Molecular Mapping of the Major Epitopes of BP180 Recognized by Herpes Gestationis Autoantibodies (1)
Herpes gestationis (HG) is a nonherpetic autoimmune disease of women characterized by the development of subepidermal inflammatory blisters during the last trimester of pregnancy. of BP180, a hemidesmosomal protein also known as BPAg2 and type XVII collagen. The BP180 antigen is also targeted by IgG autoantibodies produced by patients suffering from bullous pemphigoid (BP), an inflammatory subepidermal blistering disease most frequently observed in the elderly. The key features of HG and BP was found to be dependent on complement activation and neutrophil recruitment to the site of subepidermal blister formation. anti-BP180 autoantibodies present in the sera of HG and BP patients play a key role in the pathogenesis of these diseases.
Epitope mapping experiments were performed using a panel of BP180–glutathione S-transferase (GST) fusion proteins—NC16A (AA 490–562), NC16A1 (AA490–506), NC16A1-3 (AA 490–534), NC16A2-5 (AA507–630 and 830–855), NC16A2-4 (AA 507–548), NC16A2-3 (AA 507–534 and 836–855), NC16A2 (AA 507–520), NC16A2.5 (AA 514–532), and NC16A3 (AA521–534).
Sera from representative HG patients contain IgG autoantibodies that recognize sec180e.
Sera from all 37 HG patients contained IgG reactivity with sec180e.
All 37 HG sera reacted with fusion proteins NC16A1-5, NC16A1-3, NC16A2-5, NC16A2-4, and NC16A2-3, all five of which contain the BP180 segment AA 507–534. None of the 37 HG sera reacted with NC16A1. Major epitopes recognized by HG sera are located between AA 507 and 534.
The first and largest group of HG sera (Group I, composed of 26 of the 37 sera) reacted with NC16A2, but not with NC16A2.5 or NC16A3. The second group of HG sera (Group II, n 5 5) reacted with both NC16A2 and NC16A2.5. Group III (n 5 3) reacted with all three fusion proteins, and Group IV (n 5 3) reacted with NC16A2.5 and NC16A3 but not NC16A2. Preadsorption of Groups II and III sera with NC16A2.5 resulted in a diminution, but not elimination, of reactivity with NC16A2. Preadsorption of these same eight sera with NC16A2 reduced, but did not abolish, their immunoreactivity with NC16A2.5. The Group III sera preadsorbed against NC16A3 continued to react with NC16A2.5, although not as strongly as the unadsorbed or GST-adsorbed sera. Preadsorption of Group IV sera with NC16A3 knocked out their reactivity with NC16A2.5. After preadsorption of both Group III and IV sera with NC16A2.5, these six sera no longer reacted with NC16A3.
All 37 HG sera lost the ability to bind to the sec180e recombinant protein when immunoreactivity to NC16A was eliminated.
One NC16A site, a 14 amino acid stretch (RSILPYGDSMDRIE) designated MCW-1, was recognized by 34 of the 37 HG sera assayed, and in 26 of these cases. The Group I HG sera reacted with MCW-1, but not with NC16A2.5, thus eliminating the region of overlap (i.e., the C-terminal half of MCW-1) as the antigenic target for these 26 HG sera. Second, Groups II and III HG sera depleted of immunoreactivity with NC16A2.5 continued to react, albeit somewhat more weakly, with MCW-1. Therefore, Groups I, II, and III HG sera (which account for 34 of the 37 HG sera in this study) contain IgG autoantibodies that react with the N-terminal seven amino acid portion of MCW-1, which designates MCW-1A (RSILPYG). Two other HG-associated antigenic sites—one within the region of overlap between NC16A2.5 and NC16A3 and one located within NC16A2.5 but not in either MCW-1 or NC16A3. Four HG-associated antigenic sites were localized to a 28 amino acid segment of NC16A (AA 507–534), one of which, a seven amino acid stretch designated MCW-1A, is recognized by the vast majority of HG patients’ sera.