Yh. Song et al., LOCALIZATION OF A PERNICIOUS-ANEMIA AUTOANTIBODY EPITOPE ON THE ALPHA-SUBUNIT OF HUMAN H,K-ADENOSINE TRIPHOSPHATASE, Scandinavian journal of gastroenterology, 29(2), 1994, pp. 122-127
Four cDNA fragments encoding different portions of the Lu-subunit of h
uman H,K-adenosine triphosphatase (ATPase) were amplified by means of
the polymerase chain reaction technique, ligated into the plasmid pGEX
-2T, and expressed as glutathione S-transferase fusion proteins in Esc
herichia coli. The fragments A (residues 163-313), Ba (residues 360-79
7), Bb (residues 526-797), and C (residues 822-1031) together encompas
s 77% of the alpha-subunit and cover most of its cytosolic part. The r
eactivities of autoantibodies in the sera from patients with perniciou
s anaemia with the recombinant fusion proteins were analysed by immuno
blotting. One autoantigenic epitope was found in the NH2-terminal part
of the Ba fragment-that is, between residues 360 and 525. No epitope
was detected in the other fragments. The Ba fragment was cleaved off f
rom the glutathione S-transferase fusion protein by the action of thro
mbin and was then further purified. By means of enzyme-linked immunoso
rbent assay, 28 of 42 sera (67%) from patients with pernicious anaemia
were positive against the purified Ba fragment. The present results p
rovide a final proof that the human H,K-ATPase cu-subunit is a major a
utoantigen in the parietal cell and that the major epitope is located
between residues 360 to 525 on the cytosolic side of the secretory mem
brane.