A neural glycan HNK-1 is transferred to recipient cells via small extracellular vesicles

Mammalian cells are decorated with a large variety of glycans. Although the biosynthetic enzymes for most glycan structures have been identified, it remains unclear how glycan levels are regulated in cells. Recently, some cellular glycans and their biosynthetic enzymes were found to be loaded into a subset of small extracellular vesicles (sEVs) and transferred to recipient cells, suggesting uncharacterized sEV-mediated mechanisms for glycan remodeling. Here, we found that a brain-specific glycan involved in learning and memory, human natural killer-1 (HNK-1), and its major biosynthetic enzyme, GlcAT-P (B3GAT1), are included in sEVs. Size exclusion chromatography and immunoisolation experiments suggested that the sEVs containing GlcAT-P and glycoproteins with HNK-1 are similar in size but distinct from the tetraspanin-rich sEV subtype. We also found that GlcAT-P in the sEVs is a cleaved form and enzymatically active. Incubation of the HNK-1- and GlcAT-P-loaded sEVs rendered recipient cells positive for HNK-1, whereas sEVs containing GlcAT-P but not HNK-1 did not induce HNK-1 expression in the recipient cells, suggesting that the transfer of HNK-1 but not its biosynthetic enzyme is necessary for recipient cells to be positive for HNK-1. Our findings shed light on a non-genetic pathway for increasing the level of a specific neural glycan via sEV-mediated cell-cell communication.