High-affinity binding of the NC1 domain of collagen VII to laminin 5 and collagen IV

https://doi.org/10.1016/j.bbrc.2006.03.034Get rights and content

Abstract

Anchoring functions of collagen VII depend on its ability to form homotypic fibrils and to bind to other macromolecules to form heterotypic complexes. Biosensor-based binding assays were employed to analyze the kinetics of the NC1 domain-mediated binding of collagen VII to laminin 5, collagen IV, and collagen I. We showed that collagen VII interacts with laminin 5 and collagen IV with a Kd value of 10−9 M. In contrast, the NC1-mediated binding to collagen I was weak with a Kd value of 10−6 M. Binding assays also showed that the NC1 domain utilizes the same region to bind to both laminin 5 and collagen IV. We postulate that the ability of the NC1 domains to bind with high affinities to laminin 5 and collagen IV facilitates stabilization of the structure of the basement membrane itself and that the NC1-collagen I interaction may be less important for stabilization of the dermal–epidermal junction.

Section snippets

Materials and methods

Proteins for the binding studies. Recombinant mini-procollagen VII (mProVII) consisting of the NC1 domain, a truncated triple-helical region, and the NC2 domain was purified from cell culture media as described [13]. To express recombinant NC1 domain of human collagen VII, we employed a DNA fragment encompassing a 1–3870 bp2 region of cDNA encoding human procollagen VII (a kind gift from Dr. Y. Gache, Universite de

Proteins for biosensor binding assays

As demonstrated in Fig. 1, the NC1 domain had a predicted molecular mass of 137 kDa [12]. Electrophoresis in nonreducing conditions revealed that some of the NC1 molecules formed homotypic complexes stabilized by the disulfide bonds (Fig. 1) formed between the cysteine residues present within the NC1 domain [12]. Electron microscopy of the recombinant NC1 domains (Fig. 2) confirmed that they had a structure similar to that of the native NC1 domains seen in mProVII. Specifically, the NC1 domains

Discussion

Noncovalent interactions between macromolecules are essential for the formation of supramolecular structures of biological scaffolds that shape the architecture of tissues and organs. A macromolecular scaffold of skin is a complex structure formed by a number of molecules interacting with each other in a site-specific manner. One of the distinct morphological features of skin is that it is composed of two principal layers, namely the epidermis and dermis, separated by the basement membrane, a

Acknowledgments

We thank Dr. Yannick Gache for DNA constructs encoding the NC1 domain, Dr. David E. Birk and Biao Zuo for expert technical assistance with the electron microscopy. This study was supported by the NIH/NIAMS Grant P01-AR38923.

References (36)

Cited by (62)

  • Type VII collagen

    2023, Biochemistry of Collagens, Laminins and Elastin: Structure, Function and Biomarkers, Third Edition
View all citing articles on Scopus
View full text