The Autoantigen of Anti-p200 Pemphigoid Is an Acidic Noncollagenous N-Linked Glycoprotein of the Cutaneous Basement Membrane (1)
Indirect immunofluorescence of pa- tients’ sera demonstrates circulating IgG autoantibodies labeling the dermal side of NaCl-split normal skin. The antigenic target of these antibodies is a 200-kDa protein (p200) of the human dermis that is thought to be important for cell-matrix adhesion. While p200 was demonstrated to be im- munologically distinct from all major autoantigens of the DEJ, including bullous pemphigoid (BP) antigens 180 and 230, a6b4 integrin, laminin 5 and 6, and type VII collagen.
Fresh normal human skin was obtained from plastic surgery and incubated in phosphate-bu¡ered saline, pH 7.2 (PBS), supplemented with 1 M NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 5 mM ethylenediaminetetraacetic acid (EDTA) at 4C for 96 h. After dermal epidermal separation, the epidermis was peeled off and discarded. The epidermal surface of the dermis was extensively washed with PBS and extracted with buffer A (12.5 mM Tris-HCl, pH 7.0, 8 M urea, 6% b-mercaptoethanol, 1 mM PMSF, and 5 mM EDTA) at room temperature. After 1 h, the dermal extract was collected and centrifuged, and the supernatant was stored at -80C.
In all six sera anti-p200 reactivity was mediated by IgG4 autoantibodies. Only one serum demonstrated weak additional IgG1 and IgG2 reactivity.
Reduction of disulfide bonds with b-mercaptoethanol enables significant amounts of p200 to be solubilized by both urea concentrations, 4M and 8M. Reduction of the urea concen- tration by 50% caused roughly 50% of p200 to precipitate. Approximately 60% of originally extracted protein remained soluble in 4 M, 20% in 2 M, and 10% in 1 M urea. p200 was completely insoluble in nonurea buffers. Addition of detergents did not improve the solubility of the protein. Although cultured cells were demonstrated to produce various basement membrane proteins, including BP180, the unprocessed (200 -kDa) and processed (165 -kDa) forms of the a3 chain of laminin 5, and type VII collagen, sera from patients with anti-p200 pemphigoid did not react with any protein derived from the cellular extract or medium.
Dermal extracts were digested with bacterial collagenase and assayed for reactivity with sera from patients with anti-p200 pemphigoid and monoclonal antibody LH7.2 against human type VII collagen. Even prolonged incubation with bacterial collagenase did not result in degradation of p200. In contrast, full- length type VII collagen (290 kDa) was digested to its collagenase-resistant NC1 domain (145 kDa) within 1 h of collagenase treatment. Treatment of dermal extracts with bacterial protease V8 resulted in complete loss of p200 reactivity within 30 min after addition of the protease.
The deglycosylated protein migrated almost exactly midway between the intact p200 in dermal extracts and BP180 recognized by rabbit serum SA8010 in extracts of cultured human keratinocytes.
p200 molecules form highly insoluble S-S-dependent complexes. These complexes may represent homomultimers of p200, similar to those described for type VII collagen. Minor amounts of p200 remained soluble in the presence of low urea concentrations, whereas removal of urea resulted in complete precipitation of the protein. Various collagen types have been implicated in human autoim- mune blistering diseases. Type VII collagen is a well-characterized autoantigen of epidermolysis bullosa acquisita, type XVII col- lagen/BP180 is the common autoantigen in di¡erent pemphigoid diseases, and the a5 chain of type IV collagen has been recently described as the antigenic target in patients with a novel autoim- mune subepidermal blistering disease. Carbohydrate antigenic determinants were demonstrated to be important for autoantibody response in several diseases, including pernicious anemia, chronic Chagas’disease, rheumatoid arthritis, and endometriosis. Most sera samples from patients with linear IgA disease react with cleavage-derived neoepitopes on the LABD97 and LAD-1 autoantigens but not with the full-length BP180 molecule.
