Native Autoantigen Complex Detects Pemphigoid Autoantibodies

Abstract

Pemphigoid diseases are a group of autoimmune disorders characterized by subepidermal blistering in the skin and mucosa. Among them, mucous membrane pemphigoid (MMP) autoantibodies are characterized by targeting multiple molecules in the hemidesmosomes, including collagen XVII, laminin-332, and integrin a6/β4. Traditionally, recombinant proteins of the autoantigens have been employed to identify circulating autoantibodies by immune assays. However, developing an efficient detection system for MMP autoantibodies has been challenging because the autoantibodies have heterogeneous profiles and the antibody titers are typically low. In this study, we introduce an ELISA that takes advantage of a native autoantigen complex rather than simple recombinant proteins. We generated HaCaT keratinocytes with a DDDDK-tag knocked in at the COL17A1 locus by CRISPR/Cas9-mediated gene editing. Immunoprecipitation using the DDDDK-tag isolated a native complex that contained full-length and processed collagen XVII and integrin α6/β4. Then, we used the complex proteins to prepare an ELISA system and enrolled 55 MMP cases to validate its diagnostic performance. The sensitivity and specificity of the ELISA for detecting MMP autoantibodies were 70.9% and 86.7%, respectively, far superior to those of conventional assays. In autoimmune diseases such as MMP, in which autoantibodies target various molecules, isolating the antigen-protein complexes can help establish a diagnostic system.

Figure 1

Generation of HaCaT stably expressing endogenous DDDDK-tagged COL17. (a) Western blotting to confirm Cas9 expression in HaCaT stably expressing Cas9 (referred to as Cas9(+) HaCaT) selected according to puromycin resistance. (b) The isolated Cas9(+) clone shows a cobblestone-like appearance. Bar = 500 μm. (c) In-frame insertion of the ssDNA containing the DDDDK-tag sequence to the exon2 of COL17A1 gene after the homologous directed repair. (d) When the DDDDK-tag was successfully knocked into the COL17A1 gene, the fusion protein showed a double-positive for anti-DDDDK and anti-COL17. Western blotting showed one clone was positive for anti-DDDDK and anti-COL17. CNT: Cas9(+) HaCaT. (e) Immunofluorescence images of HaCaT. Number 12 clone showed DDDDK signals colocalized with COL17 NC16A. Bar = 50 μm. COL17, collagen XVII; ssDNA, single-strand DNA.

Figure 2

Purified nCOL17 autoantigen complex and its analysis. (a) Western blotting of Cas9(+) HaCaT and DDDDK-COL17 HaCaT using an anti-COL17 antibody shows that endogenous COL17 was detected in both Cas9(+) HaCaT and DDDDK-COL17 HaCaT cell lysates. The final samples immunoprecipitated with anti-DDDDK antibodies showed a positive band only in the DDDDK-COL17 HaCaT. (b) Coomassie Brilliant Blue staining of immunoprecipitated DDDDK-COL17 HaCaT shows not only180-kDa bands but also additional 250, 160, 130 and 120-kDa bands (arrows). (c) DDDDK-COL17 HaCaT specific bands corresponded to cell adhesion molecules detected by LC-MS/MS. (d) Comparison of rCOL17 and nCOL17 content proteins by western blotting. A complex of full-length COL17 (black arrows), processed COL17 (red arrows), ITGα6/β4, and E-cad (black arrowheads) was confirmed in nCOL17. COL17, collagen XVII; E-cad, E-cadherin; kDa, kilodalton; LC-MS/MS, liquid chromatography with tandem mass spectrometry; nCOL17, native COL17; rCOL17, recombinant COL17.

Figure 3

The diagnostic performance of rCOL17 and nCOL17 ELISAs on BP. (a) Scatter plot representation of rCOL17 ELISA index. (b) ROC analysis of rCOL17 ELISA. (c) Scatterplot representation of the nCOL17 ELISA index. (d) ROC analysis to determine a cutoff value for the nCOL17 ELISA. (e) Scatter plot of rCOL17 ELISA indices and nCOL17 ELISA indices of BP sera (n = 50). (f) The nCOL17 ELISA-preferential BP sera were positive not only for the 180-kDa of the COL17 (black arrows) but also for other extra bands (red arrows). BP, bullous pemphigoid; COL17, collagen XVII; nCOL17, native COL17; rCOL17, recombinant COL17; ROC, receiver operating characteristic; kDa, kilodalton.

Figure 4

Western blotting using BP sera with nCOL17 as a substrate. The BP sera were positive for the 180-kDa of the COL17 (black arrows) and other extra bands (red arrows). DDDDK: anti-DDDDK antibody; COL17: anti-COL17 antibody. BP, bullous pemphigoid; COL17, collagen XVII; nCOL17, native COL17; kDa, kilodalton.

Figure 5

The nCOL17 ELISA detects not only COL17 but also autoantibodies to ITGs in patients with MMP. (a) Scatter plot of rCOL17 ELISA indices and nCOL17 ELISA indices of MMP sera (n = 55). Based on the cutoff values of each of the ELISAs in the diagnosis of BP, the green region indicates positive for both ELISAs (r+n+), the red indicates positive only for the nCOL17 ELISA (r-n+), the orange indicates positive only for the nCOL17 ELISA (r-n+), and the blue indicates negative for both ELISAs (r-n-). (b) CBB staining of full-length ITGα6 (purple arrow) and ITGβ4 (green arrow). (c) Western blotting of full-length ITGα6 (purple arrow) and ITGβ4 (green arrow). (d) Western blotting using 55 MMP sera with nCOL17 as a substrate. In addition to the 180-kDa COL17 (strong: black arrow, weak: gray), other extra bands (red arrows) and approximately 205-kDa ITGβ4 (green arrow) were observed. DDDDK: anti-DDDDK antibody; COL17: anti-COL17 antibody, #1–55: MMP sera. (e) Western blotting using MMP sera with recombinant full-length ITGα6. #1–55: MMP sera, ITGα6: anti-ITGα6 antibody. The purple arrow indicates positive reactivity for ITGα6 bands. (f) Western blotting using MMP sera with recombinant full-length ITGβ4. ITGβ4: anti-ITGβ4 antibody. The green arrow indicates positive reactivity for ITGβ4 bands. #, number; BP, bullous pemphigoid; CBB, Coomassie brilliant blue; COL17, collagen XVII; nCOL17, native COL17; rCOL17, recombinant COL17; ITG, integrin; kDa, kilodalton; MMP, mucous membrane pemphigoid.

Figure 6

Western blotting using MMP sera with purified laminin-332. MMP sera reacted with the α3 chain (160-kDa and 145-kDa), β3 chain (140-kDa), and γ2 chain (105-kDa), which are indicated by red arrows. kDa, kilodalton; MMP, mucous membrane pemphigoid.

Figure 7

A summarized scheme illustrating the comparison of the nCOL17 ELISA with other available ELISAs. The nCOL17 ELISA can detect autoantibodies for various types of pemphigoid diseases. COL17, collagen XVII; nCOL17, native COL17.