Conformational epitopes of pemphigus antigens (Dsg1 and Dsg3) are calcium dependent and glycosylation independent.
The target molecule of pemphigus autoantibodies is a transmembrane desmosomal component, desmoglein 3 (Dsg3) in pemphigus vulgaris (PV) and Dsg1 in pemphigus foliaceus (PF). In this study, we examined the effects of calcium and glycosylation on the anti-genicity of the pemphigus antigens and on the generation of conformational epitopes. We used recombinant baculovirus proteins, PVIg and PFIg, which are considered to reflect accurately the native conformation of the extracellular domain of their respective proteins Dsg3 and Dsg1. These baculoproteins could immunoadsorb heterogeneous autoantibodies from the corresponding sera of PV and PF patients, completely blocking indirect immunofluorescence staining of normal human skin. Chelating calcium from the solution containing the baculoproteins using ethylenediaminetetraacetic acid (EDTA) or ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) abolished immunoadsorption by both PVIg and PFIg; however, immunoadsorption by the baculoproteins was restored after dialysis against 1 mM calcium. Nonglycosylated forms of both baculoproteins produced in the presence of tunicamycin retained their immunoadsorptive ability. Furthermore, immunoadsorption by the baculo-proteins was prevented irreversibly by treatment with low pH, high pH, and boiling, but not with the non-ionic detergent Nonidet P-40. These findings indicate that formation of the conformational epitopes on the pemphigus antigens is dependent on calcium but independent of glycosylation, and provide direct evidence that calcium plays an important role in determining the antigenic properties of the pemphigus antigens.
Amagai
M
M
Department of Dermatology, Keio University School of Medicine, Japan.
Ishii
K
K
Hashimoto
T
T
Gamou
S
S
Shimizu
N
N
Nishikawa
T
T
D000327:Adsorption; D015820:Cadherins / Q000737:chemistry / Q000276:immunology* / Q000502:physiology*; D002118:Calcium / Q000502:physiology*; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000276:immunology* / Q000502:physiology*; D051183:Desmoglein 1; D051184:Desmoglein 3; D051182:Desmogleins; D051180:Desmoplakins; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008968:Molecular Conformation; D010392:Pemphigus / Q000097:blood
D015820:Cadherins; D003598:Cytoskeletal Proteins; C495297:DSG1 protein, human; C071894:DSG3 protein, human; D051183:Desmoglein 1; D051184:Desmoglein 3; D051182:Desmogleins; D051180:Desmoplakins; D000939:Epitopes; D002118:Calcium