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Laminin 5 binds the NC-1 domain of type VII collagen.

Rousselle P, Keene DR, Ruggiero F, Champliaud MF, Rest M, Burgeson RE - J. Cell Biol. (1997)

Bottom Line: Laminin 6 (alpha3beta1gamma1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the beta3 and/or gamma2 chains of laminin 5.Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (alpha3beta2gamma1).The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie et Chimie des Protéines, Unité Propre de Recherche 412 du Centre National de la Recherche Scientifique, associée à l'Université Lyon I, 69367 Lyon Cedex 07, France.

ABSTRACT
Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this structure is the major element providing for resistance to external friction. Ultrastructurally, laminin 5 (alpha3beta3gamma2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin alpha6beta4. However, the interaction of laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. In this study we demonstrate that monomeric laminin 5 binds the NH2-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both laminin 5 and type VII collagen NC-1. Laminin 6 (alpha3beta1gamma1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the beta3 and/or gamma2 chains of laminin 5. Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (alpha3beta2gamma1). The adduction occurs between the NH2 terminus of laminin 5 and the branch point of the short arms of laminins 6 or 7. The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa. The results also support a model predicting that monomeric laminin 5 constitutes the anchoring filaments and bridges integrin alpha6beta4 with type VII collagen, and the laminin 5-6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin alpha3beta1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance.

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Inhibition of type  VII–NC-1 binding to immobilized laminin 5 by preincubation with various competitors. The test system  consisted of laminin 5 as the  immobilized phase at the  concentration of 1 μg per  well, and a fixed amount of  soluble type VII collagen– NC-1 (5 μg/ml). Type VII– NC1 was incubated at 4°C for  8 h with varying concentration of laminin 5 (•) or laminin 1–nidogen complex (○)  before testing on the insoluble ligand.
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Figure 4: Inhibition of type VII–NC-1 binding to immobilized laminin 5 by preincubation with various competitors. The test system consisted of laminin 5 as the immobilized phase at the concentration of 1 μg per well, and a fixed amount of soluble type VII collagen– NC-1 (5 μg/ml). Type VII– NC1 was incubated at 4°C for 8 h with varying concentration of laminin 5 (•) or laminin 1–nidogen complex (○) before testing on the insoluble ligand.

Mentions: The specificity of this interaction was tested by evaluating the relative binding of type VII–NC-1, laminin 1–nidogen, and collagens I-, IV-, XII-, and XIV-coated wells to soluble laminin 5. As illustrated in Fig. 3 A, only type VII– NC-1 strongly bound laminin 5. The laminin 1–nidogen complex showed background affinity for laminin 5. When type VII–NC-1 was added in solution to laminin 5–coated wells (Fig. 3 B), again NC-1 bound most strongly to laminin 5. Under these conditions, NC-1 showed no affinity for laminin 1–nidogen, but type VII–NC-1 did bind collagen IV as previously shown (Burgeson et al., 1990), but only to about half the extent of the binding to laminin 5. Of particular interest, laminin 6 showed little binding to type VII– NC-1 relative to laminin 5 even though both laminins share the same α3 subunit (Champliaud et al., 1996). Consistent with this finding, NC-1 showed diminished binding to laminin 5 when complexed with laminin 6. However, the molar amounts of laminin 5 present in the complex solutions are ∼50% the molar concentration of laminin 5 in solutions of laminin 5 alone. Since laminin 6 does not bind NC-1, the laminin 5–6 complex binds NC-1 with the same efficiency or only slightly diminished efficiency relative to laminin 5 alone. The binding of 5 μg of NC-1 to laminin 5 was inhibited by preincubation of NC-1 with increasing concentrations of laminin 5, but not by preincubation with laminin 1–nidogen (Fig. 4).


Laminin 5 binds the NC-1 domain of type VII collagen.

Rousselle P, Keene DR, Ruggiero F, Champliaud MF, Rest M, Burgeson RE - J. Cell Biol. (1997)

Inhibition of type  VII–NC-1 binding to immobilized laminin 5 by preincubation with various competitors. The test system  consisted of laminin 5 as the  immobilized phase at the  concentration of 1 μg per  well, and a fixed amount of  soluble type VII collagen– NC-1 (5 μg/ml). Type VII– NC1 was incubated at 4°C for  8 h with varying concentration of laminin 5 (•) or laminin 1–nidogen complex (○)  before testing on the insoluble ligand.
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Related In: Results  -  Collection

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Figure 4: Inhibition of type VII–NC-1 binding to immobilized laminin 5 by preincubation with various competitors. The test system consisted of laminin 5 as the immobilized phase at the concentration of 1 μg per well, and a fixed amount of soluble type VII collagen– NC-1 (5 μg/ml). Type VII– NC1 was incubated at 4°C for 8 h with varying concentration of laminin 5 (•) or laminin 1–nidogen complex (○) before testing on the insoluble ligand.
Mentions: The specificity of this interaction was tested by evaluating the relative binding of type VII–NC-1, laminin 1–nidogen, and collagens I-, IV-, XII-, and XIV-coated wells to soluble laminin 5. As illustrated in Fig. 3 A, only type VII– NC-1 strongly bound laminin 5. The laminin 1–nidogen complex showed background affinity for laminin 5. When type VII–NC-1 was added in solution to laminin 5–coated wells (Fig. 3 B), again NC-1 bound most strongly to laminin 5. Under these conditions, NC-1 showed no affinity for laminin 1–nidogen, but type VII–NC-1 did bind collagen IV as previously shown (Burgeson et al., 1990), but only to about half the extent of the binding to laminin 5. Of particular interest, laminin 6 showed little binding to type VII– NC-1 relative to laminin 5 even though both laminins share the same α3 subunit (Champliaud et al., 1996). Consistent with this finding, NC-1 showed diminished binding to laminin 5 when complexed with laminin 6. However, the molar amounts of laminin 5 present in the complex solutions are ∼50% the molar concentration of laminin 5 in solutions of laminin 5 alone. Since laminin 6 does not bind NC-1, the laminin 5–6 complex binds NC-1 with the same efficiency or only slightly diminished efficiency relative to laminin 5 alone. The binding of 5 μg of NC-1 to laminin 5 was inhibited by preincubation of NC-1 with increasing concentrations of laminin 5, but not by preincubation with laminin 1–nidogen (Fig. 4).

Bottom Line: Laminin 6 (alpha3beta1gamma1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the beta3 and/or gamma2 chains of laminin 5.Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (alpha3beta2gamma1).The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie et Chimie des Protéines, Unité Propre de Recherche 412 du Centre National de la Recherche Scientifique, associée à l'Université Lyon I, 69367 Lyon Cedex 07, France.

ABSTRACT
Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this structure is the major element providing for resistance to external friction. Ultrastructurally, laminin 5 (alpha3beta3gamma2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin alpha6beta4. However, the interaction of laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. In this study we demonstrate that monomeric laminin 5 binds the NH2-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both laminin 5 and type VII collagen NC-1. Laminin 6 (alpha3beta1gamma1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the beta3 and/or gamma2 chains of laminin 5. Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (alpha3beta2gamma1). The adduction occurs between the NH2 terminus of laminin 5 and the branch point of the short arms of laminins 6 or 7. The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa. The results also support a model predicting that monomeric laminin 5 constitutes the anchoring filaments and bridges integrin alpha6beta4 with type VII collagen, and the laminin 5-6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin alpha3beta1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance.

Show MeSH
Related in: MedlinePlus