Immunoblotting studies of linear IgA disease

Dmochowski, Marian; Hashimoto, Takashi; Bhogal, B.S.; Black, Martin M.; Zone, John J.; Nishikawa, Takeji

doi:10.1016/0923-1811(93)90038-q

Cited by 107 publications

(78 citation statements)

References 13 publications

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“…Linear IgA bullous dermatosis (LAD) is an autoimmune subepidermal blistering disease characterized by linear deposits of IgA autoantibodies at the dermal-epidermal basement membrane. Immunoblot analyses of patients' sera have demonstrated that the target antigens are heterogeneous, although a 97-kDa antigen is most frequently detected (44,45). Recent studies have shown that the autoantibodies recognize a 97-kDa and a 120-kDa polypeptide in skin extracts and a 120-kDa form in keratinocyte culture medium (46).…”

Section: Figmentioning

confidence: 99%

Cleavage of BP180, a 180-kDa Bullous Pemphigoid Antigen, Yields a 120-kDa Collagenous Extracellular Polypeptide

Hirako¹,

Usukura²,

Uematsu³

et al. 1998

Journal of Biological Chemistry

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114

124

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The hemidesmosome (HD) is a cell-to-substrate adhesion apparatus found in stratified and complex epithelia. One of the putative cell-matrix adhesion molecules present in the HD is the 180-kDa bullous pemphigoid antigen (BP180), also termed type XVII collagen. In our previous study, using a monoclonal antibody (mAb) 1337, we have detected a 120-kDa collagenase-sensitive polypeptide in the HD fraction (Uematsu, J. and Owaribe, K. (1993) Cell Struct. Funct. 18, 588 (abstr.)). The present study was undertaken to assess the relation of the 120-kDa polypeptide to this BP180. Immunofluorescence microscopy of bovine skin revealed the basement membrane zone of skin to be stained clearly with mAb 1337, whereas the lateral surfaces of basal cells, which were decorated by typical antibodies against BP180, were not. The antibody did not detect HDs in cultured cells but rather in the culture medium. These results indicate a localization of mAb 1337 antigen distinct from BP180. However, the same polypeptide was also recognized by monoclonal antibodies to the extracellular but not the cytoplasmic part of BP180, and found to react with a polyclonal antibody against the non-collagenous 16A domain of BP180. Therefore, the polypeptide was identified as an extracellular fragment of BP180. mAb 1337 immunoprecipitated the 120-kDa fragment from the medium, but not the 180-kDa molecule of BP180 extracted from cultured cells, indicating that the antibody specifically recognizes the fragment. The mAb 1337 apparently recognizes a unique epitope that is exposed or formed by the cleavage. Hence, the staining pattern observed for bovine skin demonstrated the presence of the 120-kDa extracellular fragment. Rotary shadow electron microscopy of affinity-purified 120-kDa fragments demonstrated that they have the unique molecular shape consisting of a central rod and a flexible tail, without the globular head that is present in the BP180 molecule. From these results, we conclude that mAb 1337 shows unique epitope specificity, recognizing only the 120-kDa extracellular fragment of BP180, which is constitutively cleaved on the cell surface as a 120-kDa fragment both in in vivo and in vitro.

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Section: Figmentioning

confidence: 99%

Cleavage of BP180, a 180-kDa Bullous Pemphigoid Antigen, Yields a 120-kDa Collagenous Extracellular Polypeptide

Hirako¹,

Usukura²,

Uematsu³

et al. 1998

Journal of Biological Chemistry

Self Cite

114

124

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“…Normal human epidermal extracts were derived as described previously (25). Briefly, fresh normal human skin was incubated in PBS containing 2 mM EDTA and 1 mM PMSF for 48 h at 4˚C.…”

Section: Preparation Of Epidermal Extractsmentioning

confidence: 99%

Antibodies to Pathogenic Epitopes on Type XVII Collagen Cause Skin Fragility in a Complement-Dependent and -Independent Manner

Natsuga

Nishie

Shinkuma

et al. 2012

The Journal of Immunology

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In bullous pemphigoid (BP), the most prevalent autoimmune blistering disease, type XVII collagen (COL17) is targeted by circulating autoantibodies. BP is thought to be an autoantibody-mediated complement-fixing blistering disease, and a juxtamembranous noncollagenous 16A (NC16A) domain spanning Glu490 to Arg566 was proved to be the main pathogenic region on COL17, although precise pathogenic epitopes within NC16A have not been elucidated. In this study, we showed that injection of rabbit IgG Abs targeting Asp522 to Gln545 induced skin fragility associated with in vivo deposition of IgG and complement in neonatal COL17-humanized mice. Notably, immunoadsorption of rabbit anti-NC16A IgG Ab with this epitope (Asp522 to Gln545) or the anti-NC16A IgG administered together with the peptides of this epitope as a decoy ameliorated skin fragility in the injected neonatal COL17-humanized mice compared with the anti-NC16A IgG alone even though all of the mice showed both IgG and complement deposition. These results led us to investigate an additional, complement-independent mechanism of skin fragility in the mice injected with anti-COL17 Abs. The rabbit anti-NC16A IgG depleted the expression of COL17 in cultured normal human keratinocytes, whereas immunoadsorption of the same IgG with this epitope significantly suppressed the depletion effect. Moreover, passive transfer of F(ab′)2 fragments of the human BP or rabbit IgG Abs against COL17 demonstrated skin fragility in neonatal COL17-humanized mice. In summary, this study reveals the importance of Abs directed against distinct epitopes on COL17, which induce skin fragility in complement-dependent as well as complement-independent ways.

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“…Immunoblot analysis was performed using normal human epidermal and dermal extracts as substrates, as previously described [3]. Immunoblotting of dermal extracts demonstrated that the patient’s serum contained circulating IgG autoantibodies, which reacted with a 290-kD band.…”

Section: Histological and Immunopathological Studiesmentioning

confidence: 99%

A Case of an Inflammatory Variant of Epidermolysis bullosa acquisita: Chronic Bullous Dermatosis Associated with Nonscarring Mucosal Blisters and Circulating IgG Anti-Type-VII-Collagen Antibody

et al. 1998

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A 42-year-old man showed prominent blistering lesions of the mouth and esophagus in addition to a few bullous lesions of the skin. Direct immunofluorescence microscopy revealed distinct linear deposition of IgG and C3 at the epidermal basement membrane zone where slight deposition of IgA and IgM was also observed. In direct immunoelectron-microscopic examination, antibody was detected in the sublamina densa of the basement membrane zone. Immunoblot analysis with dermal extracts demonstrated that the patient’s serum contained circulating IgG antibodies against the 290-kD protein, which comigrated with type VII collagen. The lesions healed without any scars. The results of these studies corresponded to the laboratory findings in epidermolysis bullosa acquisita (EBA), although the clinical features were distinct from classic EBA.

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Immunoblotting studies of linear IgA disease

Cited by 107 publications

References 13 publications

Cleavage of BP180, a 180-kDa Bullous Pemphigoid Antigen, Yields a 120-kDa Collagenous Extracellular Polypeptide

Cleavage of BP180, a 180-kDa Bullous Pemphigoid Antigen, Yields a 120-kDa Collagenous Extracellular Polypeptide

Antibodies to Pathogenic Epitopes on Type XVII Collagen Cause Skin Fragility in a Complement-Dependent and -Independent Manner

A Case of an Inflammatory Variant of Epidermolysis bullosa acquisita: Chronic Bullous Dermatosis Associated with Nonscarring Mucosal Blisters and Circulating IgG Anti-Type-VII-Collagen Antibody

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