Anti-neutrophil cytoplasmic antibodies (ANCA) are a group of autoantibodies predominantly targeting neutrophil antigens. ANCA can be found in many autoimmune diseases, including rheumatoid arthritis and inflammatory bowel disease, and they have as well diagnostic value in distinguishing ANCA-associated vasculidites. One of the most common antigens for ANCA is myeloperoxidase (MPO), a protein that is part of the antimicrobial defense system. MPO is typically present in the azurophilic granulocytes of neutrophils, but a large portion may also enter the extracellular space following neutrophil degranulation. It remains as of yet unclear why MPO is so frequently the target of an autoimmune response.
MPO is a glycoprotein, meaning that the peptide is post-translationally modified to contain complex sugars at specific sites. This glycosylation is typically of major influence to a protein, affecting its folding, receptor interaction, backbone accessibility, and more. MPO is heavily modified by 5 N-glycosylation sites – 10 in the mature dimer. While important for the structure and function of MPO, the full scope and relative abundance of the glycans on each site still remains to be investigated, even for healthy individuals.
As such, we describe here the most in-depth and thorough glycan characterization of neutrophil MPO to date, making use of recent advances in bottom-up glycoproteomics and native mass spectrometry. By this, we achieved, for the first time, the relative quantification of the glycans on each of the 5 sites, and reveal the considerable heterogeneity between them (Figure 1). In addition, we detected abundant glycosylation phenotypes that are uncommonly observed in the extracellular space, including phosphorylated high-mannose species and severely truncated glycans in both paucimannose size and smaller. We speculate that the atypical glycosylation found on MPO might be involved in its frequent development into self-antigen.