly of Laminin Isoforms
1997; Elsevier BV; Volume: 272; Issue: 50 Linguagem: Inglês
10.1074/jbc.272.50.31525
ISSN1083-351X
AutoresYi‐Shan Cheng, Marie-France Champliaud, Robert E. Burgeson, M. Peter Marinkovich, Peter D. Yurchenco,
Tópico(s)RNA Research and Splicing
ResumoThe α, β, and γ subunits of basement membrane laminins can combine into different heterotrimeric molecules with either three full short arms (e.g. laminins-1–4), or molecules containing one (laminins-6–9) or more (laminin-5) short arm truncations. Laminin-1 (α1β1γ1), self-assembles through a calcium-dependent thermal gelation requiring binding interactions between N-terminal short arm domains, forming a meshwork polymer thought to contribute to basement membrane architecture (Yurchenco, P. D., and Cheng, Y. S. (1993) J. Biol. Chem. 268, 17286–17299). However, it has been unclear whether other isoforms share this property, and if so, which ones. To begin to address this, we evaluated laminin-2 (α2β1γ1), laminin-4 (α2β2γ1), laminin-5 (α3Aβ3γ2), and laminin-6 (α3Aβ1γ1). The first two isoforms were found to self-aggregate in a concentration- and temperature-dependent manner and a close self-assembly relationship among laminins-1, -2, and -4 were demonstrated by: (a) polymerization of all three proteins was inhibited by EDTA and laminin-1 short arm fragments, (b) polymerization of laminin-1 was inhibited by fragments of laminins-2 and -4, (c) laminin-2 and, to a lesser degree, laminin-4, even well below their own critical concentration, co-aggregated with laminin-1, evidence for co-polymerization. Laminin-5, on the other hand, neither polymerized nor co-polymerized with laminin-1. Laminin-6 failed to co-aggregate with laminin-1 at all concentrations evaluated, evidence for a lack of a related self-assembly activity. The data support the hypothesis that all three short arms are required for self-assembly and suggest that the short arm domain structure of laminin isoforms affect their architecture-forming properties in basement membranes. The α, β, and γ subunits of basement membrane laminins can combine into different heterotrimeric molecules with either three full short arms (e.g. laminins-1–4), or molecules containing one (laminins-6–9) or more (laminin-5) short arm truncations. Laminin-1 (α1β1γ1), self-assembles through a calcium-dependent thermal gelation requiring binding interactions between N-terminal short arm domains, forming a meshwork polymer thought to contribute to basement membrane architecture (Yurchenco, P. D., and Cheng, Y. S. (1993) J. Biol. Chem. 268, 17286–17299). However, it has been unclear whether other isoforms share this property, and if so, which ones. To begin to address this, we evaluated laminin-2 (α2β1γ1), laminin-4 (α2β2γ1), laminin-5 (α3Aβ3γ2), and laminin-6 (α3Aβ1γ1). The first two isoforms were found to self-aggregate in a concentration- and temperature-dependent manner and a close self-assembly relationship among laminins-1, -2, and -4 were demonstrated by: (a) polymerization of all three proteins was inhibited by EDTA and laminin-1 short arm fragments, (b) polymerization of laminin-1 was inhibited by fragments of laminins-2 and -4, (c) laminin-2 and, to a lesser degree, laminin-4, even well below their own critical concentration, co-aggregated with laminin-1, evidence for co-polymerization. Laminin-5, on the other hand, neither polymerized nor co-polymerized with laminin-1. Laminin-6 failed to co-aggregate with laminin-1 at all concentrations evaluated, evidence for a lack of a related self-assembly activity. The data support the hypothesis that all three short arms are required for self-assembly and suggest that the short arm domain structure of laminin isoforms affect their architecture-forming properties in basement membranes. Basement membranes are animal extracellular matrices that appear early in development and that are present in nearly all tissues. They act as supportive architecture for communities of cells, providing differentiation and migration information that are transmitted through cell receptors. These matrices consist of one or more members of the laminin family, one or more members of the type IV collagen family, entactin/nidogen, and smaller amounts of other components such as perlecan (heparan sulfate proteoglycan). From studies of the extracellular matrix components originally discovered in the EHS 1The abbreviations used are: EHS, Engelbreth-Holm-Swarm; HMB, p-hydroxymecuribenzoic acid; PMSF, phenylmethylsulfonyl fluoride; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin.1The abbreviations used are: EHS, Engelbreth-Holm-Swarm; HMB, p-hydroxymecuribenzoic acid; PMSF, phenylmethylsulfonyl fluoride; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin. matrix and elsewhere, a model for the assembly and architecture of an idealized basement membrane scaffolding has been proposed (1Yurchenco P.D. Yurchenco P.D. Birk D.E. Mecham R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 351-388Crossref Google Scholar, 2Timpl R. Brown J.C. Bioessays. 1996; 18: 123-132Crossref PubMed Scopus (576) Google Scholar). Central to this model are the binding interactions of "classical" laminin, type IV collagen, and entactin/nidogen in which three groups of interactions play key roles: type IV collagen polymerization, laminin polymerization, and the stabilizing connections of entactin/nidogen. The type IV collagen network consists of triple helical monomers joined together through lateral, N-terminal, and C-terminal interactions (3Yurchenco P.D. Furthmayr H. Biochemistry. 1984; 23: 1839-1850Crossref PubMed Scopus (245) Google Scholar,4Yurchenco P.D. Ruben G.C. J. Cell Biol. 1987; 105: 2559-2568Crossref PubMed Scopus (251) Google Scholar). The N-terminal (7S) and C-terminal (NC-1) bonds become stabilized by reducible and irreducible covalent cross-links. The laminin polymer is self-assembled from monomers in a calcium-dependent, reversible, and cooperative heat-gelation with a measured critical concentration of assembly of 70–140 nm (5Yurchenco P.D. Cheng Y.S. Schittny J.C. J. Biol. Chem. 1990; 265: 3981-3991Abstract Full Text PDF PubMed Google Scholar, 6Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (223) Google Scholar). It has been found, based on biochemical and electron microscopic studies conducted with laminin-1 and laminin-1 fragments, that all three short arms are involved in polymerization and furthermore all three appear to be essential for assembly (7Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar, 8Colognato-Pyke H. O'Rear J.J. Yamada Y. Carbonetto S. Cheng Y.S. Yurchenco P.D. J. Biol. Chem. 1995; 270: 9398-9406Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Entactin/nidogen can form a stable noncovalent bridge between laminin and type IV collagen and also bind to perlecan and the fibulins (2Timpl R. Brown J.C. Bioessays. 1996; 18: 123-132Crossref PubMed Scopus (576) Google Scholar). The resulting architecture, as proposed, is that of a double polymer network bearing stabilizing entactin/nidogen cross-links.However, the different laminin and type IV collagen family isoforms recently identified may provide a molecular basis for distinct architectural variations that in turn create functional diversity. Naturally occurring and targeted mutations in the various chains of these two families illustrate functional differences. For example, mutations in the chains of laminin-5 cause skin blistering, with a loss of epidermal-dermal anchorage at hemidesmosome sites in diverse tissues. A null mutation in the β2 chain of laminin, present in glomerulus, leads to profound renal failure despite apparent compensation by the β1 chain. Mutations that affect the α2 chain of laminin-2 can cause a muscular dystrophy despite partial replacement of this chain by the α1 chain (9Xu H. Christmas P. Wu X.R. Wewer U.M. Engvall E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5572-5576Crossref PubMed Scopus (239) Google Scholar, 10Xu H. Wu X.R. Wewer U.M. Engvall E. Nat. Genet. 1994; 8: 297-302Crossref PubMed Scopus (321) Google Scholar, 11Sunada Y. Bernier S.M. Utani A. Yamada Y. Campbell K.P. Hum. Mol. Genet. 1995; 4: 1055-1061Crossref PubMed Scopus (156) Google Scholar). Two questions arise. First, how universal is the laminin self-assembly model? Second, if there are differences in self-assembly properties among different members of the laminin and collagen-IV families, what are these differences and can they explain basement membrane structural and functional heterogeneity? In the case of the laminins, we already recognize five α, three β, and two γ subunits that can combine into at least eleven heterotrimeric molecules (12Burgeson R.E. Chiquet M. Deutzmann R. Ekblom P. Engel J. Kleinman H. Martin G.R. Meneguzzi G. Paulsson M. Sanes J. Timpl R. Tryggvason K. Yamada Y. Yurchenco P.D. Matrix Biol. 1994; 14: 209-211Crossref PubMed Scopus (694) Google Scholar, 13Ryan M.C. Tizard R. VanDevanter D.R. Carter W.G. J. Biol. Chem. 1994; 269: 22779-22787Abstract Full Text PDF PubMed Google Scholar, 14Iivanainen A. Sainio K. Sariola H. Tryggvason K. FEBS Lett. 1995; 365: 183-188Crossref PubMed Scopus (115) Google Scholar, 15Miner 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, 16Miner J.H. Lewis R.M. Sanes J.R. J. Biol. Chem. 1995; 270: 28523-28526Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). At the level of domain structure, one can define three groups: laminins that contain a complete complement of domains (laminins-1, -2, -3, -4, -10, -11), laminin which lacks domains in all three short arms (laminin-5 containing the common α3A chain splice variant), and laminins which lack an α chain short arm but which retain full-sized β and γ chains (laminins-6, -7, -8, and -9). The three-short arm hypothesis for laminin assembly predicts that only the "full-sized" laminins that have three short arms would be able to readily polymerize.In this study we have evaluated the capacity of two full-sized laminin isoforms, a laminin isoform bearing extensive short arm truncations, and a laminin isoform bearing a truncation of a single short arm, to self-assemble. In developing approaches to study some laminin isoforms, we needed to overcome the problem that unlike laminin-1, the other forms of laminin can be obtained in only modest (laminins-2, -4, and -5) or only very small (laminin-6) amounts. A solution to this difficulty was accomplished by developing a specific laminin co-polymerization assay which exploits the abundance of laminin-1, using it to drive the specific polymerization of other laminin isoforms. From the analysis we found that only full-size laminins were capable of laminin-1 type self-assembly.DISCUSSIONEarlier studies have shown that laminin-1 reversibly self-assembles into a lattice-like polymer and that this polymer is present in the basement membranes of embryonal carcinoma cells, EHS tumor, and mouse placenta (1Yurchenco P.D. Yurchenco P.D. Birk D.E. Mecham R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 351-388Crossref Google Scholar, 6Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (223) Google Scholar). The assembly process is a cooperative heat-gelation in which divalent cation, in particular calcium, is required. Given evidence that type IV collagen separately polymerizes using N-terminal, C-terminal, and lateral associations into a covalently stabilized network (3Yurchenco P.D. Furthmayr H. Biochemistry. 1984; 23: 1839-1850Crossref PubMed Scopus (245) Google Scholar, 4Yurchenco P.D. Ruben G.C. J. Cell Biol. 1987; 105: 2559-2568Crossref PubMed Scopus (251) Google Scholar), and that entactin/nidogen binds firmly to both the laminin γ1 chain and to the triple helix of type IV collagen (26Mayer U. Timpl R. Yurchenco P.D. Birk D.E. Mechan R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 389-416Crossref Google Scholar), a model for the assembly and structure of an idealized basement membrane has been described (reviewed in Refs. 1Yurchenco P.D. Yurchenco P.D. Birk D.E. Mecham R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 351-388Crossref Google Scholar and26Mayer U. Timpl R. Yurchenco P.D. Birk D.E. Mechan R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 389-416Crossref Google Scholar). The model, however, is limited in that it only considers the classical basement membrane components, i.e. those first identified in the EHS tumors and several cultured cell lines. We have now characterized four laminin isoforms with respect to their ability to form a network polymer in a manner similar to laminin-1.Laminin-2 and laminin-4, both possessing three full short arms, were found to polymerize in a time-, concentration- and temperature-dependent manner. Self-assembly was cooperative with an apparent critical concentration of 0.2 μm, about twice the value (0.07 - 0.14 μm) observed for laminin-1. This self-assembly appeared to be closely related to that found for laminin-1 for several reasons. First, all polymerizations were inhibited by EDTA and N-terminal laminin-1 fragments E4 and E1′, the latter two previously shown to be specific inhibitors of laminin-1 self-assembly (6Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (223) Google Scholar, 7Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). Second, laminin-2/4 fragments inhibited laminin-1 polymerization. Third, when laminin-1 was maintained above its critical concentration, and one or both of the laminin isoforms were maintained above or below their critical concentration, co-aggregation was observed. This co-aggregation occurred with isolated laminin-2 and laminin-4, but was better between laminin-1 and -2 compared with laminin-1 and -4. A possible explanation for the difference is that laminin-1 shares two chains in common with laminin-2 but only one chain in common with laminin-4. Thus the three laminins co-polymerize and, when expressed together, can form a composite network using similar bonds between the different isoforms. Since laminin-2 and laminin-4 have α2β1γ1 and α2β2γ1 chain compositions, respectively, one can deduce that laminin-3, which has an α1β2γ1 chain composition, also polymerizes. In contrast to the full-sized isoforms, laminin-5 (α3Aβ3γ2), a rod-like molecule whose short arms lack most of their domains, was found not to polymerize at concentrations at or below 1 mg/ml, nor to co-polymerize with laminin-1. Furthermore, laminin-6 (α3Aβ1γ1), a Y-shaped laminin with two short arms, did not co-polymerize with laminin-1 and therefore, given the association between polymerization and co-polymerization, probably does not self-assemble in a manner similar to laminin-1. It is even possible, although it could not be evaluated in this study, that at higher concentrations of laminin-6 one would observe an inhibitory effect of the isoform on laminin-1, in a manner analogous to the double short arm structure E1′ (7Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar).The self-assembly behavior of these isoforms fits a prediction of a three-arm interaction model which holds that the polymer is formed by the joining of the ends of the three short arms to create a lattice-like array (6Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (223) Google Scholar, 7Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). The failure of laminin-5, which lacks most of the short arm domains (although does possess one domain 6 homologue in the β3 chain), to self-assemble is consistent with the above hypothesis. A more exacting test of the model, however, was provided by laminin-6. This laminin, like laminins-7, -8, and -9, and unlike laminins-1, -2, -4, has two rather than three full short arms and would be expected either not to polymerize/co-polymerize or to polymerize/co-polymerize poorly. The inability of laminin-6 to co-polymerize with laminin-1 argues that the three-arm assembly hypothesis is correct. Note that this non-co-polymerizing laminin shares two short arms (β1, γ1) in common with laminin-1, in contrast to the co-polymerizing laminin-2, which also shares two short arms (β1, γ1), and even the co-polymerizing laminin-4, which shares only one short arm (γ1) in common with laminin-1. Thus presence of an α chain short arm appears to be critical for laminin self-assembly. Recent studies 4H. Colognato and P. D. Yurchenco, unpublished results. reveal that laminin-2 isolated from the dystrophic dy 2Jmouse, which only lacks α2-domain VI, co-polymerizes poorly with laminin-1. This domain is of course absent in laminin-6 and suggests that loss of only the N-terminal globule of the α chain adversely affects self-assembly.The co-polymerization assay has proved to be a useful tool in the current study to evaluate the self-assembly properties of other isoforms in amounts below their critical concentrations. The property of co-polymerization furthermore suggests that where a given basement membrane contains a mixture of two or more polymerizing laminins, these laminins will form a cooperative network unless specifically prevented by other immobilizing bonds. Laminins-1, -2, and -4 share this property (Fig. 12). Laminin-5 can form a disulfide-stabilized bond to laminin-6 or laminin-7 (27Marinkovich M.P. Lunstrum G.P. Keene D.R. Burgeson R.E. J. Cell Biol. 1992; 119: 695-703Crossref PubMed Scopus (235) Google Scholar, 28Champliaud M.F. Lunstrum G.P. Rousselle P. Nishiyama T. Keene D.R. Burgeson R.E. J. Cell Biol. 1996; 132: 1189-1198Crossref PubMed Scopus (226) Google Scholar) but cannot bind to entactin/nidogen (29Mayer U. Poschl E. Gerecke D.R. Wagman D.W. Burgeson R.E. Timpl R. FEBS Lett. 1995; 365: 129-132Crossref PubMed Scopus (69) Google Scholar). Laminins-6 and -7, on the other hand, can bind to entactin/nidogen (and indirectly to the type IV collagen network), since they possess a γ1 chain. It appears that laminin-5 forms a cable-like structure connecting the hemidesmosome of the epidermis with the underlying dermis through type VII collagen and links this cabling through the rest of the basement membrane through laminins-6 and -7 (30Rousselle P. Keene D.R. Ruggiero F. Champliaud M.F. van der Rest M. Burgeson R.E. J. Cell Biol. 1997; 138: 719-728Crossref PubMed Scopus (222) Google Scholar). Finally, it is possible that laminins-5, -6, and -7 have other as yet undiscovered matrix binding interactions. It is conceivable that laminins lacking critical short arms domain could form large oligomeric or polymeric complexes using a completely different set of bonds compared with laminin-1. However, it is difficult to imagine how this would occur given the homology of the remaining short arm domains with those present in laminin-1 that are found not to participate in any detectable self-assembly. Basement membranes are animal extracellular matrices that appear early in development and that are present in nearly all tissues. They act as supportive architecture for communities of cells, providing differentiation and migration information that are transmitted through cell receptors. These matrices consist of one or more members of the laminin family, one or more members of the type IV collagen family, entactin/nidogen, and smaller amounts of other components such as perlecan (heparan sulfate proteoglycan). From studies of the extracellular matrix components originally discovered in the EHS 1The abbreviations used are: EHS, Engelbreth-Holm-Swarm; HMB, p-hydroxymecuribenzoic acid; PMSF, phenylmethylsulfonyl fluoride; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin.1The abbreviations used are: EHS, Engelbreth-Holm-Swarm; HMB, p-hydroxymecuribenzoic acid; PMSF, phenylmethylsulfonyl fluoride; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin. matrix and elsewhere, a model for the assembly and architecture of an idealized basement membrane scaffolding has been proposed (1Yurchenco P.D. Yurchenco P.D. Birk D.E. Mecham R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 351-388Crossref Google Scholar, 2Timpl R. Brown J.C. Bioessays. 1996; 18: 123-132Crossref PubMed Scopus (576) Google Scholar). Central to this model are the binding interactions of "classical" laminin, type IV collagen, and entactin/nidogen in which three groups of interactions play key roles: type IV collagen polymerization, laminin polymerization, and the stabilizing connections of entactin/nidogen. The type IV collagen network consists of triple helical monomers joined together through lateral, N-terminal, and C-terminal interactions (3Yurchenco P.D. Furthmayr H. Biochemistry. 1984; 23: 1839-1850Crossref PubMed Scopus (245) Google Scholar,4Yurchenco P.D. Ruben G.C. J. Cell Biol. 1987; 105: 2559-2568Crossref PubMed Scopus (251) Google Scholar). The N-terminal (7S) and C-terminal (NC-1) bonds become stabilized by reducible and irreducible covalent cross-links. The laminin polymer is self-assembled from monomers in a calcium-dependent, reversible, and cooperative heat-gelation with a measured critical concentration of assembly of 70–140 nm (5Yurchenco P.D. Cheng Y.S. Schittny J.C. J. Biol. Chem. 1990; 265: 3981-3991Abstract Full Text PDF PubMed Google Scholar, 6Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (223) Google Scholar). It has been found, based on biochemical and electron microscopic studies conducted with laminin-1 and laminin-1 fragments, that all three short arms are involved in polymerization and furthermore all three appear to be essential for assembly (7Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar, 8Colognato-Pyke H. O'Rear J.J. Yamada Y. Carbonetto S. Cheng Y.S. Yurchenco P.D. J. Biol. Chem. 1995; 270: 9398-9406Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Entactin/nidogen can form a stable noncovalent bridge between laminin and type IV collagen and also bind to perlecan and the fibulins (2Timpl R. Brown J.C. Bioessays. 1996; 18: 123-132Crossref PubMed Scopus (576) Google Scholar). The resulting architecture, as proposed, is that of a double polymer network bearing stabilizing entactin/nidogen cross-links. However, the different laminin and type IV collagen family isoforms recently identified may provide a molecular basis for distinct architectural variations that in turn create functional diversity. Naturally occurring and targeted mutations in the various chains of these two families illustrate functional differences. For example, mutations in the chains of laminin-5 cause skin blistering, with a loss of epidermal-dermal anchorage at hemidesmosome sites in diverse tissues. A null mutation in the β2 chain of laminin, present in glomerulus, leads to profound renal failure despite apparent compensation by the β1 chain. Mutations that affect the α2 chain of laminin-2 can cause a muscular dystrophy despite partial replacement of this chain by the α1 chain (9Xu H. Christmas P. Wu X.R. Wewer U.M. Engvall E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5572-5576Crossref PubMed Scopus (239) Google Scholar, 10Xu H. Wu X.R. Wewer U.M. Engvall E. Nat. Genet. 1994; 8: 297-302Crossref PubMed Scopus (321) Google Scholar, 11Sunada Y. Bernier S.M. Utani A. Yamada Y. Campbell K.P. Hum. Mol. Genet. 1995; 4: 1055-1061Crossref PubMed Scopus (156) Google Scholar). Two questions arise. First, how universal is the laminin self-assembly model? Second, if there are differences in self-assembly properties among different members of the laminin and collagen-IV families, what are these differences and can they explain basement membrane structural and functional heterogeneity? In the case of the laminins, we already recognize five α, three β, and two γ subunits that can combine into at least eleven heterotrimeric molecules (12Burgeson R.E. Chiquet M. Deutzmann R. Ekblom P. Engel J. Kleinman H. Martin G.R. Meneguzzi G. Paulsson M. Sanes J. Timpl R. Tryggvason K. Yamada Y. Yurchenco P.D. Matrix Biol. 1994; 14: 209-211Crossref PubMed Scopus (694) Google Scholar, 13Ryan M.C. Tizard R. VanDevanter D.R. Carter W.G. J. Biol. Chem. 1994; 269: 22779-22787Abstract Full Text PDF PubMed Google Scholar, 14Iivanainen A. Sainio K. Sariola H. Tryggvason K. FEBS Lett. 1995; 365: 183-188Crossref PubMed Scopus (115) Google Scholar, 15Miner 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, 16Miner J.H. Lewis R.M. Sanes J.R. J. Biol. Chem. 1995; 270: 28523-28526Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). At the level of domain structure, one can define three groups: laminins that contain a complete complement of domains (laminins-1, -2, -3, -4, -10, -11), laminin which lacks domains in all three short arms (laminin-5 containing the common α3A chain splice variant), and laminins which lack an α chain short arm but which retain full-sized β and γ chains (laminins-6, -7, -8, and -9). The three-short arm hypothesis for laminin assembly predicts that only the "full-sized" laminins that have three short arms would be able to readily polymerize. In this study we have evaluated the capacity of two full-sized laminin isoforms, a laminin isoform bearing extensive short arm truncations, and a laminin isoform bearing a truncation of a single short arm, to self-assemble. In developing approaches to study some laminin isoforms, we needed to overcome the problem that unlike laminin-1, the other forms of laminin can be obtained in only modest (laminins-2, -4, and -5) or only very small (laminin-6) amounts. A solution to this difficulty was accomplished by developing a specific laminin co-polymerization assay which exploits the abundance of laminin-1, using it to drive the specific polymerization of other laminin isoforms. From the analysis we found that only full-size laminins were capable of laminin-1 type self-assembly. DISCUSSIONEarlier studies have shown that laminin-1 reversibly self-assembles into a lattice-like polymer and that this polymer is present in the basement membranes of embryonal carcinoma cells, EHS tumor, and mouse placenta (1Yurchenco P.D. Yurchenco P.D. Birk D.E. Mecham R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 351-388Crossref Google Scholar, 6Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (223) Google Scholar). The assembly process is a cooperative heat-gelation in which divalent cation, in particular calcium, is required. Given evidence that type IV collagen separately polymerizes using N-terminal, C-terminal, and lateral associations into a covalently stabilized network (3Yurchenco P.D. Furthmayr H. Biochemistry. 1984; 23: 1839-1850Crossref PubMed Scopus (245) Google Scholar, 4Yurchenco P.D. Ruben G.C. J. Cell Biol. 1987; 105: 2559-2568Crossref PubMed Scopus (251) Google Scholar), and that entactin/nidogen binds firmly to both the laminin γ1 chain and to the triple helix of type IV collagen (26Mayer U. Timpl R. Yurchenco P.D. Birk D.E. Mechan R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 389-416Crossref Google Scholar), a model for the assembly and structure of an idealized basement membrane has been described (reviewed in Refs. 1Yurchenco P.D. Yurchenco P.D. Birk D.E. Mecham R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 351-388Crossref Google Scholar and26Mayer U. Timpl R. Yurchenco P.D. Birk D.E. Mechan R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 389-416Crossref Google Scholar). The model, however, is limited in that it only considers the classical basement membrane components, i.e. those first identified in the EHS tumors and several cultured cell lines. We have now characterized four laminin isoforms with respect to their ability to form a network polymer in a manner similar to laminin-1.Laminin-2 and laminin-4, both possessing three full short arms, were found to polymerize in a time-, concentration- and temperature-dependent manner. Self-assembly was cooperative with an apparent critical concentration of 0.2 μm, about twice the value (0.07 - 0.14 μm) observed for laminin-1. This self-assembly appeared to be closely related to that found for laminin-1 for several reasons. First, all polymerizations were inhibited by EDTA and N-terminal laminin-1 fragments E4 and E1′, the latter two previously shown to be specific inhibitors of laminin-1 self-assembly (6Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (223) Google Scholar, 7Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). Second, laminin-2/4 fragments inhibited laminin-1 polymerization. Third, when laminin-1 was maintained above its critical concentration, and one or both of the laminin isoforms were maintained above or below their critical concentration, co-aggregation was observed. This co-aggregation occurred with isolated laminin-2 and laminin-4, but was better between laminin-1 and -2 compared with laminin-1 and -4. A possible explanation for the difference is that laminin-1 shares two chains in common with laminin-2 but only one chain in common with laminin-4. Thus the three laminins co-polymerize and, when expressed together, can form a composite network using similar bonds between the different isoforms. Since laminin-2 and laminin-4 have α2β1γ1 and α2β2γ1 chain compositions, respectively, one can deduce that laminin-3, which has an α1β2γ1 chain composition, also polymerizes. In contrast to the full-sized isoforms, laminin-5 (α3Aβ3γ2), a rod-like molecule whose short arms lack most of their domains, was found not to polymerize at concentrations at or below 1 mg/ml, nor to co-polymerize with laminin-1. Furthermore, laminin-6 (α3Aβ1γ1), a Y-shaped laminin with two short arms, did not co-polymerize with laminin-1 and therefore, given the association between polymerization and co-polymerization, probably does not self-assemble in a manner similar to laminin-1. It is even possible, although it could not be evaluated in this study, that at higher concentrations of laminin-6 one would observe an inhibitory effect of the isoform on laminin-1, in a manner analogous to the double short arm structure E1′ (7Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar).The self-assembly behavior of these isoforms fits a prediction of a three-arm interaction model which holds that the polymer is formed by the joining of the ends of the three short arms to create a lattice-like array (6Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (223) Google Scholar, 7Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). The failure of laminin-5, which lacks most of the short arm domains (although does possess one domain 6 homologue in the β3 chain), to self-assemble is consistent with the above hypothesis. A more exacting test of the model, however, was provided by laminin-6. This laminin, like laminins-7, -8, and -9, and unlike laminins-1, -2, -4, has two rather than three full short arms and would be expected either not to polymerize/co-polymerize or to polymerize/co-polymerize poorly. The inability of laminin-6 to co-polymerize with laminin-1 argues that the three-arm assembly hypothesis is correct. Note that this non-co-polymerizing laminin shares two short arms (β1, γ1) in common with laminin-1, in contrast to the co-polymerizing laminin-2, which also shares two short arms (β1, γ1), and even the co-polymerizing laminin-4, which shares only one short arm (γ1) in common with laminin-1. Thus presence of an α chain short arm appears to be critical for laminin self-assembly. Recent studies 4H. Colognato and P. D. Yurchenco, unpublished results. reveal that laminin-2 isolated from the dystrophic dy 2Jmouse, which only lacks α2-domain VI, co-polymerizes poorly with laminin-1. This domain is of course absent in laminin-6 and suggests that loss of only the N-terminal globule of the α chain adversely affects self-assembly.The co-polymerization assay has proved to be a useful tool in the current study to evaluate the self-assembly properties of other isoforms in amounts below their critical concentrations. The property of co-polymerization furthermore suggests that where a given basement membrane contains a mixture of two or more polymerizing laminins, these laminins will form a cooperative network unless specifically prevented by other immobilizing bonds. Laminins-1, -2, and -4 share this property (Fig. 12). Laminin-5 can form a disulfide-stabilized bond to laminin-6 or laminin-7 (27Marinkovich M.P. Lunstrum G.P. Keene D.R. Burgeson R.E. J. Cell Biol. 1992; 119: 695-703Crossref PubMed Scopus (235) Google Scholar, 28Champliaud M.F. Lunstrum G.P. Rousselle P. Nishiyama T. Keene D.R. Burgeson R.E. J. Cell Biol. 1996; 132: 1189-1198Crossref PubMed Scopus (226) Google Scholar) but cannot bind to entactin/nidogen (29Mayer U. Poschl E. Gerecke D.R. Wagman D.W. Burgeson R.E. Timpl R. FEBS Lett. 1995; 365: 129-132Crossref PubMed Scopus (69) Google Scholar). Laminins-6 and -7, on the other hand, can bind to entactin/nidogen (and indirectly to the type IV collagen network), since they possess a γ1 chain. It appears that laminin-5 forms a cable-like structure connecting the hemidesmosome of the epidermis with the underlying dermis through type VII collagen and links this cabling through the rest of the basement membrane through laminins-6 and -7 (30Rousselle P. Keene D.R. Ruggiero F. Champliaud M.F. van der Rest M. Burgeson R.E. J. Cell Biol. 1997; 138: 719-728Crossref PubMed Scopus (222) Google Scholar). Finally, it is possible that laminins-5, -6, and -7 have other as yet undiscovered matrix binding interactions. It is conceivable that laminins lacking critical short arms domain could form large oligomeric or polymeric complexes using a completely different set of bonds compared with laminin-1. However, it is difficult to imagine how this would occur given the homology of the remaining short arm domains with those present in laminin-1 that are found not to participate in any detectable self-assembly. Earlier studies have shown that laminin-1 reversibly self-assembles into a lattice-like polymer and that this polymer is present in the basement membranes of embryonal carcinoma cells, EHS tumor, and mouse placenta (1Yurchenco P.D. Yurchenco P.D. Birk D.E. Mecham R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 351-388Crossref Google Scholar, 6Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (223) Google Scholar). The assembly process is a cooperative heat-gelation in which divalent cation, in particular calcium, is required. Given evidence that type IV collagen separately polymerizes using N-terminal, C-terminal, and lateral associations into a covalently stabilized network (3Yurchenco P.D. Furthmayr H. Biochemistry. 1984; 23: 1839-1850Crossref PubMed Scopus (245) Google Scholar, 4Yurchenco P.D. Ruben G.C. J. Cell Biol. 1987; 105: 2559-2568Crossref PubMed Scopus (251) Google Scholar), and that entactin/nidogen binds firmly to both the laminin γ1 chain and to the triple helix of type IV collagen (26Mayer U. Timpl R. Yurchenco P.D. Birk D.E. Mechan R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 389-416Crossref Google Scholar), a model for the assembly and structure of an idealized basement membrane has been described (reviewed in Refs. 1Yurchenco P.D. Yurchenco P.D. Birk D.E. Mecham R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 351-388Crossref Google Scholar and26Mayer U. Timpl R. Yurchenco P.D. Birk D.E. Mechan R.P. Extracellular Matrix Assembly and Structure. Academic Press, New York1994: 389-416Crossref Google Scholar). The model, however, is limited in that it only considers the classical basement membrane components, i.e. those first identified in the EHS tumors and several cultured cell lines. We have now characterized four laminin isoforms with respect to their ability to form a network polymer in a manner similar to laminin-1. Laminin-2 and laminin-4, both possessing three full short arms, were found to polymerize in a time-, concentration- and temperature-dependent manner. Self-assembly was cooperative with an apparent critical concentration of 0.2 μm, about twice the value (0.07 - 0.14 μm) observed for laminin-1. This self-assembly appeared to be closely related to that found for laminin-1 for several reasons. First, all polymerizations were inhibited by EDTA and N-terminal laminin-1 fragments E4 and E1′, the latter two previously shown to be specific inhibitors of laminin-1 self-assembly (6Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (223) Google Scholar, 7Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). Second, laminin-2/4 fragments inhibited laminin-1 polymerization. Third, when laminin-1 was maintained above its critical concentration, and one or both of the laminin isoforms were maintained above or below their critical concentration, co-aggregation was observed. This co-aggregation occurred with isolated laminin-2 and laminin-4, but was better between laminin-1 and -2 compared with laminin-1 and -4. A possible explanation for the difference is that laminin-1 shares two chains in common with laminin-2 but only one chain in common with laminin-4. Thus the three laminins co-polymerize and, when expressed together, can form a composite network using similar bonds between the different isoforms. Since laminin-2 and laminin-4 have α2β1γ1 and α2β2γ1 chain compositions, respectively, one can deduce that laminin-3, which has an α1β2γ1 chain composition, also polymerizes. In contrast to the full-sized isoforms, laminin-5 (α3Aβ3γ2), a rod-like molecule whose short arms lack most of their domains, was found not to polymerize at concentrations at or below 1 mg/ml, nor to co-polymerize with laminin-1. Furthermore, laminin-6 (α3Aβ1γ1), a Y-shaped laminin with two short arms, did not co-polymerize with laminin-1 and therefore, given the association between polymerization and co-polymerization, probably does not self-assemble in a manner similar to laminin-1. It is even possible, although it could not be evaluated in this study, that at higher concentrations of laminin-6 one would observe an inhibitory effect of the isoform on laminin-1, in a manner analogous to the double short arm structure E1′ (7Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). The self-assembly behavior of these isoforms fits a prediction of a three-arm interaction model which holds that the polymer is formed by the joining of the ends of the three short arms to create a lattice-like array (6Yurchenco P.D. Cheng Y.S. Colognato H. J. Cell Biol. 1992; 117: 1119-1133Crossref PubMed Scopus (223) Google Scholar, 7Yurchenco P.D. Cheng Y.S. J. Biol. Chem. 1993; 268: 17286-17299Abstract Full Text PDF PubMed Google Scholar). The failure of laminin-5, which lacks most of the short arm domains (although does possess one domain 6 homologue in the β3 chain), to self-assemble is consistent with the above hypothesis. A more exacting test of the model, however, was provided by laminin-6. This laminin, like laminins-7, -8, and -9, and unlike laminins-1, -2, -4, has two rather than three full short arms and would be expected either not to polymerize/co-polymerize or to polymerize/co-polymerize poorly. The inability of laminin-6 to co-polymerize with laminin-1 argues that the three-arm assembly hypothesis is correct. Note that this non-co-polymerizing laminin shares two short arms (β1, γ1) in common with laminin-1, in contrast to the co-polymerizing laminin-2, which also shares two short arms (β1, γ1), and even the co-polymerizing laminin-4, which shares only one short arm (γ1) in common with laminin-1. Thus presence of an α chain short arm appears to be critical for laminin self-assembly. Recent studies 4H. Colognato and P. D. Yurchenco, unpublished results. reveal that laminin-2 isolated from the dystrophic dy 2Jmouse, which only lacks α2-domain VI, co-polymerizes poorly with laminin-1. This domain is of course absent in laminin-6 and suggests that loss of only the N-terminal globule of the α chain adversely affects self-assembly. The co-polymerization assay has proved to be a useful tool in the current study to evaluate the self-assembly properties of other isoforms in amounts below their critical concentrations. The property of co-polymerization furthermore suggests that where a given basement membrane contains a mixture of two or more polymerizing laminins, these laminins will form a cooperative network unless specifically prevented by other immobilizing bonds. Laminins-1, -2, and -4 share this property (Fig. 12). Laminin-5 can form a disulfide-stabilized bond to laminin-6 or laminin-7 (27Marinkovich M.P. Lunstrum G.P. Keene D.R. Burgeson R.E. J. Cell Biol. 1992; 119: 695-703Crossref PubMed Scopus (235) Google Scholar, 28Champliaud M.F. Lunstrum G.P. Rousselle P. Nishiyama T. Keene D.R. Burgeson R.E. J. Cell Biol. 1996; 132: 1189-1198Crossref PubMed Scopus (226) Google Scholar) but cannot bind to entactin/nidogen (29Mayer U. Poschl E. Gerecke D.R. Wagman D.W. Burgeson R.E. Timpl R. FEBS Lett. 1995; 365: 129-132Crossref PubMed Scopus (69) Google Scholar). Laminins-6 and -7, on the other hand, can bind to entactin/nidogen (and indirectly to the type IV collagen network), since they possess a γ1 chain. It appears that laminin-5 forms a cable-like structure connecting the hemidesmosome of the epidermis with the underlying dermis through type VII collagen and links this cabling through the rest of the basement membrane through laminins-6 and -7 (30Rousselle P. Keene D.R. Ruggiero F. Champliaud M.F. van der Rest M. Burgeson R.E. J. Cell Biol. 1997; 138: 719-728Crossref PubMed Scopus (222) Google Scholar). Finally, it is possible that laminins-5, -6, and -7 have other as yet undiscovered matrix binding interactions. It is conceivable that laminins lacking critical short arms domain could form large oligomeric or polymeric complexes using a completely different set of bonds compared with laminin-1. However, it is difficult to imagine how this would occur given the homology of the remaining short arm domains with those present in laminin-1 that are found not to participate in any detectable self-assembly. We thank the assistance of the Office of Research Administration, Palo Alto Veterans Affairs Health Care System, Palo Alto, CA. We also thank Dr. Patricia Rouselle (Center National de la Recherche Scientifique, Lyon, France) for providing us with several aliquots of laminin-5-specific antibodies. Finally, we are grateful to the nursing staff at the Medical Center at Princeton and Dr. Susan Shen-Schwartz at St. Peter's Medical Center (New Brunswick, NJ) for their assistance in providing placental tissue.
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