M. Amagai et al., CONFORMATIONAL EPITOPES OF PEMPHIGUS ANTIGENS (DSG1 AND DSG3) ARE CALCIUM-DEPENDENT AND GLYCOSYLATION INDEPENDENT, Journal of investigative dermatology, 105(2), 1995, pp. 243-247
The target molecule of pemphigus autoantibodies is a transmembrane des
mosomal component, desmoglein 3 (Dsg3) in pemphigus vulgaris (PV) and
Dsg1 in pemphigus foliaceus (PF). In this study, we examined the effec
ts of calcium and glycosylation on the antigenicity of the pemphigus a
ntigens and on the generation of conformational epitopes. We used reco
mbinant baculovirus proteins, PVIg and PFIg, which are considered to r
eflect 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 immunofluorescenc
e staining of normal human skin. Chelating calcium from the solution c
ontaining the baculoproteins using ethylenediaminetetraacetic acid (ED
TA) or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraaceti
c acid (EGTA) abolished immunoadsorption by both PVIg and PFIg; howeve
r, immunoadsorption by the baculoproteins was restored after dialysis
against 1 mM calcium. Nonglycosylated forms of both baculoproteins pro
duced in the presence of tunicamycin retained their immunoadsorptive a
bility. Furthermore, immunoadsorption by the baculoproteins was preven
ted irreversibly by treatment with low pH, high pH, and boiling, but n
ot with the non-ionic detergent Nonidet P-40. These findings indicate
that formation of the conformational epitopes on the pemphigus antigen
s is dependent on calcium but independent of glycosylation, and provid
e direct evidence that calcium plays an important role in determining
the antigenic properties of the pemphigus antigens.