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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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Binding of soluble  laminin 5 to immobilized  type VII procollagen. Microtiter wells were coated with 1  μg of type VII–NC-1 purified  from amnion (•) or intact  type VII procollagen immunopurified from WISH cell  culture medium (□), or type  VII procollagen digested  with either bacterial collagenase (○) or pepsin (▪). pAb  to laminin 5 were used for  detecting the amount of  bound substrate.
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Figure 2: Binding of soluble laminin 5 to immobilized type VII procollagen. Microtiter wells were coated with 1 μg of type VII–NC-1 purified from amnion (•) or intact type VII procollagen immunopurified from WISH cell culture medium (□), or type VII procollagen digested with either bacterial collagenase (○) or pepsin (▪). pAb to laminin 5 were used for detecting the amount of bound substrate.

Mentions: To test the potential binding of laminin 5 to type VII collagen, type VII procollagen was partially purified from WISH cell (spontaneously transformed amniotic epithelial cell line) culture medium by immunoaffinity chromatography using NP-32–Sepharose. Solutions of 0–20 μg/ml of laminin 5 were incubated with multiwell plates precoated with partially purified type VII procollagen, a clostridial collagenase digest of the type VII procollagen preparation, a pepsin digest of the type VII procollagen preparation, and purified type VII collagen NC-1. Saturable binding of type VII procollagen to laminin 5 was observed (Fig. 2), indicating that the two molecules did interact either directly or indirectly. To localize the binding activity within type VII procollagen to the triple-helical or nontriple-helical domains, the procollagen solution was digested separately with pepsin or collagenase before coating multichamber wells. Incubation with pepsin completely eliminated binding of laminin 5, while removal of the triple helix augmented the interaction. These observations suggest that the laminin 5–type VII collagen interaction occurs through either NC-1 or NC-2 of type VII procollagen. Since NC-2 is proteolytically removed during anchoring fibril formation (Lunstrum et al., 1987; Bruckner-Tuderman et al., 1994), if the interaction has physiological relevance, it is most likely an interaction of NC-1 with laminin 5. Therefore, NC-1 was purified from human amnion using established methods. The product was >95% pure as judged by SDS-PAGE (Fig. 1, lane 2) and by rotary shadowing (Bachinger et al., 1990) (not shown). The majority of the purified NC-1 remained trimeric as shown by electrophoretic separation without disulfide bond reduction (Fig. 1, lane 3). NC-1 showed comparable binding to laminin 5 as was seen with collagenase-digested type VII procollagen. The results show a direct interaction of laminin 5 with type VII collagen mediated by the large NC-1 domain. We believe that the difference in binding seen between pure NC-1 or collagenase-treated type VII procollagen and intact type VII procollagen is not significant as it most likely results from removal of collagenous contaminants (primarily type VI collagen aggregates) from the procollagen preparation by collagenase digestion thereby increasing the effective NC-1 concentration.


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)

Binding of soluble  laminin 5 to immobilized  type VII procollagen. Microtiter wells were coated with 1  μg of type VII–NC-1 purified  from amnion (•) or intact  type VII procollagen immunopurified from WISH cell  culture medium (□), or type  VII procollagen digested  with either bacterial collagenase (○) or pepsin (▪). pAb  to laminin 5 were used for  detecting the amount of  bound substrate.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2141627&req=5

Figure 2: Binding of soluble laminin 5 to immobilized type VII procollagen. Microtiter wells were coated with 1 μg of type VII–NC-1 purified from amnion (•) or intact type VII procollagen immunopurified from WISH cell culture medium (□), or type VII procollagen digested with either bacterial collagenase (○) or pepsin (▪). pAb to laminin 5 were used for detecting the amount of bound substrate.
Mentions: To test the potential binding of laminin 5 to type VII collagen, type VII procollagen was partially purified from WISH cell (spontaneously transformed amniotic epithelial cell line) culture medium by immunoaffinity chromatography using NP-32–Sepharose. Solutions of 0–20 μg/ml of laminin 5 were incubated with multiwell plates precoated with partially purified type VII procollagen, a clostridial collagenase digest of the type VII procollagen preparation, a pepsin digest of the type VII procollagen preparation, and purified type VII collagen NC-1. Saturable binding of type VII procollagen to laminin 5 was observed (Fig. 2), indicating that the two molecules did interact either directly or indirectly. To localize the binding activity within type VII procollagen to the triple-helical or nontriple-helical domains, the procollagen solution was digested separately with pepsin or collagenase before coating multichamber wells. Incubation with pepsin completely eliminated binding of laminin 5, while removal of the triple helix augmented the interaction. These observations suggest that the laminin 5–type VII collagen interaction occurs through either NC-1 or NC-2 of type VII procollagen. Since NC-2 is proteolytically removed during anchoring fibril formation (Lunstrum et al., 1987; Bruckner-Tuderman et al., 1994), if the interaction has physiological relevance, it is most likely an interaction of NC-1 with laminin 5. Therefore, NC-1 was purified from human amnion using established methods. The product was >95% pure as judged by SDS-PAGE (Fig. 1, lane 2) and by rotary shadowing (Bachinger et al., 1990) (not shown). The majority of the purified NC-1 remained trimeric as shown by electrophoretic separation without disulfide bond reduction (Fig. 1, lane 3). NC-1 showed comparable binding to laminin 5 as was seen with collagenase-digested type VII procollagen. The results show a direct interaction of laminin 5 with type VII collagen mediated by the large NC-1 domain. We believe that the difference in binding seen between pure NC-1 or collagenase-treated type VII procollagen and intact type VII procollagen is not significant as it most likely results from removal of collagenous contaminants (primarily type VI collagen aggregates) from the procollagen preparation by collagenase digestion thereby increasing the effective NC-1 concentration.

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.

Show MeSH