Identification of a Novel Non-desmoglein Autoantigen in Pemphigus Vulgaris

Abstract

Pemphigus vulgaris (PV) is an autoimmune bullous disease of the skin and mucous membranes characterized by the presence of circulating and tissue-bound autoantibodies against keratinocyte cell surface antigens, specifically desmoglein (Dsg) 1 and 3. The pathogenic role of anti-Dsg antibodies is well-established, while the mechanism of blister formation is only partly defined. We have applied a previously developed method for the efficient immortalization of IgG+ memory B cells to identify novel target antigens in PV. A human monoclonal antibody reactive with a hitherto unreported non-Dsg antigen was isolated. Immunoprecipitation and immunoblotting studies with keratinocyte extracts indicated α-catenin as the putative antigen, then confirmed by immunoblotting on the recombinant protein. Four of ten PV sera reacted with recombinant α-catenin. Although the isolated human monoclonal antibody was per se unable to dissociate keratinocyte monolayers and also to synergize with a pathogenic antibody in vitro, further studies are warranted to assess its possible in vivo contribution in the multifactorial pathogenesis and heterogeneous manifestations of PV disease.

Keywords: autoantibodies; memory B cells; mucous membranes; non-desmoglein autoantigens; pemphigus vulgaris; skin; α-catenin.

Figure 1

Intercellular staining pattern of hMab PVF144. PVF144 binds a membrane associated epithelial antigen showing a typical intercellular staining pattern on permeabilized HaCaT keratinocytes (nuclear counterstain is obtained with DAPI) (20X) (A), human skin (40X) (B) guinea pig (40X) (C), and monkey esophagus (40X) (D).

Figure 2

PVF144 binds a membrane-associated epithelial antigen: α-catenin. PVF144 immunoprecipitates (IP) an unknown antigen of 100 kDa from radiolabeled normal human keratinocyte extracts (A). Immunoblotting (IB) experiments on keratinocyte extracts by using PVF144 and commercial antibodies suggest that α-catenin could be the putative antigen of 100 kDa: PVF144 and a monoclonal murine anti-α-catenin antibody (Ab) react with a keratinocyte antigen of similar molecular weight (100 kDa) (B). Anti-α-catenin commercial antibody reacts to α-catenin from keratinocyte extracts by IB (lane 1); PVF144 immunoprecipitates α-catenin recognized by the commercial anti-α-catenin antibody by IB (lane 2) and, viceversa, anti-α-catenin antibody immunoprecipitates α-catenin recognized by PVF144 by IB (lane 3). The faint reactivity observed in lane 3 could be related to the epitope recognized (C). IB performed with a recombinant GST-tagged human α-catenin (120 kDa) confirms that the target of PVF144 is α-catenin. The lower bands are likely degradation products (D).

Figure 3

Almost half of PV sera specifically react with recombinant α-catenin. Immunoblotting with sera obtained from 10 pemphigus vulgaris (PV) patients shows that 4 of 10 sera (4, 8, 9, 10) react with the novel epithelial antigen, while 3 healthy donor sera (11, 12, 13), representative of 10 sera analyzed, show only a background signal. The positive control (C+) is the commercial anti-α-catenin antibody. The background signal, could be also due to reactivity to the tag protein (GST). Quantification and normalization of bands using ImageJ analysis (data not shown) have confirmed the reported results (see Materials and Methods section).

Figure 4

PVF144 is not able to dissociate a keratinocyte monolayer even in the presence of suboptimal concentrations of PVB28. A representative keratinocyte dissociation experiment. Primary human keratinocytes, seeded to confluence, were incubated with PVF144, an unrelated human Mab (negative control) and, as positive controls, the human pathogenic Mab PVB28 (7) and the murine pathogenic Mab AK23 (14), both Dsg3-specific (A). To investigate synergistic potential of cloned antibody we have employed PVF144 (10 μg/ml) together with optimal (4 μg/ml) and suboptimal concentrations (1 μg/ml and 0.25 μg/ml) of the pathogenic anti-Dsg3 antibody PVB28 without obtaining any difference in ability of PVB28, with or without PVF144, to dissociate the keratinocyte monolayer (B).