Non-lysosomal degradation of singly phosphorylated oligosaccharides initiated by the action of a cytosolic endo-β-N-acetylglucosaminidase

Abstract Phosphorylated oligosaccharides (POSs) are produced by the degradation of dolichol-linked oligosaccharides (DLOs) by an unclarified mechanism in mammalian cells. Although POSs are exclusively found in the cytosol, their intracellular fates remain unclear. Our findings indicate that POSs are catabolized via a non-lysosomal glycan degradation pathway that involves a cytosolic endo-β-N-acetylglucosaminidase (ENGase). Quantitative and structural analyses of POSs revealed that ablation of the ENGase results in the significant accumulation of POSs with a hexasaccharide structure composed of Manα1,2Manα1,3(Manα1,6)Manβ1,4GlcNAcβ1,4GlcNAc. In vitro ENGase assays revealed that the presence of an α1,2-linked mannose residue facilitates the hydrolysis of POSs by the ENGase. Liquid chromatography-mass spectrometric analyses and fluorescent labeling experiments show that such POSs contain one phosphate group at the reducing end. These results indicate that ENGase efficiently hydrolyzes POSs that are larger than Man4GlcNAc2-P, generating GlcNAc-1-P and neutral Gn1-type free oligosaccharides. These results provide insight into important aspects of the generation and degradation of POSs.