Some, but Not All, Glycine Substitution Mutations inCOL7A1 Result in Intracellular Accumulation of Collagen VII, Loss of Anchoring Fibrils, and Skin Blistering

Hammami-Hauasli, Nadja; Schumann, Hauke; Raghunath, Michael; Kilgus, Oliver; Lüthi, Urs; Luger, Thomas A.; Bruckner‐Tuderman, Leena

doi:10.1074/jbc.273.30.19228

Cited by 74 publications

(67 citation statements)

References 36 publications

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“…Consistent with this notion, our studies demonstrated that this mutation, similar to R2063W, also leads to destabilization of the triple helix and makes the mutant C7 more susceptible to protease digestion. It is interesting to note that four other C7 mutations, G2006D, G2034R, G2015E, and R2008G, within exon 73 next to the hinge region have also been shown to interfere with protein folding in a dominant negative manner and cause intracellular accumulation of the mutant molecules (15,40). However, we did not detect any difference in the secretion of these two mutant proteins when compared with wild type C7.…”

Section: Figure 9 Migration Of Human Keratinocytes On Various C7 Mutcontrasting

confidence: 44%

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

Woodley

Hou

Martín

et al. 2008

Journal of Biological Chemistry

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Type VII collagen (C7) is a major component of anchoring fibrils, structures that mediate epidermal-dermal adherence. Mutations in gene COL7A1 encoding for C7 cause dystrophic epidermolysis bullosa (DEB), a genetic mechano-bullous disease. The biological consequences of specific COL7A1 mutations and the molecular mechanisms leading to DEB clinical phenotypes are unknown. In an attempt to establish genotype-phenotype relationships, we generated four individual substitution mutations that have been associated with recessive DEB, G2049E, R2063W, G2569R, and G2575R, and purified the recombinant mutant proteins. All mutant proteins were synthesized and secreted as a 290-kDa mutant C7 ␣ chain at levels similar to wild type C7. The G2569R and G2575R glycine substitution mutations resulted in mutant C7 with increased sensitivity to protease degradation and decreased ability to form trimers. Limited proteolytic digestion of mutant G2049E and R2063W proteins yielded aberrant fragments and a triple helix with reduced stability. These two mutations next to the 39-amino acid helical interruption hinge region caused local destabilization of the triple-helix that exposed an additional highly sensitive proteolytic site within the region of the mutation. Our functional studies demonstrated that C7 is a potent pro-motility matrix for skin human keratinocyte migration and that this activity resides within the triple helical domain. Furthermore, G2049E and R2063W mutations reduced the ability of C7 to support fibroblast adhesion and keratinocyte migration. We conclude that known recessive DEB C7 mutations perturb critical functions of the C7 molecule and likely contribute to the clinical phenotypes of DEB patients.Type VII collagen (C7) 3 resides within the basement membrane zone beneath stratified squamous epithelium (1, 2). C7 is a major component of anchoring fibrils, structures that attach the epidermal basement membrane of the skin to the underlying papillary dermis (3, 4). In hereditary dystrophic epidermolysis bullosa (DEB), anchoring fibrils are diminutive and/or reduced in number (5-7). DEB is caused by mutations in the COL7A1 gene encoding C7 (8 -11).C7 is a homotrimer composed of three identical ␣ chains. Each ␣ chain has a 145-kDa central collagenous triple-helical segment characterized by repeating Gly-X-Y amino acid sequences. The helical central domain is flanked by a large 145-kDa amino-terminal, noncollagenous domain (NC1) and a small 34-kDa carboxyl-terminal non-collagenous domain (NC2) (4, 12). Within the papillary dermis, C7 molecules form antiparallel, tail-to-tail dimers stabilized by disulfide bonding through a small carboxyl-terminal NC2 overlap between two C7 molecules. A portion of the NC2 domain is then proteolytically removed (13,14). The antiparallel dimers then aggregate laterally to form anchoring fibrils which interact with microfibrils, collagen fibrils, and micro-thread-like fibers within the papillary dermis. These interactions are thought to secure the epidermis and its underlying basement membra...

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Section: Figure 9 Migration Of Human Keratinocytes On Various C7 Mutcontrasting

confidence: 44%

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

Woodley

Hou

Martín

et al. 2008

Journal of Biological Chemistry

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“…60 It has been postulated that some glycine substitution mutations interfere with the formation of the stable triple helix of type VII collagen, preventing their secretion into the extracellular space, and thus leading to the intracellular accumulation of type VII collagen. 61 …”

Section: Consequences Of Splice-junction Mutationsmentioning

confidence: 52%

Epidermolysis bullosa. II. Type VII collagen mutations and phenotype-genotype correlations in the dystrophic subtypes

Varki

Sadowski

Uitto

et al. 2006

Journal of Medical Genetics

244

228

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Background: The dystrophic forms of epidermolysis bullosa (DEB), a group of heritable blistering disorders, show considerable phenotypic variability, and both autosomal dominant and autosomal recessive inheritance can be recognised. DEB is derived from mutations in the type VII collagen gene (COL7A1), encoding a large collagenous protein that is the predominant, if not exclusive, component of the anchoring fibrils at the dermalepidermal junction. Methods: The Dystrophic Epidermolysis Bullosa Research Association Molecular Diagnostics Laboratory (Philadelphia, Pennsylvania, USA), established in 1996, has analysed more than 1000 families with different forms of epidermolysis bullosa, among them 332 families with DEB. DNA specimens were subjected to mutation analysis by polymerase chain reaction (PCR) amplification of all 118 exons and flanking intronic sequences of COL7A1, followed either by heteroduplex scanning and sequencing of the PCR products demonstrating heteroduplexes or by direct nucleotide sequencing. Results: 355 mutant alleles out of the anticipated 438 (81.1%) were disclosed. Among these mutations, a total of 242 mutations were distinct and 138 were novel, previously unreported mutations. No evidence of mutations in any other gene was obtained. Discussion: Examination of the mutation database suggested phenotype-genotype correlations, contributing to the improved subclassification of DEB with prognostic implications. The mutation information also forms the basis for accurate genetic counselling and prenatal diagnosis in families at risk for recurrence.

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“…Unlike many other glycine substitution mutations in the genes for skin basement membrane collagens (20,21,40), the mutation G627V causes a drastic reduction of the conformational stability of the Col15 domain. The mutated fragment was partially digested by trypsin already at 4°C and completely abolished at 16°C.…”

Section: Discussionmentioning

confidence: 99%

Collagen XVII Is Destabilized by a Glycine Substitution Mutation in the Cell Adhesion Domain Col15

Tasanen

Eble

Aumailley

et al. 2000

Journal of Biological Chemistry

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Collagen XVII is a hemidesmosomal transmembrane molecule important for epithelial adhesion in the skin. It exists in two forms, as a full-length protein and as a soluble ectodomain that is shed from the keratinocyte surface by furin-mediated proteolysis. To obtain information on the conformation and the functions of this unusual collagen, its largest collagenous domain, Col15, was expressed in a eukaryotic episomal expression system and purified by DEAE and fast protein liquid-Mono S chromatography. The protein was triple-helical (T m of 26.5°C) when produced in cultures containing ascorbic acid. When the vitamin supply was limited, the 4-hydroxyproline content was reduced from 74 to 9%, which, in turn, resulted in a drastic reduction of the stability of the triple helix. The glycine substitution mutation G627V associated with junctional epidermolysis bullosa, a human blistering skin disease, also had a striking effect on thermal stability of rCol15 causing partial unfolding already at 4°C. Col15 promoted cell adhesion of epithelial and fibroblastic cell lines with a ␤1 integrinmediated mechanism. In concert with this, in acquired autoimmune blistering skin diseases, circulating IgG and IgA autoantibodies were found to target rCol15r.Collagens are a family of closely related, although genetically distinct, extracellular matrix proteins. Each collagen consists of three polypeptide chains, ␣ chains, which contain a characteristic repeating "collagenous" triplet amino acid sequence -Gly-Xaa-Yaa-, where Xaa and Yaa denote amino acids other than glycine. In all collagen types, the ␣ chains also have non-collagenous domains of varying sizes (1). Typically, the three polypeptide chains are twisted around each other into a collagen triple helix; however, only suggestive data for this exist in the recently characterized transmembrane collagens, types XIII and XVII (for review, see Ref.2). Collagen XVII, also known as the 180-kDa bullous pemphigoid antigen, or BP180, is a structural component of hemidesmosomes, multiprotein complexes that mediate the adhesion of epidermal keratinocytes to the underlying basement membrane (3, 4). The cDNA sequence codes for a type II integral transmembrane protein of 1497 amino acids, with an intracellular domain of 560 amino acids, a short transmembrane stretch, and an extracellular collagenous domain of 914 amino acids with multiple interruptions. The length of the individual collagenous subdomains varies from 14 to 242 amino acid residues (5). Recently, it was established that collagen XVII exists as two molecular forms, i.e. as a full-length transmembrane homotrimer of three 180-kDa ␣1(XVII) chains and as a 120-kDa soluble form that corresponds to the extracellular domain and is presumably released from the cell surface through furin-mediated proteolytic processing (6, 7). In some situations also a shorter, approximately 90 -100-kDa fragment, has been observed (3, 4, 6). Very little is known about the molecular shape of collagen XVII under physiological conditions. Rotary shadowing ele...

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Some, but Not All, Glycine Substitution Mutations inCOL7A1 Result in Intracellular Accumulation of Collagen VII, Loss of Anchoring Fibrils, and Skin Blistering

Cited by 74 publications

References 36 publications

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

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

Epidermolysis bullosa. II. Type VII collagen mutations and phenotype-genotype correlations in the dystrophic subtypes

Collagen XVII Is Destabilized by a Glycine Substitution Mutation in the Cell Adhesion Domain Col15

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