Primary membranous nephropathy (pMN) is a leading cause of nephrotic syndrome in adults. In most cases, this autoimmune kidney disease is associated with autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) expressed on kidney podocytes, but the mechanisms leading to glomerular damage remain elusive. Here, we developed a cell culture model using human podocytes and found that anti-PLA2R1–positive pMN patient sera or isolated IgG4, but not IgG4-depleted sera, induced proteolysis of the 2 essential podocyte proteins synaptopodin and NEPH1 in the presence of complement, resulting in perturbations of the podocyte cytoskeleton. Specific blockade of the lectin pathway prevented degradation of synaptopodin and NEPH1. Anti-PLA2R1 IgG4 directly bound mannose-binding lectin in a glycosylation-dependent manner. In a cohort of pMN patients, we identified increased levels of galactose-deficient IgG4, which correlated with anti-PLA2R1 titers and podocyte damage induced by patient sera. Assembly of the terminal C5b-9 complement complex and activation of the complement receptors C3aR1 or C5aR1 were required to induce proteolysis of synaptopodin and NEPH1 by 2 distinct proteolytic pathways mediated by cysteine and aspartic proteinases, respectively. Together, these results demonstrated a mechanism by which aberrantly glycosylated IgG4 activated the lectin pathway and induced podocyte injury in primary membranous nephropathy.

Mucin type O‐glycosylation is one of the most diverse types of glycosylation, playing essential roles in tissue development and homeostasis. In complex organisms, O‐GalNAc glycans comprise a substantial proportion of the glycocalyx, with defined functions in hemostatic, gastrointestinal, and respiratory systems. Furthermore, O‐GalNAc glycans are important players in host–microbe interactions, and changes in O‐glycan composition are associated with certain diseases and metabolic conditions, which in some instances can be used for diagnosis or therapeutic intervention. Breakthroughs in O‐glycobiology have gone hand in hand with the development of new technologies, such as advancements in mass spectrometry, as well as facilitation of genetic engineering in mammalian cell lines. High‐throughput O‐glycoproteomics have enabled us to draw a comprehensive map of O‐glycosylation, and mining this information has supported the definition and confirmation of functions related to site‐specific O‐glycans. This includes protection from proteolytic cleavage, as well as modulation of binding affinity or receptor function. Yet, there is still much to discover, and among the important next challenges will be to define the context‐dependent functions of O‐glycans in different stages of cellular differentiation, cellular metabolism, host–microbiome interactions, and in disease. In this review, we present the achievements and the promises in O‐GalNAc glycobiology driven by technological advances in analytical methods, genetic engineering, and systems biology.

The in-depth, high-sensitivity characterization of the glycome from complex biological samples, such as biofluids and tissues, is of utmost importance in basic biological research and biomarker discovery. Major challenges often arise from the vast structural diversity of glycans in combination with limited sample amounts. Here, we present a method for the highly sensitive characterization of released N-glycans by combining a capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) approach with linkage-specific derivatization of sialic acids and uniform cationic reducing end labelling of all glycans. This method allows the analysis of glycans at the attomole level, provides information on sialic acid isomers and enables the in-depth characterization of complex samples, even when available in minute amounts.