The Recombinant Expression of Full-length Type VII Collagen and Characterization of Molecular Mechanisms Underlying Dystrophic Epidermolysis Bullosa

Mei Chen,Fritz K Costa,Christopher R Lindvay,Yuan-Ping Han,David T Woodley

doi:10.1074/jbc.m108779200

Mei Chen, Fritz K Costa + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m108779200

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Abstract

Type VII collagen is a major component of anchoring fibrils, attachment structures that mediate dermal-epidermal adherence in human skin. Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous disorder caused by mutations in the type VII collagen gene and perturbations in anchoring fibrils. In this study, we produced recombinant human type VII collagen in stably transfected human 293 cell clones and purified large quantities of the recombinant protein from culture media. The recombinant type VII collagen was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation. Purified type VII collagen bound to fibronectin, laminin-5, type I collagen, and type IV collagen and also supported human dermal fibroblast adhesion. In an attempt to establish genotype-phenotype relationships, we generated two individual substitution mutations that have been associated with recessive DEB, R2008G and G2749R, and purified the recombinant mutant proteins. The G2749R mutation resulted in mutant type VII collagen with increased sensitivity to protease degradation and decreased ability to form trimers. The R2008G mutation caused the intracellular accumulation of type VII collagen. We conclude that structural and functional studies of in vitro generated type VII collagen mutant proteins will aid in correlating genetic mutations with the clinical phenotypes of DEB patients.

Highlights

Type VII collagen resides within the basement membrane zone (BMZ)1 beneath stratified squamous epithelium [1, 2]
We demonstrated that a truncated type VII minicollagen, containing the intact noncollagenous NC1 and NC2 and half of the central helical collagenous domain, supported fibroblast attachment at levels similar to those of NC1 alone
In a study by Christiano and colleagues [24], a family with members who had recessive Dystrophic epidermolysis bullosa (DEB) was described in which a gene defect in type VII collagen was identified as a single amino acid substitution from arginine to glycine at residue 2008 within the triple-helical domain

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—The human embryonic kidney cell line 293 (ATCC, Rockville, MD) was routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 (1:1) supplemented with 10% fetal bovine serum. The full-length human type VII cDNA was assembled from CMV/NC1, three cDNA clones described previously [18], and four newly generated cDNA fragments by standard subcloning techniques and recombinant PCR. 96-well microtiter plates were coated by incubation with 100 ␮l of various ECM proteins (20 ␮g/ml) or purified recombinant NC1 and type VII collagen (20 ␮g/ml) in 20 mM carbonate buffer, pH 9.3, at 4 °C for overnight and were blocked with PBS containing 0.5 ␮g/ml heat-inactivated BSA at 37 °C for 1 h. Protease Digestion—Purified recombinant type VII collagen or mutant type VII collagen was incubated with trypsin (Sigma) at an enzyme-to-substrate ratio of 1:10 by weight in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl at 14 °C for 2 h or with pepsin in 0.1 M acetic acid at 4 °C for 2 h or overnight and analyzed by SDS-PAGE, followed by immunoblot analysis with a polyclonal antibody against the collagenous domain of type VII collagen. After culture for 72 h, the conditioned medium was collected

Recombinant Type VII Collagen Expression

RESULTS

DISCUSSION