Glycoproteome remodelling and granule-specificN-glycosylation accompany neutrophil granulopoiesis

Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we perform comprehensive spatiotemporalN-glycoproteome profiling of isolated granule populations from blood-derived neutrophils and during their maturation from bone marrow-derived progenitors using glycomics-assisted glycoproteomics. Interestingly, the granules of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, peculiar highly truncatedN-glycosylation in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodelling underpins the promyelocytic-to-metamyelocyte transition and that remodelling is driven primarily by changes in protein expression and less by the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation ofN-glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with high glycosylation efficiencies of the azurophilic granule proteins. Our study provides spatiotemporal insights into the complex neutrophilN-glycoproteome featuring an intriguing granule-specificN-glycosylation formed by dynamic remodelling during myeloid progenitor-to-neutrophil maturation.Key pointsSystems glycobiology reveals that profoundN-glycoproteome remodelling accompanies early neutrophil granulopoiesisPrecision glycoproteomics produces detailed cartography of neutrophils that exhibit site-, protein- and granule-specific N-glycosylation