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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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Western blot analysis of the complex of laminin 5 and NC-1 produced in  solution. Laminin 5 alone  (lanes A1 and B1), NC-1  alone (lanes A2 and B2), a  mixture of laminin 5 and NC-1  (lanes A3 and B3), or a mixture of laminin 5 and heat denatured NC-1 (lanes A4 and B4) were immunoprecipitated with  anti-α3 mAb (BM-165). The contents of the precipitates were  separated by SDS-PAGE after disulfide bond reduction and visualized by Western analysis using (A) polyclonal anti-laminin 5  or (B) monoclonal anti-NC-1 (mAb NP-32).
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Figure 6: Western blot analysis of the complex of laminin 5 and NC-1 produced in solution. Laminin 5 alone (lanes A1 and B1), NC-1 alone (lanes A2 and B2), a mixture of laminin 5 and NC-1 (lanes A3 and B3), or a mixture of laminin 5 and heat denatured NC-1 (lanes A4 and B4) were immunoprecipitated with anti-α3 mAb (BM-165). The contents of the precipitates were separated by SDS-PAGE after disulfide bond reduction and visualized by Western analysis using (A) polyclonal anti-laminin 5 or (B) monoclonal anti-NC-1 (mAb NP-32).

Mentions: To be certain that the binding of laminin 5 to type VII collagen NC-1 was not artifactually influenced by the solid-phase conditions, purified laminin 5 was coincubated in solution with and without NC-1, and with heat-denatured NC-1, and then complexed and uncomplexed laminin 5 was isolated by immunoprecipitation using anti-laminin α3 (BM-165). The immunoprecipitation products were separated by SDS-PAGE after disulfide bond reduction. Laminin 5 and NC-1 were identified by Western blot analysis using polyclonal anti-laminin 5 and monoclonal anti-NC-1 (NP32). Immunoprecipitated laminin 5 in the absence of NC-1 gave the expected pattern by Western analysis (Fig. 6, lane A1) and did not react with anti-NC-1 (Fig. 6, lane B1). Immunoprecipitation of NC-1 in the absence of laminin 5 produced no product (Fig. 6, lanes A2 and B2). Both laminin 5 (Fig. 6, lane A3) and NC-1 (Fig. 6, lane B3) were immunoprecipitated from the mixture of the two, while denatured NC-1 did not precipitate with laminin 5 (Fig. 6, lanes A4 and B4). The results obtained using these conditions confirm the solid-phase binding data and again emphasize the importance of the native conformation of NC-1.


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)

Western blot analysis of the complex of laminin 5 and NC-1 produced in  solution. Laminin 5 alone  (lanes A1 and B1), NC-1  alone (lanes A2 and B2), a  mixture of laminin 5 and NC-1  (lanes A3 and B3), or a mixture of laminin 5 and heat denatured NC-1 (lanes A4 and B4) were immunoprecipitated with  anti-α3 mAb (BM-165). The contents of the precipitates were  separated by SDS-PAGE after disulfide bond reduction and visualized by Western analysis using (A) polyclonal anti-laminin 5  or (B) monoclonal anti-NC-1 (mAb NP-32).
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Figure 6: Western blot analysis of the complex of laminin 5 and NC-1 produced in solution. Laminin 5 alone (lanes A1 and B1), NC-1 alone (lanes A2 and B2), a mixture of laminin 5 and NC-1 (lanes A3 and B3), or a mixture of laminin 5 and heat denatured NC-1 (lanes A4 and B4) were immunoprecipitated with anti-α3 mAb (BM-165). The contents of the precipitates were separated by SDS-PAGE after disulfide bond reduction and visualized by Western analysis using (A) polyclonal anti-laminin 5 or (B) monoclonal anti-NC-1 (mAb NP-32).
Mentions: To be certain that the binding of laminin 5 to type VII collagen NC-1 was not artifactually influenced by the solid-phase conditions, purified laminin 5 was coincubated in solution with and without NC-1, and with heat-denatured NC-1, and then complexed and uncomplexed laminin 5 was isolated by immunoprecipitation using anti-laminin α3 (BM-165). The immunoprecipitation products were separated by SDS-PAGE after disulfide bond reduction. Laminin 5 and NC-1 were identified by Western blot analysis using polyclonal anti-laminin 5 and monoclonal anti-NC-1 (NP32). Immunoprecipitated laminin 5 in the absence of NC-1 gave the expected pattern by Western analysis (Fig. 6, lane A1) and did not react with anti-NC-1 (Fig. 6, lane B1). Immunoprecipitation of NC-1 in the absence of laminin 5 produced no product (Fig. 6, lanes A2 and B2). Both laminin 5 (Fig. 6, lane A3) and NC-1 (Fig. 6, lane B3) were immunoprecipitated from the mixture of the two, while denatured NC-1 did not precipitate with laminin 5 (Fig. 6, lanes A4 and B4). The results obtained using these conditions confirm the solid-phase binding data and again emphasize the importance of the native conformation of NC-1.

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