α and β Subunits of the Gastric H/K -ATPase Are Concordantly Targeted by Parietal Cell Autoantibodies Associated with Autoimmune Gastritis

Abstract

We have previously shown that parietal cell autoantibodies predominantly react with a 60-90 kDa gastric autoantigen^1,2, subsequently identified as the (3 subunit of the gastric H⁺/K⁺-ATPase (EC 3.6.1.3) (proton pump)^3-5 whereas Karlsson et al⁶ showed that these autoantibodies primarily target the 95 kDa α subunit of the pump. In view of these discordant results, we have reassessed the reactivity of parietal cell autoantibodies with the two subunits of the gastric H⁺/K⁺-ATPase. We show here that all 26 parietal cell autoantibody-positive sera immunoblot both subunits under appropriate, but mutually exclusive, conditions. Thus, reactivity of anti- parietal cell autoantibodies with the 95 kDa α subunit is optimal when the SDS-PAGE is carried out with samples which are reduced but not boiled. Whereas reactivity with the 60-90 kDa β subunit is optimal with samples which are boiled but not reduced. Autoantibody reactivity with the β subunit is critically dependent on the presence of a full complement of N-linked glycans since partially deglycosylated protein, and recombinant β subunit expressed in COS cells, bearing high mannose N-glycans, failed to bind to the autoantibody. These studies also suggest that B cell auto-epitopes are located on the lumenal domain of the β subunit. Reactivity of parietal cell autoantibodies with a bacterial fusion protein incorporating the catalytic cytoplasmic domain of the α subunit suggests the presence of auto-epitopes in this region of the molecule.