TRANSPORT TO ENDOPLASMIC-RETICULUM BY SIGNAL PEPTIDE, BUT NOT PROTEOLYTIC PROCESSING, IS REQUIRED FOR FORMATION OF CONFORMATIONAL EPITOPES OF PEMPHIGUS-VULGARIS ANTIGEN (DSG3)

Desmoglein 3 is the autoimmune target of pemphigus vulgaris. Most, if not all, pathogenic autoantibodies are raised against conformational e pitopes on desmoglein 3. In this study, we examined whether posttransl ational modification in endoplasmic reticulum is required for the prop er three-dimensional structure formation of recombinant pemphigus vulg aris antigen. Previously, we have produced by baculovirus expression a secreted form of desmoglein 3, PVIg, which represents equivalent conf ormational 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 co nstructed a mutant PVIg molecule, PVIg-fXa, whose putative endoproteol ytic cleavage site was replaced by the recognition sequence of serum c oagulation factor Xa, PVIg-fXa was produced without proteolytic proces sing; 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, P VIg-Delta sig, which lacks the signal peptide and prosequence. PVIg-De lta sig was not secreted and accumulated in the cytosol, PVIg-Delta si g failed to show the immunoadsorption, Together with our previous find ing 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 t hey need to be formed in the endoplasmic reticulum, and emphasize the importance of conformation of the antigen in pathogenic autoantibody b inding.