Human autoantibodies against the 230-kD bullous pemphigoid antigen (BPAG1) bind only to the intracellular domain of the hemidesmosome, whereas those against the 180-kD bullous pemphigoid antigen (BPAG2) bind along the plasma membrane of the hemidesmosome in normal human and swine skin.

Abstract

Bullous pemphigoid (BP) is a blistering skin disease in which autoantibodies develop to hemidesmosomal components of the epidermal basement membrane zone, including two major antigenic proteins of the 230-kD antigen (BPAG1 ) and the 180-kD antigen (BPAG2). The present study demonstrated the precise ultrastructural localization of the epitopes for autoantibodies against BPAG1 and BPAG2 in normal skin. Autoantibodies against either BPAG1 or BPAG2 were affinity-purified using nitrocellulose membrane, which was blotted with SDS-PAGEfractionated antigens from human epidermal extract as the immunoabsorbent. Postembedding, immunogold electron microscopy was performed after skin was processed by rapid freezing and freeze substitution fixation without chemical fixatives. Purified autoantibodies against BPAG1 bound only to the intracellular domain of the hemidesmosome, and 80% of the gold labeling was within 40-140 nm from the plasma membrane (mean distance 91 nm inside). In contrast, the autoantibodies against BPAG2 bound along the plasma membrane of the hemidesmosome, and 80% of the gold labeling was within 10 nm outside to 50 nm inside the cells (mean distance 12 nm inside). These results suggest that the autoantibodies against BPAG1 and BPAG2 react with the epitopes localizing in distinct regions of the hemidesmosome complex, and may play different roles in the blister formation in patients with

New insights into the immunoultrastructural organization of cutaneous basement membrane zone molecules

“…Pre-embedding immunoperoxidase EM also revealed the absence of detectable laminin 5 beneath the melanocytes (52). These data indicate that laminin 5 is closely related to or is co-localized with hemidesmosomes (52), and that the results correlate well with the previous finding that melanocytes do not express hemidesmosome-associated molecules, such as 230-kD BPAG1 and 180-kD BPAG2 (39,55).…”

“…In contrast, the autoantibodies against BPAG2 bound along the plasma membrane of the hemidesmosome and 80% of the gold labeling was within 10 nm outside to 50 nm inside the cells (39). The differing locations of BPAG1 and BPAG2 suggests that BPAG2, the transmembranous molecule which is directly exposed to autoantibodies in vivo, may play a more significant role than BPAG1 in the pathogenesis of BP blister formation (39).…”

“…Post-embedding immuno-EM with cryofixation and cryosubstitution without chemical fixatives demonstrated that the sera from patients with BP reacted with both the intracellular and extracellular domains of the hemidesmosome (13), whereas rabbit polyclonal and human monoclonal antibodies against BPAG1 bound only to the intracellular domain of the hemidesmosome (37,38). Quantitative analysis revealed that affinity-purified autoantibodies against BPAG1 bound only to the intracellular domain of the hemidesmosome, and that 80% of the gold labeling was observed to be within 40-140 nm from the plasma membrane (39). In contrast, the autoantibodies against BPAG2 bound along the plasma membrane of the hemidesmosome and 80% of the gold labeling was within 10 nm outside to 50 nm inside the cells (39).…”

“…Quantitative analysis revealed that affinity-purified autoantibodies against BPAG1 bound only to the intracellular domain of the hemidesmosome, and that 80% of the gold labeling was observed to be within 40-140 nm from the plasma membrane (39). In contrast, the autoantibodies against BPAG2 bound along the plasma membrane of the hemidesmosome and 80% of the gold labeling was within 10 nm outside to 50 nm inside the cells (39). The differing locations of BPAG1 and BPAG2 suggests that BPAG2, the transmembranous molecule which is directly exposed to autoantibodies in vivo, may play a more significant role than BPAG1 in the pathogenesis of BP blister formation (39).…”

“…A previous study showed that the majority (80%) of affinity-purified BPAG2 autoantibodies obtained from BP patients bound along the plasma membrane of the hemidesmosome within 10 nm outside to 50 nm inside the cells (39). Thus the location of this molecule was considered to be restricted along the plasma membrane.…”