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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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Localization of  laminin 5 to anchoring  plaques. Laminin 5 is localized to anchoring plaques in  human skin using mAb  BM165 and 5-nm (A, arrow)  or 1 nm silver-enhanced colloidal gold (B–D). Labeling  of the lamina lucida and anchoring plaques is intense using silver-enhanced 1 nm colloidal gold in comparison to  5 nm colloidal gold. Using a  mixture of mAb BM165  (anti-laminin α3) and pAb  3959 (anti-type VII collagen  NC-1), laminin 5 is seen to  colocalize with type VII collagen in anchoring plaques  (E, 1-nm silver-enhanced  particles specific for mAb  BM165 [small arrow] and 10-nm silver-enhanced particles  are specific for pAb 3959  [large arrow]; F, 5-nm particles specific for mAb BM165  (small arrow) and 15-nm particles specific for pAb 3959  [large arrow]. Bar, 100 nm.
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Figure 9: Localization of laminin 5 to anchoring plaques. Laminin 5 is localized to anchoring plaques in human skin using mAb BM165 and 5-nm (A, arrow) or 1 nm silver-enhanced colloidal gold (B–D). Labeling of the lamina lucida and anchoring plaques is intense using silver-enhanced 1 nm colloidal gold in comparison to 5 nm colloidal gold. Using a mixture of mAb BM165 (anti-laminin α3) and pAb 3959 (anti-type VII collagen NC-1), laminin 5 is seen to colocalize with type VII collagen in anchoring plaques (E, 1-nm silver-enhanced particles specific for mAb BM165 [small arrow] and 10-nm silver-enhanced particles are specific for pAb 3959 [large arrow]; F, 5-nm particles specific for mAb BM165 (small arrow) and 15-nm particles specific for pAb 3959 [large arrow]. Bar, 100 nm.

Mentions: Fresh neonatal foreskin was immunolabeled as previously described (Sakai and Keene, 1994). Briefly, fresh tissue was rinsed in PBS and then submersed overnight in mAb BM-165 (anti-laminin α3) diluted 1:5 in PBS or a mixture of BM-165 and rabbit pAb 3959 (anti-type VII collagen) at a ratio of 1:1:4 parts PBS. After an extensive rinse in PBS, the tissue was submersed overnight in secondary antibodies conjugated to different size gold particles (Amersham International, Little Chalfont, UK) as described in the legend for Fig. 9. Silver enhancement was performed on samples labeled with 1 nm gold. The samples were rinsed extensively in PBS and 0.1 M cacodylate buffer and then fixed, dehydrated, and embedded in Spurr's epoxy resin. 90-nm thick sections, which included the epithelium, papillary, and reticular dermis, were stained in uranyl acetate and lead citrate and examined using a Philips EM 410 transmission electron microscope.


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)

Localization of  laminin 5 to anchoring  plaques. Laminin 5 is localized to anchoring plaques in  human skin using mAb  BM165 and 5-nm (A, arrow)  or 1 nm silver-enhanced colloidal gold (B–D). Labeling  of the lamina lucida and anchoring plaques is intense using silver-enhanced 1 nm colloidal gold in comparison to  5 nm colloidal gold. Using a  mixture of mAb BM165  (anti-laminin α3) and pAb  3959 (anti-type VII collagen  NC-1), laminin 5 is seen to  colocalize with type VII collagen in anchoring plaques  (E, 1-nm silver-enhanced  particles specific for mAb  BM165 [small arrow] and 10-nm silver-enhanced particles  are specific for pAb 3959  [large arrow]; F, 5-nm particles specific for mAb BM165  (small arrow) and 15-nm particles specific for pAb 3959  [large arrow]. Bar, 100 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2141627&req=5

Figure 9: Localization of laminin 5 to anchoring plaques. Laminin 5 is localized to anchoring plaques in human skin using mAb BM165 and 5-nm (A, arrow) or 1 nm silver-enhanced colloidal gold (B–D). Labeling of the lamina lucida and anchoring plaques is intense using silver-enhanced 1 nm colloidal gold in comparison to 5 nm colloidal gold. Using a mixture of mAb BM165 (anti-laminin α3) and pAb 3959 (anti-type VII collagen NC-1), laminin 5 is seen to colocalize with type VII collagen in anchoring plaques (E, 1-nm silver-enhanced particles specific for mAb BM165 [small arrow] and 10-nm silver-enhanced particles are specific for pAb 3959 [large arrow]; F, 5-nm particles specific for mAb BM165 (small arrow) and 15-nm particles specific for pAb 3959 [large arrow]. Bar, 100 nm.
Mentions: Fresh neonatal foreskin was immunolabeled as previously described (Sakai and Keene, 1994). Briefly, fresh tissue was rinsed in PBS and then submersed overnight in mAb BM-165 (anti-laminin α3) diluted 1:5 in PBS or a mixture of BM-165 and rabbit pAb 3959 (anti-type VII collagen) at a ratio of 1:1:4 parts PBS. After an extensive rinse in PBS, the tissue was submersed overnight in secondary antibodies conjugated to different size gold particles (Amersham International, Little Chalfont, UK) as described in the legend for Fig. 9. Silver enhancement was performed on samples labeled with 1 nm gold. The samples were rinsed extensively in PBS and 0.1 M cacodylate buffer and then fixed, dehydrated, and embedded in Spurr's epoxy resin. 90-nm thick sections, which included the epithelium, papillary, and reticular dermis, were stained in uranyl acetate and lead citrate and examined using a Philips EM 410 transmission electron microscope.

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