Epitopes in the Linker Subdomain Region of Envoplakin Recognized by Autoantibodies in Paraneoplastic Pemphigus Patients Bingxin Zhang, Rui Zheng, Jing.

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Epitopes in the Linker Subdomain Region of Envoplakin Recognized by Autoantibodies in Paraneoplastic Pemphigus Patients Bingxin Zhang, Rui Zheng, Jing Wang, Dingfang Bu, Xuejun Zhu Journal of Investigative Dermatology Volume 126, Issue 4, Pages 832-840 (April 2006) DOI: 10.1038/sj.jid.5700198 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Sequence alignment of recombinant linker domains of envoplakin, periplakin, desmoplakin, and BPAG1 we used as the antigens for ELISA. Black shaded letters, identical amino acids; gray shaded and light gray shaded letters, highly conservative and conservative amino-acid changes among the four proteins. Underlined letters are sequence of peptide no. 8 used as the probe to examine the specific antigen receptor on surface membrane of B lymphocytes in tumor sections. Journal of Investigative Dermatology 2006 126, 832-840DOI: (10.1038/sj.jid.5700198) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunoblotting of PNP sera to the recombinant linker subdomains of envoplakin, periplakin, desmoplakin, and BPAG1. 1–12: sera from PNP cases 1–12; PV: serum from a PV patient; PF: serum from a PF patient; BP: serum from a BP patient; N: serum from a normal individual. Journal of Investigative Dermatology 2006 126, 832-840DOI: (10.1038/sj.jid.5700198) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Affinity of PNP sera binding to the 12 peptides covering the linker subdomain of envoplakin by ELISA. The affinity of serum binding to the 12 peptides is presented as net OD490. The short and horizontal line in each column marks the 2 × net OD490 of upper 95% confidence interval from normal controls. The upper figure of panel a shows the results of the 12 PNP sera. Numbers above the lines denote the case numbers of PNP, and sera with net OD490 below the upper limit are only indicated as circles in columns. The lower figure of panel a is the results of the controls. Squares, rhombuses, and triangles represent sera from PV, PF, and BP, respectively. Panel b is the results of competitive ELISA. The upper figure of panel b used PNP sera pre-incubated with the peptides same as those coated on the wells. The lower figure used the PNP sera pre-incubated with peptides PERI-L, DESM-L, or BPAG1-L to react with peptide no. 8 on the wells. Serum of case 1 was not available any more in this test. Journal of Investigative Dermatology 2006 126, 832-840DOI: (10.1038/sj.jid.5700198) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Dot blot of PNP sera to the peptide no. 1–12. Sera of cases 1 and 3 were not available any more for this test. N: serum from a normal individual. Journal of Investigative Dermatology 2006 126, 832-840DOI: (10.1038/sj.jid.5700198) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Cells in tumor sections stained by peptide no. 8 and anti-CD20 antibody. Double staining of tumor cells by peptide no. 8 or the control peptide (green) and anti-human CD20 antibody (red) was observed by confocal laser scanning microscope. (a–c) A tumor section from case 8 was immunostained by peptide no. 8 and anti-human CD20 antibody, showing the (a) green staining, (b) red staining, and (c) combination of the two images. Mixed green and red staining appears orange in the merged figure. Peptide no. 8 staining locates on the surface of tumor cells and fits well with the CD20 staining, suggesting that the specific membrane immunoglobulin receptors locate predominately on CD20+ cells. (d–f) A lymphoid follicle structure from case 12 was shown, it was double stained by peptide no. 8 and anti-human CD20 antibody. (g–i) The tonsil section was immunostained by peptide no. 8 and anti-human CD20 antibody. (j–l) The section was immunostained by the control peptide and anti-human CD20 antibody. No green staining beyond background was detected in (g) and (j), suggesting that the binding of peptide no. 8 to the surface of CD20+ cells is specific. (m–o) Peptide no. 8 was pre-absorbed by a serum from PNP case 8 and then immunostained the tumor section from the same PNP case. The green staining became very weak. Journal of Investigative Dermatology 2006 126, 832-840DOI: (10.1038/sj.jid.5700198) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Peptide sequence for the localization of areas of major epitopes to PNP autoantibodies. Domain structure of envoplakin is shown at the upper line. N, amino-terminal globular domain; R, central coiled-coil rod domain; C, C-subdomain; L, linker subdomain (Ruhrberg et al., 1996). Peptides are aligned to show their correspondent positions in the linker subdomain of envoplakin. Journal of Investigative Dermatology 2006 126, 832-840DOI: (10.1038/sj.jid.5700198) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions