Loss of the Asn333 high-mannose N-glycan impairs ACP3 folding and drives disease-associated dysfunction.

Missense mutations disrupting N-glycosylation motifs can perturb protein folding and trafficking, yet the site-specific contributions of individual N-glycans to proteostasis remain poorly defined. Here, we delineated the functional hierarchy of three N-glycosylation sites in human prostatic acid phosphatase (ACP3). Glycoproteomic analysis showed that Asn94 and Asn220 carry predominantly complex-type N-glycans, whereas Asn333 is enriched in high-mannose structures. Glycan-deficient N94Q and N220Q variants were efficiently secreted and retained phosphatase activity, whereas N333Q and T335M accumulated intracellularly, formed endoplasmic reticulum aggregates, and lacked enzymatic activity. Molecular dynamics simulations revealed that the Asn333 high-mannose glycan forms persistent hydrogen-bond networks with surrounding residues and exhibits markedly reduced conformational flexibility, supporting a non-redundant structural role. Individuals harboring a heterozygous p.T335M variant presented severe neurodevelopmental abnormalities, linking Asn333 glycan loss to proteostasis failure in vivo. These findings establish a site-specific glycan-protein stabilizing mechanism with broad implications in glycoprotein folding and human disease.