A disease-associated glycine substitution in BP180 (type XVII collagen) leads to a local destabilization of the major collagen triple helix

Olague-Marchan, Mónica; Twining, Sally S.; Hacker, Mary K.; McGrath, John A.; Díaz, Luis A.; Giudice, George J.

doi:10.1016/s0945-053x(00)00070-6

Cited by 13 publications

(12 citation statements)

References 40 publications

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“…19,24 The relative positions of the BP180 recombinant and fusion proteins are indicated in Fig 1 and are referred to by the first and last amino acid (AA) of the selected protein: AA 490 to 1497 (sec180), AA 490 to 566 (NC16A), and outside the NC16A domain, AA 567 to 1497 (delNC16A), AA 1080 to 1107, AA 1280 to 1315, AA 1331 to 1404, AA 1365 to 1413, and AA 1365 to 1458.…”

Section: Methodsmentioning

confidence: 99%

Missing the target: Characterization of bullous pemphigoid patients who are negative using the BP180 enzyme-linked immunosorbant assay

Fairley

Bream

Fullenkamp

et al. 2013

Journal of the American Academy of Dermatology

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Background Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by autoanti-bodies specific for the 180-kd BP antigen-2 (BP180) (also termed “type XVII collagen”) protein. The BP180 enzyme-linked immunosorbent assay (ELISA) is specific for the immunodominant NC16A domain of the protein. However, we and others have observed patients whose reactivity to BP180 is exclusive of the NC16A domain (referred to henceforth as non-NC16A BP). Objective We sought to determine the incidence of non-NC16A BP and identify regions of reactivity within the BP180 protein. Methods Sera from 51 patients who met the clinical and histologic criteria for BP were screened for NC16A reactivity by ELISA. Sera that were negative by ELISA were screened for IgG reactivity to an epidermal extract, recombinant BP180 protein, and subregions of BP180, by immunoblot. Demographic and clinical data were also collected on all patients. Results Four sera (7.8%) were negative using the BP180 ELISA but positive for IgG reactivity to the extracellular domain of BP180. Further mapping identified 4 regions outside of NC16A recognized by these sera: amino acid (AA) 1280 to 1315, AA 1080 to 1107, AA 1331 to 1404, and AA 1365 to 1413. One of these sera also had IgE specific for NC16A. One patient had an atypical presentation with lesions limited to the lower aspect of the legs and scarring of the nail beds. Limitations The small total number of patients with non-NC16A BP limits the identification of demographic or clinical correlates. Conclusion It is significant that 7.8% of sera from patients with new BP react to regions of BP180 exclusively outside of NC16A and, thus, would not be identified using the currently available BP180 ELISA.

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Section: Methodsmentioning

confidence: 99%

Missing the target: Characterization of bullous pemphigoid patients who are negative using the BP180 enzyme-linked immunosorbant assay

Fairley

Bream

Fullenkamp

et al. 2013

Journal of the American Academy of Dermatology

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“…Participants gave their written informed consent. BP180 constructs A recombinant protein comprising the entire BP180 ectodomain (designated sec180) was generated using the pCEP4 eukaryotic expression system (Invitrogen, Carlsbad, California), as previously reported (Olague-Marchan et al, 2000). Briefly, the cDNA encoding the amino-terminal signal sequence and precursor peptide from human desmoglein-1 was inserted into the multiple cloning region of the pCEP4 vector.…”

Section: Methodsmentioning

confidence: 99%

“…The portion of BP180 encoded by pCEP4-sec180 extends from Glu-490 to the C-terminus, Pro-1497 (note: amino acid numbering begins with Met-36 from GenBank M91669). The pCEP4-180DN16A construct was generated via a standard PCR-based mutagenesis protocol (Olague-Marchan et al, 2000) using pCEP4-sec180 as the template. This truncated construct encodes all but the NC16A region of the BP180 ectodomain (Gly-567 through Pro-1497).…”

Section: Methodsmentioning

confidence: 99%

Mapping the Binding Sites of Anti-BP180 Immunoglobulin E Autoantibodies in Bullous Pemphigoid

Fairley

Giudice

2005

Journal of Investigative Dermatology

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Bullous pemphigoid (BP) is a subepidermal blistering disease characterized by autoantibodies against the hemidesmosomal protein BP180 (BPAg2, type XVII collagen). NC16A, a non-collagenous stretch of the BP180 ectodomain, is the primary target of pathogenic immunoglobulin (Ig)G autoantibodies and IgE class autoantibodies. This study further characterized the IgE-reactive regions of BP180. Of the ten sera from untreated BP patients, eight contained IgE reactive with the entire BP180 ectodomain. The IgE in four of these eight sera reacted with NC16A, whereas in the remaining four sera IgE immunoreactivity was restricted to sites downstream of NC16A. In contrast, IgG reactivity to NC16A was detected in nine of the ten BP sera, and in the remaining serum, IgG, as well as IgE, reacted exclusively with non-NC16A sites on the BP180 ectodomain. Fine mapping of the antigenic sites within NC16A revealed very similar reactivity patterns for IgE and IgG, with NC16A subregion-2 being the major site recognized by both isotypes. Eight of the untreated BP patients were tested for histamine release from their basophils in response to NC16A. Antigen-specific histamine release was observed only in those patients with detectable circulating IgE directed against NC16A (three of eight). Future studies will investigate the pathogenic relevance of anti-BP180 IgE.

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“…The hypothesis is that the mutations may lead to inhibition of physiological ligand binding or proteolytic degradation of the mutant protein and to skin blistering. When these glycine substitutions were generated into a recombinantly expressed Col15 or full-length ectodomain, the thermal stability of the Col15 domain was significantly decreased (Olague-Marchan et al, 2000;Tasanen et al, 2000a, b).…”

Section: Discussionmentioning

confidence: 99%

Molecular Mechanisms of Junctional Epidermolysis Bullosa: Col15 Domain Mutations Decrease the Thermal Stability of Collagen XVII

Väisänen

Has

Franzke

et al. 2005

Journal of Investigative Dermatology

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Mutations in the collagen XVII gene, COL17A1, are associated with junctional epidermolysis bullosa. Most COL17A1 mutations lead to a premature termination codon (PTC), whereas only a few mutations result in amino acid substitutions or deletions. We describe here two novel glycine substitutions, G609D and G612R, and a splice site mutation resulting in a deletion of three Gly-X-Y amino acid triplets. In order to investigate the molecular pathomechanisms of non-PTC mutations, G609D and G612R and two previously known substitutions, G627V and G633, and deletion of the amino acids 779-787 were introduced into recombinant collagen XVII. The thermal stability of the mutated collagens was assessed using trypsin digestions at incremental temperatures. All the four glycine substitutions significantly destabilized the ectodomain of collagen XVII, which manifested as 16 degrees C-20 degrees C lower T(m) (midpoint of the helix-to-coil transition). These results were supported by secondary structure predictions, which suggested interruptions of the collagenous triple helix within the largest collagenous domain, Col15. In contrast, deletion of the three full Gly-X-Y triplets, amino acids 779-787, had no overall effect on the stability of the ectodomain, as the deletion was in register with the triplet structure and also generated compensatory changes in the NC15 domain.

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A disease-associated glycine substitution in BP180 (type XVII collagen) leads to a local destabilization of the major collagen triple helix

Cited by 13 publications

References 40 publications

Missing the target: Characterization of bullous pemphigoid patients who are negative using the BP180 enzyme-linked immunosorbant assay

Missing the target: Characterization of bullous pemphigoid patients who are negative using the BP180 enzyme-linked immunosorbant assay

Mapping the Binding Sites of Anti-BP180 Immunoglobulin E Autoantibodies in Bullous Pemphigoid

Molecular Mechanisms of Junctional Epidermolysis Bullosa: Col15 Domain Mutations Decrease the Thermal Stability of Collagen XVII

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