Cloning of Hamster Type XVII Collagen cDNA, and Pathogenesis of Anti-Type XVII Collagen Antibody and Complement in Hamster Bullous Pemphigoid Katsushi.

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Cloning of Hamster Type XVII Collagen cDNA, and Pathogenesis of Anti-Type XVII Collagen Antibody and Complement in Hamster Bullous Pemphigoid Katsushi Yamamoto, Naokazu Inoue, Riako Masuda, Akira Fujimori, Toshiyuki Saito, Shinobu Imajoh-Ohmi, Hiroshi Shinkai, Hisako Sakiyama Journal of Investigative Dermatology Volume 118, Issue 3, Pages 485-492 (March 2002) DOI: 10.1046/j.0022-202x.2001.01683.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Hamster type XVII collagen cDNA and the deduced amino acid sequence. The deduced amino acid sequence revealed that hamster type XVII collagen consists of a large NC domain (NC-1), amino acids 1–569, and a C-terminal collagenous domain consisting of 14 separate segments with a Gly-X-Y repeating sequence (underlined). The NC-1 domain is predicted to contain the transmembrane domain (amino acids 472–493, bold and underlined). The six potential N-glycosylation sites are shown in bold face. Journal of Investigative Dermatology 2002 118, 485-492DOI: (10.1046/j.0022-202x.2001.01683.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Direct comparison of the hamster, human, mouse, and canine deduced amino acid sequences of type XVII collagen. The hamster transmembrane domain (bold) is identical to those of human, mouse, and canine. The antigenic site on type XVII collagen was not conserved among hamster, human, mouse, and canine (box). Journal of Investigative Dermatology 2002 118, 485-492DOI: (10.1046/j.0022-202x.2001.01683.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Reactivity of antipeptide 1191 antisera and antipeptide 1190 antisera with the 180 kDa protein of hamster skin extracts. Hamster epidermal extract known to contain type XVII collagen was separated on a 7.5% SDS polyacrylamide gel, and transferred to a PVP membrane. The membrane was cut and incubated with rabbit anti-1191 peptide antisera, anti-1190 antisera, or control rabbit sera. Journal of Investigative Dermatology 2002 118, 485-492DOI: (10.1046/j.0022-202x.2001.01683.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Separation of the DEJ of hamsters administered with anti-1191 IgG. Neonatal hamsters were intraperitoneally injected with IgG at doses from 0.04 to 5.0 mg anti-1191 IgG per g body weight. The epidermis shown in this figure was obtained from a hamster injected with 1.0 mg anti-1191 IgG per g body weight. The epidermis was easily lifted away from the dermis with tweezers 24 h after injection (arrows). Journal of Investigative Dermatology 2002 118, 485-492DOI: (10.1046/j.0022-202x.2001.01683.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Histologic examination of hamster skin injected with anti-1191 IgG or control IgG. The skins of hamsters injected with 5.0 mg anti-1191 IgG per g body weight (a - c) or 5.0 mg control IgG per g body weight (d- f) were frozen in OCT compound and sectioned. The sections were stained with hematoxylin and eosin (a, d), anti-rabbit IgG (b, e), or anti-human C3 (c, f). The skins of hamsters injected with anti-1191 IgG showed a subepidermal cleft (a, arrows), an accumulation of IgG in the basement membrane zone over a subepidermal cleft (b, arrows), and a linear deposition of C3 in the basement membrane zone (c, arrows). The skins of hamsters injected with control IgG did not show any pathologic change (d, e, f). Neither IgG (e) nor C3 (f) were observed. Scale bar: 30 µm. Journal of Investigative Dermatology 2002 118, 485-492DOI: (10.1046/j.0022-202x.2001.01683.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effects of anti-1191 IgG and complement on DEJ separation in vitro. Skins from neonatal hamsters were incubated with fresh anti-1191 antisera (a - c), purified anti-1191 IgG plus DMEM medium (d- f), fresh anti-1191 antisera preincubated with CVF (g- i), heat-inactivated fresh anti-1191 antisera plus normal rabbit serum (j- l), purified anti-1191 IgG plus fresh normal rabbit sera (m- o), and purified anti-1191 IgG plus fresh normal rabbit sera preincubated with CVF (p- r). Sections were stained with hematoxylin and eosin (a, d, g, j, m, p), anti-rabbit IgG (b, e, h, k, n, q), or anti-human C3 (c, f, i, l, o, r). Skins incubated with fresh anti-1191 antisera, heat-inactivated fresh anti-1191 antisera plus fresh normal rabbit sera, and purified anti-1191 IgG plus fresh normal rabbit sera showed a subepidermal cleft (a, j, m, arrows), IgG deposition (b, k, n, arrows), and C3 deposition in the basement membrane zone over the subepidermal cleft (c, l, o, arrows). The skins incubated with anti-1191 IgG, fresh anti-1191 antisera preincubated with CVF, and purified anti-1191 IgG plus normal rabbit sera preincubated with CVF showed no pathologic change in the skin (d, g, p). Although IgG deposition was observed in the basement membrane zone (e, h, q), no C3 deposition was detected (f, i, r). Scale bar: 30 µm (a, d-k, m, n, p, q), 12 µm (b, c, l, o, r). Journal of Investigative Dermatology 2002 118, 485-492DOI: (10.1046/j.0022-202x.2001.01683.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions