Map of the neuronal O-glycoproteome reveals driver functions in the regulated secretory pathway

Impairments in protein glycosylation, including O-GalNAc-type glycosylation, are linked to severe developmental disorders with prominent neurological involvement. However, the role of this glycosylation pathway at a cellular level is not yet fully understood. Here, we report a comprehensive map of GalNAc-type O-glycoproteins (>800) and O-glycosites (>4000) from neuronal tissues and cell lines and identify abundant O-glycosites within major classes of proteins involved in neuroplasticity, including axon guidance, membrane remodeling, and regulated vesicular secretion. Applying the map, we demonstrate that the regulated secretory pathway constitutes highly O-glycosylated proteins including Chromogranin A, a key player in dense core granulogenesis, and that correct O-glycosylation is important for its multimerization. Concurrently, genetically engineered neuronal cell lines deficient in O-glycosylation exhibit altered capacity for storing neurotransmitter noradrenaline and present enlarged neurotransmitter-containing dense core granules. Collectively, this map provides the foundation for uncovering critical roles for O-glycosylation in regulating neuroplasticity and provides evidence that dense core granule content is regulated by this pathway. Subjects: Granin, glycosylation, glycosaminoglycans, dense core granules (DCG), perineuronal net (PNN), mucin, central nervous system (CNS), neuron, neuroplasticity, neurotransmitter.