cDNA Cloning of the 210-kDa Paraneoplastic Pemphigus Antigen Reveals that Envoplakin Is a Component of the Antigen Complex

Abstract

Although the 210 and 190-kDa proteins are the most frequently detected antigens reacting with sera of patients with paraneoplastic pemphigus (PNP) in immunoblot analysis, there is still uncertainty as to the nature of these PNP antigens. To isolate and characterize a cDNA clone encoding the 210-kDa PNP antigen, we screened a human keratinocyte lambda gt 11 cDNA expression library by the immunoperoxidase method with serum IgG from a PNP patient. The IgG used for the immunoscreening of a keratinocyte cDNA expression library recognized 210- and 190-kDa antigens by immunoblotting. A single clone, called here the PNP clone, producing a fusion protein that reacted strongly with the patient's IgG, was further characterized. Only the PNP patient's IgG, but not IgG from a normal control, pemphigus foliaceus, or pemphigus vulgaris patients, bound the plaques of this positive clone. Furthermore, PNP IgG affinity purified on plaques of this clone, but not unrelated clones, bound to keratinocyte cell surfaces by immunofluorescence and reacted with the 210-kDa PNP antigen by immunoblotting. EcoRI digestion of the clone's cDNA insert demonstrated a 1.4-kbp fragment. This cDNA insert was placed into a M13 mp 18 vector and sequenced. Sequence analysis revealed that the cDNA insert of the PNP clone encodes a part of the central rod domain and the COOH-terminal C domain of envoplakin, a newly defined precursor of the cornified envelope that is homologous to desmoplakin. This result demonstrates that the 210-kDa PNP antigen is envoplakin and PNP is an autoimmune disease that produces autoantibodies against intermediate filament-associated proteins in desmosomes and hemidesmosomes, desmoplakin, bullous pemphigoid antigen 1 (BPAG 1), and envoplakin.

Envoplakin and Periplakin Are the Paraneoplastic Pemphigus Antigens.

“…This further supports the previous result by Kim et al that immunoscreening a keratinocyte cDNA library with a PNP serum cloned a partial cDNA corresponding to envoplakin [30]. Joly et al showed that PNP sera react with a 185 kDa protein that co-migrates with the protein detected by a human IgM monoclonal antibody F12 [33], which has been established from lymphocytes of a pemphigus vulgaris patient [34], and it will be interesting to determine whether F12 reacts with periplakin.…”

“…In fact, Kim et al (1997) have recently reported that by immunoscreening a keratinocyte cDNA library with a PNP serum they cloned a partial cDNA conesponding to envoplakin, which strongly suggests that the 210 kDa protein is envoplakin [30]. In the present study, using polyclonal antibodies specific to envoplakin and periplakin and sera of Immunoblot analysis…”

Antibodies against desmoglein 3 (pemphigus vulgaris antigen) are present in sera from patients with paraneoplastic pemphigus and cause acantholysis in vivo in neonatal mice.

“…The 210-kD antigen recognized by immunoprecipitation was found to be a doublet, of which the slower migrating band was desmoplakin II. The faster migrating band, which is recognized by immunoblotting (8), was later identified by cDNA cloning as envoplakin (9). Envoplakin is a novel precursor protein of the cornified envelope that is homologous to desmoplakin (10).…”

Paraneoplastic Pemphigus: An Unusual Neoplasm Association

“…Immunofluorescence reveals typical binding of both IgG and C3 in a focal, intercellular epidermal pattern and linear granular pattern at the basement membrane zone. Immunoprecipitation and immunoblotting reveal several characteristic target antigens of molecular weights 250, 230, 210, 190 and 170 kDa corresponding to desmoplakin, the 230-kDa bullous pemphigoid (BP) antigen BP230, envoplakin, periplakin and transmembrane protein [5][6][7][8][9]. In addition,a pathogenic role has also been suggested for antidesmoglein (Dsg) antibodies [10].…”