Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.24.219956v1?rss=1 Authors: Tjondro, H. C., Ugonotti, J., Kawahara, R., Chatterjee, S., Loke, I., Chen, S., Soltermann, F., Hinneburg, H., Parker, B. L., Venkatakrishnan, V., Dieckmann, R., Grant, O. C., Bylund, J., Rodger, A., Woods, R. J., Struwe, W. B., Karlsson-Bengtsson, A., Thaysen-Andersen, M. Abstract: Myeloperoxidase (MPO), an important glycoprotein in neutrophil-mediated immunity, produces microbicidal hypohalous acids, but the underpinning glycobiology remains elusive. Deep characterisation of neutrophil-derived MPO (nMPO) using advanced mass spectrometry demonstrated that under-processed oligomannosidic-, truncated paucimannosidic- and hyper-truncated N-acetyl-{beta}-D-glucosamine (GlcNAc) core-type asparagine-linked glycans decorate the protein. Occlusion of Asn355 and Asn391 and sterical hindrance of Asn323- and Asn483-glycans located in the MPO dimerisation zone were found to shape the local glycan processing thereby providing a molecular basis for the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed extreme molecular complexity of dimeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a lower-abundance monomer. Longitudinal profiling of maturing, mature, granule-separated, and pathogen-activated neutrophils demonstrated that MPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and rapidly degranulated, but surprisingly carries uniform glycosylation across conditions. Complete proMPO-to-MPO maturation evidently occur during early/mid-stage granulopoiesis. The conserved Asn355- and Asn391-sequons displayed elevated GlcNAc signatures and higher oxidation and chlorination activity of the secretory vesicle/plasma membrane-resident MPO relative to MPO from other granules. Endoglycosidase H-treated nMPO displaying Asn355-/Asn391-GlcNAcylation recapitulated the activity gain and showed increased thermal stability and enhanced polypeptide flexibility relative to untreated nMPO as measured by activity assays, circular dichroism and molecular dynamics. Endoglycosidase H-treated nMPO also demonstrated an elevated ceruloplasmin-mediated inhibition relative to nMPO. Modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO:ceruloplasmin interface are critical for contact-free inhibition. We report on novel roles of the peculiar MPO glycosylation providing new insight into neutrophil glycobiology. Copy rights belong to original authors. Visit the link for more info