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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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Electrophoretic analysis of immunoaffinity-purified  laminin 5 from cell culture media and type VII collagen–NC-1  from collagenase extracts of amniotic membranes. 2 μg of materials affinity purified on mAb 6/F12 from SCC25 cell culture medium (lane 1), and 2 μg of materials affinity purified on mAb NP-32 from collagenase extracts of amniotic membranes (lane 2)  were separated by SDS-PAGE after reduction on a 3–7.5% gradient polyacrylamide gel and visualized by Coomassie blue staining. Consistent with previous results, the reduced bands representing subunits of laminin 5 (α3, 165 kD; β3, 140 kD; γ2, 155 and  105 kD) and type VII collagen–NC-1 (150 kD). The electrophoretic migration position of purified NC-1 before disulfide-bond reduction is shown in lane 3. Migration positions of the molecular weight markers are indicated to the left of each gel.
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Figure 1: Electrophoretic analysis of immunoaffinity-purified laminin 5 from cell culture media and type VII collagen–NC-1 from collagenase extracts of amniotic membranes. 2 μg of materials affinity purified on mAb 6/F12 from SCC25 cell culture medium (lane 1), and 2 μg of materials affinity purified on mAb NP-32 from collagenase extracts of amniotic membranes (lane 2) were separated by SDS-PAGE after reduction on a 3–7.5% gradient polyacrylamide gel and visualized by Coomassie blue staining. Consistent with previous results, the reduced bands representing subunits of laminin 5 (α3, 165 kD; β3, 140 kD; γ2, 155 and 105 kD) and type VII collagen–NC-1 (150 kD). The electrophoretic migration position of purified NC-1 before disulfide-bond reduction is shown in lane 3. Migration positions of the molecular weight markers are indicated to the left of each gel.

Mentions: Laminin 5 was purifed from the culture medium of SCC25 cells. The preparation was homogeneous as judged by SDS-PAGE (Fig. 1, lane 1), and contained the proteolytically processed α3 chain at 165 kD, the unprocessed β3 chain (140 kD), and unprocessed γ2 chain (155 kD), which fail to resolve on this gel, and the fully processed γ2 chain (105 kD). Solid phase protein binding assays were first optimized for signal strength and specificity using the documented affinity of collagen IV for the laminin 1–nidogen complex (Aumailley et al., 1989; and data not shown). The assay demonstrated saturable binding of collagen IV to laminin–nidogen, four- to fivefold stronger than to collagen I. Under these conditions, laminin 5, collagens XII and XIV, and the NC-1 domain of collagen VII showed minimal binding.


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)

Electrophoretic analysis of immunoaffinity-purified  laminin 5 from cell culture media and type VII collagen–NC-1  from collagenase extracts of amniotic membranes. 2 μg of materials affinity purified on mAb 6/F12 from SCC25 cell culture medium (lane 1), and 2 μg of materials affinity purified on mAb NP-32 from collagenase extracts of amniotic membranes (lane 2)  were separated by SDS-PAGE after reduction on a 3–7.5% gradient polyacrylamide gel and visualized by Coomassie blue staining. Consistent with previous results, the reduced bands representing subunits of laminin 5 (α3, 165 kD; β3, 140 kD; γ2, 155 and  105 kD) and type VII collagen–NC-1 (150 kD). The electrophoretic migration position of purified NC-1 before disulfide-bond reduction is shown in lane 3. Migration positions of the molecular weight markers are indicated to the left of each gel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141627&req=5

Figure 1: Electrophoretic analysis of immunoaffinity-purified laminin 5 from cell culture media and type VII collagen–NC-1 from collagenase extracts of amniotic membranes. 2 μg of materials affinity purified on mAb 6/F12 from SCC25 cell culture medium (lane 1), and 2 μg of materials affinity purified on mAb NP-32 from collagenase extracts of amniotic membranes (lane 2) were separated by SDS-PAGE after reduction on a 3–7.5% gradient polyacrylamide gel and visualized by Coomassie blue staining. Consistent with previous results, the reduced bands representing subunits of laminin 5 (α3, 165 kD; β3, 140 kD; γ2, 155 and 105 kD) and type VII collagen–NC-1 (150 kD). The electrophoretic migration position of purified NC-1 before disulfide-bond reduction is shown in lane 3. Migration positions of the molecular weight markers are indicated to the left of each gel.
Mentions: Laminin 5 was purifed from the culture medium of SCC25 cells. The preparation was homogeneous as judged by SDS-PAGE (Fig. 1, lane 1), and contained the proteolytically processed α3 chain at 165 kD, the unprocessed β3 chain (140 kD), and unprocessed γ2 chain (155 kD), which fail to resolve on this gel, and the fully processed γ2 chain (105 kD). Solid phase protein binding assays were first optimized for signal strength and specificity using the documented affinity of collagen IV for the laminin 1–nidogen complex (Aumailley et al., 1989; and data not shown). The assay demonstrated saturable binding of collagen IV to laminin–nidogen, four- to fivefold stronger than to collagen I. Under these conditions, laminin 5, collagens XII and XIV, and the NC-1 domain of collagen VII showed minimal binding.

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