Three Novel Homozygous Point Mutations and a New Polymorphism in the COL17A1 Gene: Relation to Biological and Clinical Phenotypes of Junctional Epidermolysis Bullosa

Schumann, Hauke; Hammami-Hauasli, Nadja; Pulkkinen, Leena; Mauviel, Alain; Küster, Wolfgang; Lüthi, Urs; Owaribe, Katsushi; Uitto, Jouni; Bruckner‐Tuderman, Leena

doi:10.1086/515463

Cited by 75 publications

(77 citation statements)

References 29 publications

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“…3 and 4). Most are null mutations resulting in premature termination codons and, subsequently, in nonsense-mediated mRNA decay, whereas some missense mutations and small deletions have been reported (5)(6)(7)(8)(9)(10).…”

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confidence: 99%

C-terminal Truncation Impairs Glycosylation of Transmembrane Collagen XVII and Leads to Intracellular Accumulation

Franzke

Has

Schulte

et al. 2006

Journal of Biological Chemistry

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Collagen XVII, a type II transmembrane protein in hemidesmosomes, is involved in the anchorage of stratified epithelia to the underlying mesenchyme. Its functions are regulated by ectodomain shedding, and its genetic defects lead to epidermal detachment in junctional epidermolysis bullosa (JEB), a heritable skin fragility syndrome, but the molecular disease mechanisms remain elusive. Here we used a spontaneously occurring homozygous COL17A1 deletion mutant in JEB to discern glycosylation of collagen XVII. The mutation truncated the distal ectodomain and positioned the only N-glycosylation site 34 amino acids from the newly formed C terminus, which impaired efficient N-glycosylation. Immunofluorescence staining of authentic JEB keratinocytes and of COS-7 cells transfected with the mutant indicated intracellular accumulation of collagen XVII precursor molecules. Cell surface biotinylation and quantification of ectodomain shedding demonstrated that only about 15% of the truncated collagen XVII reached the cell surface. The cell surface-associated molecules were N-glycosylated in a normal manner, in contrast to the molecules retained within the cells, indicating that N-glycosylation of the ectodomain is required for targeting of collagen XVII to the plasma membrane and that reduced accessibility of the N-glycosylation site negatively regulates this process. Functional consequences of the strong reduction of collagen XVII on the cell surface included scattered deposition of cell adhesion molecule laminin 5 into the extracellular environment and, as a consequence of faulty collagen XVII-laminin ligand interactions, aberrant motility of the mutant cells.Collagen XVII belongs to the family of collagenous transmembrane proteins, which function both as cell membrane receptors and as extracellular matrix components and are involved in a broad spectrum of biological events, ranging from cell adhesion to host defense (1). The group members typically are trimers of identical polypeptides, ␣-chains, which contain an N-terminal intracellular domain, a single transmembrane stretch, and a large extracellular C terminus with one or more collagenous subdomains, i.e. a structure characteristic of type II transmembrane proteins. The ectodomain is shed from the cell surface by metalloproteinases of the ADAM (a disintegrin and metalloprotease domain) family to yield a shorter form of the molecule, which remains stable in the extracellular space after the release (2). As a component of the hemidesmosomes, collagen XVII is involved in the anchorage of stratified epithelia in the skin, the mucous membranes, or the eye to the underlying mesenchyme.Mutations in the collagen XVII gene, COL17A, cause JEB, a genodermatosis characterized by mechanically induced detachment of the epidermis from the dermis. To date, over 30 different COL17A1 mutations have been reported (Human Gene Mutation Database, Cardiff University; Refs. 3 and 4). Most are null mutations resulting in premature termination codons and, subsequently, in nonsense-mediated mRNA d...

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C-terminal Truncation Impairs Glycosylation of Transmembrane Collagen XVII and Leads to Intracellular Accumulation

Franzke

Has

Schulte

et al. 2006

Journal of Biological Chemistry

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“…The role of collagen XVII in maintaining adhesion is supported by studies on pathological skin conditions. Mutations in the collagen XVII gene, COL17A1, lead to junctional epidermolysis bullosa, a hereditary blistering skin disease with epidermal detachment from the basement membrane (15)(16)(17)(18)(19)(20)(21). Tissue-bound and circulating IgG and IgA autoantibodies to collagen XVII are also associated with skin blistering in autoimmune bullous skin diseases (22)(23)(24)(25)(26)(27)(28).…”

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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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“…Most COL17A1 mutations described to date are nonsense and deletion mutations leading to premature termination of translation and absence of type XVII collagen from the skin (Darling et al, 1997;Floeth et al, 1998;Gatalica et al, 1997;Jonkman et al, 1997;McGrath et al, 1995McGrath et al, , 1996aMcGrath et al, , 1996bScheffer et al, 1997;Schumann et al, 1997;Shimizu et al, 1998 887 acceptor splice-site mutations (Chavanas et al, 1997;Darling et al, 1998;Pulkkinen et al, 1999), one donor splice-site mutation (Pulkkinen et al, 1999), and four missense mutations (Floeth et al, 1998;McGrath et al, 1996a;Schumann et al, 1997;Tasanen et al, 2000) have been reported. In only one (Schumann et al, 1997) of the patients could type XVII collagen be immunologically detected in skin or cultured keratinocytes. The phenotype of JEB-nH caused by type XVII collagen deficiency may vary from generalized blistering with alopecia, also known as generalized atrophic benign epidermolysis bullosa (GABEB) (Hashimoto et al, 1976;Hintner and Wolff, 1982;Jonkman et al, 1996), to mild localized blistering without loss of hair (Floeth et al, 1998;Mazzanti et al, 1998;Schumann et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

“…In only one (Schumann et al, 1997) of the patients could type XVII collagen be immunologically detected in skin or cultured keratinocytes. The phenotype of JEB-nH caused by type XVII collagen deficiency may vary from generalized blistering with alopecia, also known as generalized atrophic benign epidermolysis bullosa (GABEB) (Hashimoto et al, 1976;Hintner and Wolff, 1982;Jonkman et al, 1996), to mild localized blistering without loss of hair (Floeth et al, 1998;Mazzanti et al, 1998;Schumann et al, 1997).In this study, we report for the first time homozygosity for a donor splice-site mutation in COL17A1 leading to multiple aberrant mRNA in a patient with generalized JEB-nH. The most abundant mRNA has an internal deletion of 390 bp, which leads to the expression of a shortened nonfunctional type XVII collagen polypeptide with a 130 amino acid deletion in the carboxy terminal half.…”

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confidence: 98%

Truncated Type XVII Collagen Expression in a Patient with Non-Herlitz Junctional Epidermolysis Bullosa Caused by a Homozygous Splice-Site Mutation

Leusden

Pas

Gedde‐Dahl

et al. 2001

Laboratory Investigation

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SUMMARY:Type XVII collagen (180-kDa bullous pemphigoid antigen) is a structural component of hemidesmosomes.Mutations in the type XVII collagen gene (COL17A1) have been established to be the molecular basis of non-Herlitz junctional epidermolysis bullosa (JEB-nH), an inherited skin blistering disorder. Here we report for the first time truncated type XVII collagen expression, caused by homozygosity for a COL17A1 donor splice-site mutation (4261ϩ1 g 3 c), which was identified by PCR amplification on genomic DNA. By RT-PCR and sequencing of cDNA derived from mRNA from the patient's cultured keratinocytes, we provide evidence of cryptic splicing and exon skipping, most abundantly of exon 52. JEB-nH patients with COL17A1 splice-site mutations resulting in an exon skip often have no immunologically detectable type XVII collagen. However, in our patient with the generalized atrophic benign epidermolysis bullosa subtype, a small amount of type XVII collagen was detectable in the skin, and immunoblotting of cultured keratinocytes revealed that the 180-kDa protein was 10 kDa shorter. We hypothesize that the function of this truncated type XVII collagen polypeptide, which is expressed at low levels, is impaired, explaining the JEB-nH phenotype. (Lab Invest 2001, 81:887-894).T ype XVII collagen (180-kDa bullous pemphigoid antigen; BP180, BPAG2), encoded by the COL17A1 gene, plays a critical role in adhesion of basal keratinocytes to the epidermal basement membrane zone (EBMZ). Its expression is restricted to hemidesmosomes, morphological structures that mediate the adhesion of basal keratinocytes to the underlying EBMZ in stratified and pseudostratified epithelia. Type XVII collagen interacts with other hemidesmosomal components (eg, the ␣6␤4 integrin and 230-kDa bullous pemphigoid antigen [BP230]), and thereby plays a role in the assembly and stabilization of hemidesmosomes (Hopkinson and Jones, 2000;Schaapveld et al, 1998). The molecule is a type II transmembrane protein whose carboxy terminal end is located outside the cell. This extracellular part contains 15 collagenous domains (COL1-15), interspaced by noncollagenous domains (NC1-16) (Giudice et al, 1992), which gives the molecule a certain flexibility. By electron microscopy, type XVII collagen is seen as a rigid central rod (COL15) followed by a movable tail (Hirako et al, 1996). Like other collagens, the molecule is able to trimerize by self assembly in such a way that the extracellular fragment forms a triple helix, ␣1[XVII] 3 (Balding et al, 1997;Hirako et al, 1996;Pas et al, 1999). The length of the rod domain is sufficient to traverse the lamina lucida, and the flexible carboxyl terminus has been seen within the lamina densa (Shimizu, 1998). Its as yet unidentified ligand(s) may reside within the lamina densa or lamina lucida. A candidate ligand is laminin 5, which is mainly localized in the upper lamina densa (Shimizu, 1998). An interaction between the extracellular domain of type XVII collagen and laminin 5 has been reported (Reddy et al, 1998). The globular...

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Three Novel Homozygous Point Mutations and a New Polymorphism in the COL17A1 Gene: Relation to Biological and Clinical Phenotypes of Junctional Epidermolysis Bullosa

Cited by 75 publications

References 29 publications

C-terminal Truncation Impairs Glycosylation of Transmembrane Collagen XVII and Leads to Intracellular Accumulation

C-terminal Truncation Impairs Glycosylation of Transmembrane Collagen XVII and Leads to Intracellular Accumulation

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

Truncated Type XVII Collagen Expression in a Patient with Non-Herlitz Junctional Epidermolysis Bullosa Caused by a Homozygous Splice-Site Mutation

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