The protease inhibitor alpha-2-macroglobulin-like-1 is the p170 antigen recognized by paraneoplastic pemphigus autoantibodies in human

Abstract

Background: Paraneoplastic pemphigus (PNP) is a devastating autoimmune blistering disease, involving mucocutaneous and internal organs, and associated with underlying neoplasms. PNP is characterized by the production of autoantibodies targeting proteins of the plakin and cadherin families involved in maintenance of cell architecture and tissue cohesion. Nevertheless, the identity of an antigen of Mr 170,000 (p170), thought to be critical in PNP pathogenesis, has remained unknown.

Methodology/principal findings: Using an immunoprecipitation and mass spectrometry based approach, we identified p170 as alpha-2-macroglobuline-like-1, a broad range protease inhibitor expressed in stratified epithelia and other tissues damaged in the PNP disease course. We demonstrate that 10 PNP sera recognize alpha-2-macroglobuline-like-1 (A2ML1), while none of the control sera obtained from patients with bullous pemphigoid, pemphigus vulgaris, pemphigus foliaceus and normal subjects does.

Conclusions/significance: Our study unravels a broad range protease inhibitor as a new class of target antigens in a paraneoplastic autoimmune multiorgan syndrome and opens a new challenging investigation avenue for a better understanding of PNP pathogenesis.

Figure 1. Analysis of keratinocyte proteins immunoprecipitated by PNP auto-antibodies.

Normal sera (N1 and N2) or PNP sera (1–12) were used to immunoprecipitate proteins from radiolabelled keratinocyte extracts (A) or culture medium (B). Immunoprecipitates were denatured under reducing conditions, separated on 6% SDS-PAGE, and autoradiographed. p170 migration level is indicated by a star. PNP sera 1 and 2 immunoprecipitated negligible amounts of p170. BP230, desmoplakin (DPI, DPII), envoplakin (EN) and periplakin (PP) migration levels are indicated in panel A. In panel B, the non specific band also recognized by the control sera (N1, N2) with a slightly faster electrophoretic migration than p170 is indicated (ns).

Figure 2. Denaturation conditions affect the recognition of p170 by PNP auto-antibodies by immunoblotting.

Extracts (20 µg protein) from undifferentiated keratinocytes (lane 1), or differentiated for 4 days (lane 2) and 8 days (lane 3) were denatured in protein sample buffer containing or not 2-mercaptoethanol, separated on 8% SDS-PAGE and transferred onto nitrocellulose membrane for Western blot analysis using PNP serum 3 (A), anti-A2ML1 antibody (B), and PNP serum 2 (C). Sera numbering corresponds to that of Figure 1. Ponceau staining of the membranes is indicated as loading control. p170 migration level is indicated by a star. Envoplakin (EN) and periplakin (PP) migration levels are indicated.

Figure 3. Anti-A2ML1 antibodies recognize p170 immunoprecipitated by PNP sera.

Reduced samples immunoprecipitated from radiolabelled keratinocyte extracts with normal (N1, N2) or PNP sera (1 to 12) (see Fig. 1) were separated on 8% SDS-PAGE and transferred onto nitrocellulose membrane. This membrane was autoradiographed (A) and analyzed by immunoblotting using anti-A2ML1 antibodies (B).

Figure 4. PNP sera immunoprecipitate recombinant A2ML1 expressed in HEK 293T.

PNP (A, 1–12), normal (A and B, N1-N10), pemphigus vulgaris (PV1-PV4) and bullous pemphigoid sera (B, BP1-BP4) were used to immunoprecipitate proteins from extracts of HEK 293T cells transfected with pISb05, encoding recombinant A2ML1-c-myc. PNP sera numbering corresponds to that of Figure 1. Reduced immunoprecipitates were separated on 8% SDS-PAGE and analyzed by immunoblot using anti-c-myc antibody. p170 migration level is indicated by a star.

Figure 5. Adsorption of PNP sera with recombinant A2ML1 prevents PNP serum reactivity with p170.

Nickel resin loaded with HEK 293T extracts expressing (ADS) or not (MOCK) A2ML1-Flag-HIS₈ was incubated with PNP sera (diluted 1/10). Sera numbering corresponds to that of Figure 1. (A) Treated sera were used to carry out the Western blot analysis of undifferentiated (ND) or differentiated for 8 days (Diff) human primary keratinocyte extracts (20 µg/lane), separated after non-reducing denaturation on 6% SDS-PAGE. The migration position of p170 is marked by a star. (B) Treated sera were used to immunoprecipitate proteins from 8-day-differentiated primary human keratinocytes. Reduced immunoprecipitates were separated on 6% SDS-PAGE, and analyzed by Western blotting using anti-A2ML1 antibody (A2ML1). The same membrane was reprobed with anti-envoplakin (EN). The one or two bands corresponding to envoplakin (17) are marked by a square. The sequential probing of the membrane without stripping explains the signal at p170 level when probed with the anti-envoplakin antibody.

Figure 6. The NH₂-half of A2ML1 is preferentially recognized by PNP antibodies.

PNP sera (1–12) and normal serum (N1) (see Fig. 1) were used to immunoprecipitate recombinant A2ML1^1–889-c-myc (A) or A2ML1^990–1454-c-myc (B) expressed in transfected HEK293T cells. Immunoprecipitates were separated by SDS-PAGE after reducing (A) or non-reducing (B) denaturation to avoid the co-migration of the protein of interest with the antibodies used for the immunoprecipitation and analyzed by Western blotting using anti-c-myc antibody. As a control A2ML1^990–1454-c-myc was immunoprecipitated in parallel with anti-c-myc antibodies (c-myc) and run together with the other samples. Exposure time of the c-myc lane was 2 sec versus 7 min for the others.