Dataset Information

Identification of novel target antigens in Membranous Nephorphathy

ABSTRACT: Membranous nephropathy (MN) is an autoimmune kidney disease with the clinical hallmark of causing high level proteinuria. MN is caused by circulating antibodies binding to targeting antigens on the surface of podocytes â€“ specialized endothelial cells contributing to the formation of the glomerular filtration barrier. Histomorphologically MN is characterized by a deposition of IgG along the glomerular basement membrane (GBM) â€“ predominantly IgG4 in primary MN â€“ as well as an accumulation of the respective target antigen, thickening of the GBM and effacement of podocyte foot processes. The predominant MN target antigen in 70-80% of MN patients is the phospholipase A2 receptor 1. Additional target antigens, which occur with lower frequencies, have recently been described. The aim of the study was the identification of a novel MN target antigen, which fulfills the typical criteria of MN target antigen: being a podocytic membrane protein, which is recognized by IgG4 subclass specific autoantibodies. Performing a mass spectrometry analysis based on a TMT-based relative quantification approach of immunoprecipitated samples resulted in the identification of Netrin G1 (NTNG1) as a novel MN target antigen. The results were validated using immunohistochemistry, Western Blot and ELISA. The molecular characterization of patients with MN will allow a better diagnosis and clinical management of these patients in the future.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma, Kidney, Renal Glomerulus

DISEASE(S): Membranous Glomerulonephritis

SUBMITTER: Frank Stein

LAB HEAD: Prof. Rolf A.K. Stahl

PROVIDER: PXD032713 | Pride | 2022-08-03

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	F105687.dat	Other
	Lara_210312_P1572_JS_LR_TMT6_PD1of30_R1.raw	Raw
	P1572_Lara_210312_P1572_JS_LR_TMT6_PD1of30_R1_results_20210315_1428_peptides.txt	Txt
	P1572_Lara_210312_P1572_JS_LR_TMT6_PD1of30_R1_results_20210315_1428_proteins.txt	Txt
	P1572_Lara_210312_P1572_JS_LR_TMT6_PD1of30_R1_results_20210315_1428_summary.txt	Txt

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Publications

Netrin G1 Is a Novel Target Antigen in Primary Membranous Nephropathy.

Reinhard Linda L Machalitza Maya M Wiech Thorsten T Gröne Hermann-Josef HJ Lassé Moritz M Rinschen Markus M MM Ferru Nicoletta N Bräsen Jan Hinrich JH Drömann Friederike F Rob Peter Maria PM Sethi Sanjeev S Hoxha Elion E Stahl Rolf A K RAK

Journal of the American Society of Nephrology : JASN 20220819 10

<h4>Background</h4>Primary membranous nephropathy (MN) is caused by circulating autoantibodies binding to antigens on the podocyte surface. PLA<sub>2</sub>R1 is the main target antigen in 70%-80% of cases, but the pathogenesis is unresolved in 10%-15% of patients.<h4>Methods</h4>We used native western blotting to identify IgG4 autoantibodies, which bind an antigen endogenously expressed on podocyte membranes, in the serum of the index patient with MN. These IgG4 autoantibodies were used to immun ...[more]

PMID: 35985817

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Project description:Background: In recent years, an innovative strategy using laser microdissection and mass spectrometry markedly expanded the landscape of antigens associated with membranous nephropathy (MN). Specific associations with phenotypes, diseases and sometimes reversible triggers led to a novel antigen-based classification of MN, paving the way for precision medicine and stressing the need for more routine use of proteomics in MN. Methods: To explore the proteomic landscape of human glomeruli and identify podocyte antigens and disease mechanisms in MN, we expanded the original technique to an integrative approach combining laser capture microdissection, next-generation mass spectrometry and computational analysis. Next to conventional data-dependent acquisition (DDA), we used and assessed the diagnostic yield of the more comprehensive data-independent acquisition (DIA) mass spectrometry, which enables the detection and quantification of every peptide in a sample irrespective of its level of abundance or m/z value. Our proteomic pipeline was applied to residual material from kidney biopsies in 64 individuals, including 31 healthy controls; 5 disease controls; 5 PLA2R-associated MN; and 23 PLA2R-negative MN. Results: Unbiased analyses confirmed the significant enrichment in PLA2R, IgG4 and complement proteins in glomeruli from patients with PLA2R-MN compared with healthy and disease controls, while molecular characterization of complement fragments provided evidence for complement activation in PLA2R-MN. Compared to DDA, DIA mass spectrometry increased the number of glomerular proteins (~3800 vs. ~1200) identified in healthy glomeruli; allowed the detection all known antigens except NELL1 in normal glomeruli; and increased the detection rate of podocyte antigens from 50% to >80% in PLA2R-negative MN. Conclusions: This proof-of-concept study suggests that an integrative approach combining laser microdissection, DIA mass spectrometry and computational biology is a powerful tool, with translational potential, to identify podocyte antigens and unravel disease mechanisms in MN.

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