J Biol Chem. 2026 Jul 13:113329. doi: 10.1016/j.jbc.2026.113329. Online ahead of print.
ABSTRACT
N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (NAGPA), also known as the uncovering enzyme, catalyzes the final step in mannose-6-phosphate (M6P) signal generation for lysosomal enzyme trafficking. Despite its central role, the enzyme’s architecture, catalytic mechanism, and the contribution of its C-terminal region remain incompletely defined. Here, we combine solution biophysics, cryo-electron microscopy, structural modeling, and a quantitative cell-based assay to characterize human NAGPA. We show that NAGPA forms a noncovalent dimer in solution, resolving prior uncertainty regarding disulfide-linked higher-order assemblies. The structure reveals an elongated dimer composed of two catalytic cores and two C-terminal EGF-like stalks, semi-rigid in nature, that likely position the catalytic domains ∼5 nm from the membrane. A structure determined in the presence of the substrate analog GlcNAc-1-phosphate captures GlcNAc and phosphate in the active site, identifying an invariant DGGGS motif that is critical for substrate recognition and enzyme catalysis. Based on these observations, we propose a substrate-assisted SNi-like mechanism for cleavage of the glycosidic C-O bond between GlcNAc and M6P. Functional assays show that the membrane-tethered full-length NAGPA is more active than the isolated catalytic core, and that mutations in the hinge linking the catalytic domain to the C-terminal stalk reduce activity. Together, these findings establish a structural and mechanistic framework for understanding NAGPA function in lysosomal enzyme targeting.
PMID:42442503 | DOI:10.1016/j.jbc.2026.113329