Azurocidin is loaded into small extracellular vesicles via its N‐linked glycosylation and promotes intravasation of renal cell carcinoma cells

Azurocidin (AZU1) is an antimicrobial protein secreted by neutrophils that acts as a chemoattractant for monocytes and macrophages and a permeabilizer of vascular endothelial cells. We previously identified AZU1 to be specifically present in extracellular vesicles (EVs) obtained from renal cell carcinoma (RCC) tissues. Here, we examined the relationship between N‐linked glycosylation and AZU1 loading into small EVs (SEVs). Inhibition of N‐linked glycosylation by introducing mutations in three glycosylation sites inhibited AZU1 loading into SEVs. Furthermore, SEVs released from AZU1‐wild‐type cells increased the Ca2+ concentration in endothelial cells and the endothelial permeability, whereas SEVs released from AZU1‐mutant cells had no significant effect. Anti‐AZU1 antibodies diminished the effect of SEVs on endothelial cell sheets. Collectively, we found that N‐linked glycosylation of AZU1 directs its loading into SEVs, thereby enabling AZU1‐positive SEVs to function as potent permeabilizers of endothelial cells and leading to enhanced transendothelial migration of RCC cells.