Characterization of disease-causing PV antibodies affinity purified on rDsg3-Ig-His. A: RT-PCR amplification of the sequence encompassing the junction region in the rDsg3-Ig-His baculovirus with a forward primer matching the Dsg 3 sequence and a reverse primer matching the human Fc IgG1 sequence. A PCR product appeared at the expected size when genomic DNA was isolated from infected Sf9 cells (lane 2), but not from noninfected insect cells (lane 3). The PCR product size was estimated using the 100 bp DNA ladder run in parallel (lane 1). B: Western blotting of the affinity-purified rDsg3-Ig-His baculoprotein used in absorption experiments. The chimeric protein migrated with an apparent molecular weight of 105 kd where it was recognized by a PV serum (lane 1). No protein band can be seen in the control lane in which primary antibody was omitted (lane 2). C: Genotyping of a Dsg 3−/− (Dsg3^null) mouse versus a Dsg 3+/− mouse. The Dsg3^null mice were used both as a recipient in passive transfer experiments and as a source of epidermal proteins in immunoblotting experiments. Note: both the Neo band (220 bp) and the Dsg3 band (500 bp) were amplified from Dsg 3+/− mouse, whereas amplification of the Dsg 3 knockout mouse DNA gave only the Neo band. D: Immunoblotting characterization of PV IgGs eluted from rDsg3-Ig-His after absorption of the PV serum-coded PRC-45, using epidermal proteins from a Dsg3^null mouse as a substrate. The eluted PV IgGs specifically recognized polypeptides with apparent molecular weights of 38, 55, 60, 80, 85, 110, 120, 130, 140, 170, and 190 kd (lane 1). A 115-kd protein was excluded because it was nonspecifically visualized in a negative control experiment in which the primary antibody was omitted (lane 2). Positions of the molecular weight markers in kd are shown on the right.

Visualization of keratinocyte α9 AChR—a novel PV antigen. A and B: Specific blocking of the immunostaining of the cell membranes of keratinocytes by FITC-conjugated rabbit anti-α9 monoepitopic antibody because of preincubation with the PV serum PRC-45. The strong intercellular staining of the cryostat section of monkey esophagus resulting from anti-α9 antibody binding to the cell membrane (A) was completely eliminated in the specimen preincubated with PV (B) but not normal human serum (not shown). Scale bars, 50 μm. C: Characterization of the affinity-purified monoepitopic anti-α9 AChR rabbit antibody. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved membrane protein extract of cultured human oral keratinocytes was used as the substrate for immunoblotting. The anti-α9 antibody recognized a protein that migrated with an apparent molecular weight of 50 kd (lane 1). The specificity of this single-band antibody binding was illustrated by the ability to abolish the staining in control experiments, in which a strip of the immunoblotting membrane was exposed to the anti-α9 antibody that had been preincubated overnight with 150 nmol of the peptide immunogens (lane 2). In another set of control experiments, no staining was detected when the primary antibody was omitted (lane 3). The molecular weight markers in kd are shown on the right. D–F: Visualization of α9 AChR on the keratinocyte cell membrane. In a series of indirect immunofluorescence experiments, nonpermeabilized specimens of normal human attached gingiva (D) and a second passage preconfluent (E) or confluent (F) monolayers of normal human gingival keratinocytes were incubated with immunoaffinity-purified rabbit anti-α9 AChR antibody, and its binding was visualized by using secondary, FITC-conjugated donkey anti-rabbit IgG antibody, as detailed in the Material and Methods. The antibody visualized α9 AChR on the cell membrane of keratinocytes, producing a characteristic intercellular, or pemphigus-like fishnet staining of the epithelium (D). In keratinocyte monolayers, the antibody decorated the cell surface of preconfluent cultured keratinocytes (E), and was found clustered at the sites of cell-to-cell contacts in confluent cultures (F), indicating that α9 AChRs are localized predominantly to the areas of keratinocyte cell membrane associated with cell-to-cell contacts. Scale bars, 50 μm.

Pemphigus-like acantholysis in a monolayer of second passage human oral keratinocytes treated with immunoaffinity-purified rabbit anti-α9 AChR monoepitopic antibody. The phase-contrast images were obtained in monolayers, grown to confluence in serum-free keratinocyte growth medium in a standard 6-well cell culture plate, before (A), and 15 (B), 30 (C), 45 (D), 60 (E), and 75 (F) minutes after the addition of 10 μg/ml of anti-α9 AChR antibody. Note: separation of individual keratinocytes from neighboring cells is followed by peripheral cytoplasmic blebbing and cell rounding-up. The pictures were taken from the same microscopic field before and after addition of antibody. No changes in cell morphology were observed in the monolayer treated with the equal amount of pre-immune serum from the same rabbit (not shown). Scale bar, 50 μm.

Agarose gel electrophoresis of overlapping portions of the human α9 AChR mRNA amplified in the course of step-by step RT-PCR experiments. Total RNA was extracted from second passage human gingival keratinocytes and used as a template in RT-PCR experiments detailed in the Material and Methods section. The PCR products amplified from DNase-treated RNA samples (lanes 2) appeared at expected sizes, which were deduced from rat α9 sequence. The DNase nontreated, nonreverse-transcribed RNA samples (lanes 1), which contained some residual genomic DNA, were used to test the efficacy of DNase I treatment in the negative control experiments (lanes 3) in which the RT step was omitted. The absence of the bands with the expected sized in lanes 1 of the “products 1–3” indicates that the primers amplified genomic sequences containing introns, whereas the presence of the band with the expected size in lane 1 of the “product 4” indicates that the primers amplified an intronless genomic sequence. An additional negative control experiment included the use of water instead of the RNA template (lanes 4) to control for any possible contamination with the genetic material throughout the entire procedure. No PCR products were obtained in either negative control experiment, ie, no bands in lanes 3 or 4.

The complete reading frame of human α9 AChR. A: Schematic representation of the overlapping sequences of human α9 AChR cDNAs amplified in the course of step-by-step RT-PCR experiments, showing positions of the nucleotide sequences of the amplified products. B: The nucleotide sequence and the predicted amino acid sequence of human α9 AChR. The putative signal sequence and the membrane-spanning regions (USR) are indicated. Positions of the peptides used to generate monoepitopic anti-α9 antibody are underlined. Double-underlined are the cysteines in the positions 133, 147, 197, and 198, which can provide reducible double-disulfide bridges, and the negatively charged Asp 136 and 143, Thr 138, Gln 145 and 146, which also can be involved in formation of the ACh binding pocket. C: Amino acid sequence alignment of rat and human α9 AChR homologues. The dots indicate identical residues of the two protein sequences exhibiting 92% similarity.