Desmoglein 3 is the autoimmune target of pemphigus vulgaris. Most, if not all, pathogenic autoantibodies are raised against conformational epitopes on desmoglein 3. In this study, we examined whether posttranslational modification in endoplasmic reticulum is required for the proper three-dimensional structure formation of recombinant pemphigus vulgaris antigen. Previously, we have produced by baculovirus expression a secreted form of desmoglein 3. PVIg, which represents equivalent conformational epitopes of the native antigen and showed that PVIg is able to immunoadsorb heterogeneous autoantibodies from pemphigus vulgaris patients' sera. To elucidate the role of proteolytic processing, we constructed a mutant PVIg molecule, PVIg-fXa, whose putative endoproteolytic cleavage site was replaced by the recognition sequence of serum coagulation factor Xa. PVIg-fXa was produced without proteolytic processing; however, exogenous treatment of PVIg-fXa with factor Xa resulted in cleavage of the prosequence. Interestingly, not only the processed PVIg-fXa, but also the unprocessed form, showed the immunoadsorptive activity. Furthermore, to elucidate the role of endoplasmic reticulum signal peptide at the amino terminus, we constructed another mutant, PVIg-Δsig which lacks the signal peptide and prosequence. PVIg-Δsig was not secreted and accumulated in the cytosol. PVIg-Δsig failed to show the immunoadsorption. Together with our previous finding on the role of glycosylation, these observations indicate that the conformational epitopes of the recombinant pemphigus antigen are not affected either by glycosylation or proteolytic processing, although they need to be formed in the endoplasmic reticulum, and emphasize the importance of conformation of the antigen in pathogenic autoantibody binding.
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