Bullous pemphigoid and cicatricial pemphigoid autoantibodies react with ultrastructurally separable epitopes on the BP180 ectodomain: evidence that BP180 spans the lamina lucida.

The BP180 antigen is a hemidesmosomal glycoprotein that is recognized by autoantibodies associated with three autoimmune disorders, bullous pemphigoid (BP), herpes gestationis (HG), and cicatricial pemphigoid (CP). BP and HG sera have been shown to recognize a common extracellular site located near the membrane-spanning domain of this protein, whereas CP sera react predominantly with a distinct site near the C terminus. In the current study, the main immunogenic sites on the BP180 ectodomain were ultrastructurally localized using six BP sera, four CP sera, and two rabbit antisera. The immunolocalization pattern of BP sera was largely restricted to the upper lamina lucida region immediately subjacent to the epidermal hemidesmosome and closely resembled that of a rabbit antiserum directed against the NC16A (membrane-proximal) domain of BP180. CP sera, on the other hand, exhibited a lower lamina lucida/lamina densa labeling pattern that was strikingly similar to that of rabbit antibodies to the BP180 C-terminal region. Finally, antibodies to the BP180 C-terminal region co-localized with an anti-laminin-5 antibody in the anchoring filament zone. These findings strongly suggest that the BP180 extracellular domain exists in an extended conformation, with the C terminus of this protein projecting into the lamina densa. These data support the hypothesis that BP180 contributes to the structure and function of the anchoring filaments. Differences in the ultrastructural mapping of BP and CP autoantibodies appear to correlate with epitope mapping data, which, together, may help to explain the clinical heterogeneity observed in this group of bullous disorders.

Tight clustering of extracellular BP180 epitopes recognized by bullous pemphigoid autoantibodies.

Bullous pemphigoid is a blistering skin disease associated with autoantibodies against the BP180 antigen, a transmembrane component of the hemidesmosome. Anti-BP180 antibodies have been demonstrated to be pathogenic in a passive transfer mouse model. One extracellular site on human BP180 (MCW-1) was previously shown to be recognized by 50-60% of bullous pemphigoid sera. To facilitate the identification of additional autoantibody-reactive epitopes, recombinant forms of the BP180 ectodomain were generated using both bacterial and mammalian expression systems. One recombinant protein, sec180e, that was expressed in COS-1 cells and that contained the entire BP180 ectodomain, provided us with a tool to detect conformational epitopes. Bullous pemphigoid sera immunoadsorbed against the major noncollagenous NC16A domain no longer reacted with sec180e, indicating that autoantibody reactivity to the BP180 ectodomain is restricted to the NC16A region. Immunoblot analysis of bullous pemphigoid sera immunoadsorbed with a series of recombinant NC16A peptides revealed the presence of three novel autoantigenic sites that, along with the MCW-1 epitope, are clustered within the N-terminal 45 amino acid stretch of NC16A. All 15 bullous pemphigoid sera tested reacted with a recombinant protein containing this BP180 segment. No disease-associated epitopes were detectable within the remaining 28 amino acids of NC16A. Thus, bullous pemphigoid patient autoantibodies react with a set of epitopes on the BP180 ectodomain that are highly clustered. This autoantibody-reactive region on human BP180 shows overlap with the corresponding murine BP180 site that is targeted by antibodies that are pathogenic in the mouse model of bullous pemphigoid. These findings suggest new directions for the development of diagnostic and therapeutic tools for this disease.

Cicatricial pemphigoid autoantibodies react with multiple sites on the BP180 extracellular domain.

Cicatricial pemphigoid (CP) is an autoimmune blistering disease that primarily affects mucosal tissues. Autoantibodies to laminin-5 have previously been detected in certain patients with a CP-like disease; however, individuals that exhibit this reactivity profile apparently represent a small subset of CP patients. In the present investigation, 0 of 18 CP sera showed reactivity with laminin-5 by immunoblotting. In contrast, 18 of 23 CP sera (78%) recognized a 180-kDa epidermal antigen that, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, co-migrated with BP180, a hemidesmosomal glycoprotein associated with two other autoimmune blistering diseases, bullous pemphigoid and herpes gestationis. To investigate further the CP autoimmune response, various segments of human BP180 were expressed as bacterial fusion proteins and assayed by immunoblotting for reactivity with CP patients' sera. The results of this investigation demonstrated that the BP180 autoantigen is indeed a major target of CP autoantibodies. Further, two distinct CP-reactive sites were identified on the extracellular domain of the BP180 protein, one located in the non-collagenous (NC) 16A domain (at or near the previously defined autoantibody-reactive site recognized by bullous pemphigoid and herpes gestationis sera) and the other in the carboxy-terminal region of this protein. Sixteen of 23 CP sera (70%) reacted with one or both of these antigenic sites of BP180. Other immunologic data suggested that BP180 may harbor additional CP-reactive sites. In conclusion, there are now three bullous diseases, bullous pemphigoid, herpes gestationis, and cicatricial pemphigoid, that are known to be associated with an autoimmune response against the extracellular domain of the BP180 antigen.

Analysis of antigens targeted by circulating IgG and IgA autoantibodies in 50 patients with cicatricial pemphigoid.

In this study we investigated sera from 50 typical cicatricial pemphigoid (CP) patients. By indirect immunofluorescence on 1 M NaCl-split human skin sections, IgG of 17 sera and IgA of 22 sera reacted with the epidermal side of the split, while IgG of two sera reacted with the dermal side. These latter two sera were later confirmed to be anti-epiligrin CP. By immunoblotting of epidermal extracts, IgG of 14 sera reacted with the 230 kD bullous pemphigoid (BP) antigen (BP230). IgG of 15 sera and IgA of 11 sera reacted with the 180 kD BP antigen (BP180). Interestingly, a bacterial fusion protein containing the BP180 NC16a domain was recognized by IgG of 18 sera but not by IgA of any sera. Fusion proteins containing the C-terminal region of BP180 were recognized by IgG of 20 sera, but it was detected by IgA of only two sera. Our results suggest that, although CP sera show very low titers of autoantibodies, a considerable number of sera contain IgG antibodies to BP180 (either NC16a or C-terminal domain), confirming previous studies. In addition, we showed that greater numbers of IgA antibodies react with BP180, seemingly with different types of epitopes from those for IgG antibodies. Because the specificity of IgG antibodies is not very different from those in BP, IgA antibodies may play a specific role for the development of characteristic clinical features in CP. Future studies should elucidate the pathogenic role of the IgA antibodies in CP.

L-cone pigment genes expressed in normal colour vision.

To directly test the hypothesis that only two pigment genes are expressed from the X-chromosome array, we examined expressed M and L pigment gene sequences from > 100 male eye donors. In this sample, there were eight men who expressed high levels of more than one L pigment gene in addition to M pigment genes. The fact that these eyes expressed both L and M pigment genes at significant levels suggests they were from men with normal colour vision. We reject the hypothesis that only two pigment genes from one X-chromosome array can be expressed.

Pigment gene expression in protan color vision defects.

We screened 150 male eye donors and identified four who did not have or express L pigment genes, consistent with each of them having a congenital protan color vision defect. One donor was identified as a protanope because he had and expressed a single X-chromosome photopigment gene that encoded an M pigment. Three were categorized as protanomalous because each expressed significant levels of genes specifying two spectrally different M pigments. The first gene in each of the protanomalous arrays was expressed the most and encoded an M pigment that differed in amino acid sequence from M pigments in color normal men.

A recombinant form of the human BP180 ectodomain forms a collagen-like homotrimeric complex.

BP180 is a glycoprotein constituent of the epidermal anchoring complex and a major antigenic target of autoantibodies associated with bullous pemphigoid, a blistering skin disease. The C-terminal extracellular domain of BP180 contains 15 domains composed of Gly-X-Y tandem repeats, which are predicted to form collagen-like triple helices. To facilitate the structural analysis of this protein, the extracellular region of human BP180 was expressed as a secreted protein (sec180e) in transiently transfected COS-1 cells. Gel filtration and sedimentation analyses demonstrated that sec180e exists in two forms: a globular monomeric form and a high-molecular mass multimeric form with an elongated conformation. Pulse-chase and cross-linking experiments established that the sec180e complex is a stable homotrimeric structure which assembles prior to secretion from the cell. On the basis of its calculated molecular mass, the oligomeric state of the sec180e complex is 3.25. With a Stokes radius of 13.6 nm, a sedimentation coefficent of 6.5 S, and a frictional ratio of 3.01, the sec180e protein appears to be highly extended (length to width ratio is between 52 and 60), yet is more flexible than a rigid rod. BP180 isolated from human epidermis was also shown to exist in a high-molecular mass complex which, like sec180e and other collagenous proteins, is SDS-stable but heat-labile. These findings strongly suggest that the BP180 ectodomain exists as an elongate, flexible homotrimer. This trimerization is likely to result from the formation of stable collagen triple-helical and coiled-coil type structures and does not depend upon the presence of the cytoplasmic or transmembrane domains of this protein.

BP180 gene delivery in junctional epidermolysis bullosa.

Epidermolysis bullosa (EB) comprises a family of inherited blistering skin diseases for which current therapy is only palliative. Junctional EB (JEB) involves dissociation of the dermal-epidermal junction and results from mutations in a number of genes that encode vital structural proteins, including BP180 (type XVII collagen/BPAG2). In order to develop a model of corrective gene delivery for JEB, we produced a retroviral expression vector for wild-type human BP180 and used it to restore BP180 protein expression to primary keratinocytes from BP180-negative patients with generalized atrophic JEB. Restoration of full-length BP180 protein expression was associated with adhesion parameter normalization of primary JEB keratinocytes in vitro. These cells were then used to regenerate human skin on immune-deficient mice. BP180 gene-transduced tissue demonstrated restoration of BP180 gene expression at the dermal-epidermal junction in vivo while untransduced regenerated JEB skin entirely lacked BP180 expression. These findings provide a basis for future efforts to achieve gene delivery in human EB skin tissue.