Structural characterization and insights into the formation of <i>N-</i> acetylglucosaminylasparagine and its derivatives in NGLY1-deficient models and patients

Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals), a widely conserved amidase in eukaryotes, catalyzes the removal of N-glycans from glycoproteins and contributes to the quality control system for nascent glycoproteins. Since the first report of a patient with an autosomal recessive genetic disorder caused by NGLY1 deficiency in 2012, over 150 cases have been identified globally. Among the potential biomarkers for NGLY1 deficiency, Asn-linked mono/oligosaccharides-Asn-GlcNAc and Asn-HexNAc-Hex-NeuAc-have emerged as the most consistently and markedly elevated molecules in the plasma or urine of affected patients. This study examined the Asn-GlcNAc biosynthetic pathway, demonstrating that cytosolic endo-β-N-acetylglucosaminidase (ENGase), the proteasome, and peptidases are essential for its generation. NGLY1-deficient models and patients exhibited accumulation of novel elongated forms of Asn-GlcNAc, including Asn-GlcNAc-GalNAc, Asn-GlcNAc-Gal, and Asn-GlcNAc-Gal-NeuAc, in cells, culture supernatant, plasma, and urine. Our findings indicate that Asn-GlcNAc and Asn-oligosaccharides (Asn-OSs) may serve as promising diagnostic tools for NGLY1 deficiency.