Discovery of seven novel putative antigens in membranous nephropathy and membranous lupus nephritis identified by mass spectrometry

Abstract

Multiple autoantigens have been identified in membranous nephropathy (MN) by tissue-based proteomics. However, antigenic targets of disease are unknown for over 10% of patients with MN and over half of those with membranous lupus nephritis (MLN). Here, we identified multiple new targets in PLA2R-/THSD7A-/EXT-/NELL1-quadruple negative MN biopsies through mass spectrometry of immune complexes recovered from biopsy tissue of patients with MN. Patients with MN negative for these four antigens were identified from Arkana Laboratories case archives. Protein G immunoprecipitation recovered immune complexes from frozen biopsy tissue from 142 quadruple-negative cases and 278 cases of known antigen type, followed by interrogation by mass spectrometry. Potential putative antigens were confirmed through paraffin immunofluorescence and co-localization with IgG within immune deposits. Consecutive series of 165 cases of PLA2R-negative MN and 142 MLN biopsies were screened to determine the frequency for each potential antigen. Seven putative antigens were discovered within immune complexes from biopsies of patients with MN including FCN3, CD206, EEA1, SEZ6L2, NPR3, MST1, and VASN. Peptides from these proteins were not enriched in the 278 cases of known antigen type. Between three to 30 unique peptides were detected for each new target. Frequencies of each biomarker, determined by staining consecutive case series, ranged from under 1 to 4.9%. NPR3 and CD206 were only positive in index cases. All cases showed co-localization of IgG within the immune deposits. Thus, seven putative antigens were newly identified in MN and MLN. Due to the number of antigens identified, it is becoming impractical to type PLA2R-negative MN or MLN cases through immunostaining alone. A multiplex approach is needed for subtyping of these diseases.

Keywords: antigen; biomarker; kidney biopsy; mass spectrometry; membranous nephropathy.

Figure 1.

Cohort diagram of MN cases from A-B) Mass spectrometry cohorts; C) Consecutive case series of idiopathic MN patients; D) Consecutive case series of MLN patients.

Figure 2.

Mass spectrometry data to support identification of a putative antigen in MN, using VASN as an example. A) Scaffold exclusivity plot demonstrating VASN peptides show significantly increased normalized exclusive intensity in the VASN index case compared to other MN samples; B) Protein heat map demonstrating VASN was highly enriched in the index case compared to other MN samples; C) Sequence coverage for VASN (unique peptides highlighted in yellow).

Figure 3.. Biomarker protein abundances in MN cases of known antigen type.

A) Mass spectrometry data from 279 MN cases are shown and are grouped together based on immunostaining results. The abundances of the indicated proteins are plotted as Z-scores. Abundances of the index biomarker proteins are relatively low in MN cases of known antigen type (PLA2R⁺, THSD7A⁺, EXT⁺, NELL1⁺), and abundances of canonical antigens are low in the index cases. Peptides of the known antigen types are highlighted with an increased size within the dot plots. B) Protein abundance of FCN3 in index case and controls. C) Protein abundance of CD206 in index case and controls.

Figure 4.

Co-localization of antigen candidates with IgG immune deposits. A) SEZ6L2; B) VASN; C) EEA1; D) MST1; E) NPR3; F) FCN3; G) CD206.

Figure 5.

Frequency of autoantigens in A) ‘Idiopathic’ MN; and B) Membranous lupus nephritis. Frequencies for NCAM1, HTRA1, SEMA3B, and PCDH7 are those previously reported and were not examined within this study.