Characterization of Molecular Mechanisms Underlying Mutations in Dystrophic Epidermolysis Bullosa Using Site-directed Mutagenesis

Woodley, David T.; Hou, Yingping; Martín, Sabrina; Li, Wei; Chen, Mei

doi:10.1074/jbc.m709452200

Cited by 36 publications

(32 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Interestingly, co-expression of WT and G2015E-collagen VII, in which the glycine substitution is only three triplets away from G2006D, resulted in reduced thermal stability and a T m 34°C, but no accumulation in the endoplasmic reticulum. These observations are in keeping with genotype-phenotype correlation studies (28,29,17), which have not yielded a direct correlation between the thermal stability of collagen VII mutants and the DEB phenotype, although the relation between conformational heterogeneity of collagen VII and disease severity has not been mapped in a systematic manner.…”

Section: Discussionsupporting

confidence: 51%

“…It has been reported to promote fibroblast adhesion by two cell binding sites, one in the NC-1 and one in the triple helical domain (19,17). Therefore, we compared the ability of WT collagen VII and the mutants to promote adhesion and spreading of normal human keratinocytes and fibroblasts.…”

Section: Co-expression Of Wt and Mutant Collagen VII Leads To Formatimentioning

confidence: 99%

“…Coverslips were coated with 20 g/ml purified rColVII in 20 mM carbonate buffer, pH 9.3, or with 20 g/ml bovine serum albumin as a negative control, overnight at 4°C and blocked with 0.5 g/ml bovine serum albumin for 1 h at 37°C. Then 1 ϫ 10 5 human fibroblasts in serum-free DMEM or 1.5 ϫ 10 5 human keratinocytes in KGM without supplements were added and incubated for 2 h at 37°C (17). After washing with PBS, the cells were fixed with 2% paraformaldehyde for 15 min at room temperature and stained with DAPI and rhodamine-labeled phalloidin (Chemicon) for 30 min.…”

Section: Clinical and Biological Phenotypes Of Col7a1 Mutations-mentioning

confidence: 99%

See 2 more Smart Citations

Dominant-negative Effects of COL7A1 Mutations Can be Rescued by Controlled Overexpression of Normal Collagen VII

Fritsch

Spassov

Elfert

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

Dominant-negative interference by glycine substitution mutations in the COL7A1 gene causes dominant dystrophic epidermolysis bullosa (DDEB), a skin fragility disorder with mechanically induced blistering. Although qualitative and quantitative alterations of the COL7A1 gene product, collagen VII, underlie DDEB, the lack of direct correlation between mutations and the clinical phenotype has rendered DDEB less amenable to therapeutic targeting. To delineate the molecular mechanisms of DDEB, we used recombinant expression of wildtype (WT) and mutant collagen VII, which contained a naturally occurring COL7A1 mutation, G1776R, G2006D, or G2015E, for characterization of the triple helical molecules. The mutants were co-expressed with WT in equal amounts and could form heterotrimeric hybrid triple helices, as demonstrated by affinity purification and mass spectrometry. The thermal stability of the mutant molecules was strongly decreased, as evident in their sensitivity to trypsin digestion. The helix-to-coil transition, T m , of the mutant molecules was 31-34°C, and of WT collagen VII 41°C. Co-expression of WT with G1776R-or G2006D-collagen VII resulted in partial intracellular retention of the collagen, and mutant collagen VII had reduced ability to support cell adhesion. Intriguingly, controlled overexpression of WT collagen VII gradually improved the thermal stability of the collective of collagen VII molecules. Co-expression in a ratio of 90% WT:10% mutant increased the T m to 41°C for G1776R-collagen VII and to 39°C for G2006D-and G2015E-collagen VII. Therefore, increasing the expression of WT collagen VII in the skin of patients with DDEB can be considered a valid therapeutic approach.Mutations in the collagen VII gene, COL7A1, cause dystrophic epidermolysis bullosa (DEB), 3 a heritable skin fragility disorder characterized by mechanically induced blistering of the skin and mucosa, and excessive scarring (1). DEB is classified into clinical subtypes with dominant or recessive inheritance (2), and so far more than 400 different COL7A1 mutations are known, which underlie a broad spectrum of clinical presentations. Collagen VII is the major molecular constituent of anchoring fibrils in the skin. These centro-symmetrically banded fibrils extend from the epidermal basement membrane into the underlying dermal stroma and connect the epidermis to the dermis. Collagen VII is synthesized as three identical pro-␣1(VII) polypeptide chains, which are hydroxylated and glycosylated in a coordinated manner and then fold into triple-helical procollagen VII in the endoplasmic reticulum (ER). The procollagen, which contains a central collagenous triple-helix flanked by two non-collagenous domains, NC-1 and NC-2, is secreted into the extracellular space, where the C-terminal NC-2 propeptide is proteolytically removed by bone morphogenetic protein-1 (3). Subsequently, mature collagen VII undergoes a multistep fibril polymerization process to form the anchoring fibrils (4).The pathology in DDEB has been thought to result from negative ...

show abstract

Section: Discussionsupporting

confidence: 51%

Section: Co-expression Of Wt and Mutant Collagen VII Leads To Formatimentioning

confidence: 99%

Section: Clinical and Biological Phenotypes Of Col7a1 Mutations-mentioning

confidence: 99%

See 1 more Smart Citation

Dominant-negative Effects of COL7A1 Mutations Can be Rescued by Controlled Overexpression of Normal Collagen VII

Fritsch

Spassov

Elfert

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…One of the most severe forms is recessive dystrophic epidermolysis bullosa, caused by loss-of-function mutations in the collagen type VII (C7) gene ( COL7A1 ) 2-6. These mutations result in severely diminished expression of C7, a collagen localized at the dermal–epidermal junction.…”

mentioning

confidence: 99%

Bone Marrow Transplantation for Recessive Dystrophic Epidermolysis Bullosa

Wagner¹,

Ishida‐Yamamoto²,

McGrath³

et al. 2010

N Engl J Med

Self Cite

329

297

View full text Add to dashboard Cite

Background Recessive dystrophic epidermolysis bullosa is an incurable, often fatal mucocutaneous blistering disease caused by mutations in COL7A1, the gene encoding type VII collagen (C7). On the basis of preclinical data showing biochemical correction and prolonged survival in col7−/− mice, we hypothesized that allogeneic marrow contains stem cells capable of ameliorating the manifestations of recessive dystrophic epidermolysis bullosa in humans. Methods Between October 2007 and August 2009, we treated seven children who had recessive dystrophic epidermolysis bullosa with immunomyeloablative chemotherapy and allogeneic stem-cell transplantation. We assessed C7 expression by means of immunofluorescence staining and used transmission electron microscopy to visualize anchoring fibrils. We measured chimerism by means of competitive polymerase-chain-reaction assay, and documented blister formation and wound healing with the use of digital photography. Results One patient died of cardiomyopathy before transplantation. Of the remaining six patients, one had severe regimen-related cutaneous toxicity, with all having improved wound healing and a reduction in blister formation between 30 and 130 days after transplantation. We observed increased C7 deposition at the dermal–epidermal junction in five of the six recipients, albeit without normalization of anchoring fibrils. Five recipients were alive 130 to 799 days after transplantation; one died at 183 days as a consequence of graft rejection and infection. The six recipients had substantial proportions of donor cells in the skin, and none had detectable anti-C7 antibodies. Conclusions Increased C7 deposition and a sustained presence of donor cells were found in the skin of children with recessive dystrophic epidermolysis bullosa after allogeneic bone marrow transplantation. Further studies are needed to assess the long-term risks and benefits of such therapy in patients with this disorder. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT00478244.)

show abstract

“…This may be part of the reason that the carriers of c.1006_1007delCA mutation (patients’ mother) or c.IVS5568+1delG mutation (patients’ father and younger sister) did not demonstrate any phenotype. Also, it has been clarified that the same mutation on COL7A1 gene may result in a different phenotype of either dominant or recessive DEB [16], [34], [35], strongly suggesting that not only the mutation location, but also the nature of the mutation functionally affects helix formation, protein folding, thermal stability, intracellular transport, secretion, and assembly into anti-parallel dimmers or anchoring fibrils [18], [36], [37].…”

Section: Discussionmentioning

confidence: 99%

Two Novel Mutations on Exon 8 and Intron 65 of COL7A1 Gene in Two Chinese Brothers Result in Recessive Dystrophic Epidermolysis Bullosa

et al. 2012

View full text Add to dashboard Cite

Dystrophic epidermolysis bullosa is an inherited bullous dermatosis caused by the COL7A1 gene mutation in autosomal dominant or recessive mode. COL7A1 gene encodes type VII collagen – the main component of the anchoring fibrils at the dermal–epidermal junction. Besides the 730 mutations reported, we identified two novel COL7A1 gene mutations in a Chinese family, which caused recessive dystrophic epidermolysis bullosa (RDEB). The diagnosis was established histopathologically and ultrastructurally. After genomic DNA extraction from the peripheral blood sample of all subjects (5 pedigree members and 136 unrelated control individuals), COL7A1 gene screening was performed by polymerase chain reaction amplification and direct DNA sequencing of the whole coding exons and flanking intronic regions. Genetic analysis of the COL7A1 gene in affected individuals revealed compound heterozygotes with identical novel mutations. The maternal mutation is a 2-bp deletion at exon 8 (c.1006_1007delCA), leading to a subsequent reading frame-shift and producing a premature termination codon located 48 amino acids downstream in exon 9 (p.Q336EfsX48), consequently resulting in the truncation of 2561 amino acids downstream. This was only present in two affected brothers, but not in the other unaffected family members. The paternal mutation is a 1-bp deletion occurring at the first base of intron 65 (c.IVS5568+1delG) that deductively changes the strongly conserved GT dinucleotide at the 5′ donor splice site, results in subsequent reading-through into intron 65, and creates a stop codon immediately following the amino acids encoded by exon 65 (GTAA→TAA). This is predicted to produce a truncated protein lacking of 1089 C-terminal amino acids downstream. The latter mutation was found in all family members except one of the two unaffected sisters. Both mutations were observed concurrently only in the two affected brothers. Neither mutation was discovered in 136 unrelated Chinese control individuals. This study reveals novel disease-causing mutations in the COL7A1 gene.

show abstract

Characterization of Molecular Mechanisms Underlying Mutations in Dystrophic Epidermolysis Bullosa Using Site-directed Mutagenesis

Cited by 36 publications

References 38 publications

Dominant-negative Effects of COL7A1 Mutations Can be Rescued by Controlled Overexpression of Normal Collagen VII

Dominant-negative Effects of COL7A1 Mutations Can be Rescued by Controlled Overexpression of Normal Collagen VII

Bone Marrow Transplantation for Recessive Dystrophic Epidermolysis Bullosa

Two Novel Mutations on Exon 8 and Intron 65 of COL7A1 Gene in Two Chinese Brothers Result in Recessive Dystrophic Epidermolysis Bullosa

Contact Info

Product

Resources

About