Fig 2: Similarities between PLA2R epitope and THSD7A. (A) Sequence alignment between the known major PLA2R CysR epitope peptide in bold (P28mer, aa38-65) and the potential THSD7A epitope peptide (T28mer, aa75-102) located in the TSR1 domain. Identical amino acids found across both sequences are highlighted in red. (B) Schematic of the extracellular domains of THSD7A comprising of 11 thrombospondin repeats (TSR), the first TSR domain (TSR1), the extracellular domains of PLA2R and the N-terminus CysR domain from PLA2R. Silver stained SDS-PAGE gel of purified recombinant THSD7A (180 kDa), TSR1 (17 kDa), PLA2R (180 kDa) and CysR (30 kDa) proteins under reducing conditions. (C) Western blotting analysis of denatured PLA2R, CysR, THSD7A and TSR1 proteins under non reduced and reduced conditions using a pool of five human anti-PLA2R positive sera and a pool of five anti-THSD7A sera. (D) Slot blotting analysis of non denatured PLA2R, CysR domain, THSD7A and TSR1 fragments under non reduced conditions. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: Results of Western blot analyses. In Western blot analyses serum of the patient was used as primary antibody. The Western blots, which were performed under (a) non-reducing, “standard” conditions (SDS present); and (b) non-reducing, “native” conditions (SDS absent) do not give any detectable THSD7A-specific signal. For the positive control in A and B, serum from a THSD7A-antibody positive MN patient was used. (c) Western blot analysis under reducing, “standard” conditions (SDS present) gave a Western blot signal at the expected height for rTHSD7A, but not for HGE. Also for lung tissue extract no signal was obtained (not shown). For the positive control, rabbit anti-human THSD7A was used. For all three analyses, serum from a healthy donor served as negative control.

Fig 4: Immunofluorescence co-staining. Representative photographs of THSD7A-expressing HEK cells co-stained with FITC-coupled anti-human IgG (in green) and Cy3-coupled anti-rabbit IgG (in red). (a–e): as primary antibody the following was used: (a) a commercial rabbit anti-THSD7A; (b) serum from the presented patient case; (c) a combination of rabbit anti-THSD7A with serum from the patient case; (d) serum from a patient with THSD7A-associated MN and (e) a combination of rabbit anti-THSD7A with serum from a patient with THSD7A-associated MN. Co-staining experiments reveal that serum of the patient case (c) and a patient with THSD7A-associated MN (e) co-localize with the THSD7A-specific rabbit antibody, showing that both sera specifically recognize THSD7A on the THSD7A-expressing HEK cells. (f) Representative photograph of THSD7A-expressing HEK cells assayed with serum from a patient with diabetic nephropathy but no membranous nephropathy and visualized with FITC-coupled anti-human IgG.

Fig 5: A mouse monoclonal antibody shows similar binding specificity to both epitope peptides. (A) Characterization of a mouse monoclonal raised against PLA2R. Western blot analysis of PLA2R and THSD7A variants using mouse Moab 20-2-6 under reducing and non reducing conditions. Slot blotting analysis of PLA2R, CysR, THSD7A and TSR1 purified proteins, P28mer and T28mer peptides reduced and non reduced incubated with Moab 20-2-6. (B) SPR analysis of the binding between Moab 20-2-6 and captured P28mer (left) or T28mer (right). Sensorgrams derived from injections of different concentrations of Moab 20-2-6 PLA2R-specific antibody over immobilized peptides. Kinetics data were fitted to a Langmuir 1:1 interaction model with mass transfer.