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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: 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.

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 laminin 5–type VII  collagen–NC-1 complex immunoaffinity purified from  collagenase extracts of amniotic membranes. Type VII  collagen–NC-1 was first affinity purified on mAb NP-32  from collagenase extracts of  amniotic membranes. Eluted  fractions of NC-1 were  pooled, dialyzed against  PBS, and passed over a mAb  6F12 affinity chromatography column. Bound materials were immunoblotted with polyclonal anti-laminin 5 (pLm5; lane 3), and  anti-NC-1 (mAb NP-32, lane 4). The bound material shows the  pattern obtained for purified laminin 5 (lane 1), which does not  contain NC-1 (lane 2) and for purified NC-1 (not shown, but  identical to lane 6). The nonbound fraction contains NC-1 only  (lane 6) but no laminin 5 (lane 2).
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Figure 8: Western blot analysis of the laminin 5–type VII collagen–NC-1 complex immunoaffinity purified from collagenase extracts of amniotic membranes. Type VII collagen–NC-1 was first affinity purified on mAb NP-32 from collagenase extracts of amniotic membranes. Eluted fractions of NC-1 were pooled, dialyzed against PBS, and passed over a mAb 6F12 affinity chromatography column. Bound materials were immunoblotted with polyclonal anti-laminin 5 (pLm5; lane 3), and anti-NC-1 (mAb NP-32, lane 4). The bound material shows the pattern obtained for purified laminin 5 (lane 1), which does not contain NC-1 (lane 2) and for purified NC-1 (not shown, but identical to lane 6). The nonbound fraction contains NC-1 only (lane 6) but no laminin 5 (lane 2).

Mentions: If laminin 5 and type VII collagen NC-1 interact in vivo, it may be possible to solubilize the complex if the tissue is solubilized under nondenaturing conditions. NC-1 was prepared as a collagenase digest of amnion and partially purified by NP32 (anti-NC-1) immunoaffinity chromatography. If a complex exists in this mixture, it should be retained by 6F12 (anti-laminin β3) immunoaffinity columns. Thus, partially purified NC-1 eluted from the anti-NC-1 column was applied to a column containing anti-β3. The flow-through (Fig. 8, lanes 2 and 6, nonbound) is Western blotted by anti-NC-1 (Fig. 8, lane 6) but not by anti-laminin 5 (Fig. 8, lane 2) indicating that the nonbound fraction contains only NC-1 and is equivalent to the NC-1 used in the binding studies described above. If complexed laminin 5 and NC-1 are present in the extract, they should be bound by the anti-β3 column. As seen in Fig. 8, lanes 3 and 4, the materials eluted from the anti-β3 column are Western blotted by both anti-laminin 5 (Fig. 8, lane 3) and anti-NC-1 (Fig. 8, lane 4) indicating that the bound fraction contains both NC-1 and laminin 5.


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 laminin 5–type VII  collagen–NC-1 complex immunoaffinity purified from  collagenase extracts of amniotic membranes. Type VII  collagen–NC-1 was first affinity purified on mAb NP-32  from collagenase extracts of  amniotic membranes. Eluted  fractions of NC-1 were  pooled, dialyzed against  PBS, and passed over a mAb  6F12 affinity chromatography column. Bound materials were immunoblotted with polyclonal anti-laminin 5 (pLm5; lane 3), and  anti-NC-1 (mAb NP-32, lane 4). The bound material shows the  pattern obtained for purified laminin 5 (lane 1), which does not  contain NC-1 (lane 2) and for purified NC-1 (not shown, but  identical to lane 6). The nonbound fraction contains NC-1 only  (lane 6) but no laminin 5 (lane 2).
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Related In: Results  -  Collection

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Figure 8: Western blot analysis of the laminin 5–type VII collagen–NC-1 complex immunoaffinity purified from collagenase extracts of amniotic membranes. Type VII collagen–NC-1 was first affinity purified on mAb NP-32 from collagenase extracts of amniotic membranes. Eluted fractions of NC-1 were pooled, dialyzed against PBS, and passed over a mAb 6F12 affinity chromatography column. Bound materials were immunoblotted with polyclonal anti-laminin 5 (pLm5; lane 3), and anti-NC-1 (mAb NP-32, lane 4). The bound material shows the pattern obtained for purified laminin 5 (lane 1), which does not contain NC-1 (lane 2) and for purified NC-1 (not shown, but identical to lane 6). The nonbound fraction contains NC-1 only (lane 6) but no laminin 5 (lane 2).
Mentions: If laminin 5 and type VII collagen NC-1 interact in vivo, it may be possible to solubilize the complex if the tissue is solubilized under nondenaturing conditions. NC-1 was prepared as a collagenase digest of amnion and partially purified by NP32 (anti-NC-1) immunoaffinity chromatography. If a complex exists in this mixture, it should be retained by 6F12 (anti-laminin β3) immunoaffinity columns. Thus, partially purified NC-1 eluted from the anti-NC-1 column was applied to a column containing anti-β3. The flow-through (Fig. 8, lanes 2 and 6, nonbound) is Western blotted by anti-NC-1 (Fig. 8, lane 6) but not by anti-laminin 5 (Fig. 8, lane 2) indicating that the nonbound fraction contains only NC-1 and is equivalent to the NC-1 used in the binding studies described above. If complexed laminin 5 and NC-1 are present in the extract, they should be bound by the anti-β3 column. As seen in Fig. 8, lanes 3 and 4, the materials eluted from the anti-β3 column are Western blotted by both anti-laminin 5 (Fig. 8, lane 3) and anti-NC-1 (Fig. 8, lane 4) indicating that the bound fraction contains both NC-1 and laminin 5.

Bottom Line: 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.

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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