Site-specific characterization of mannose-6-phosphate-containing N-glycans on recombinant idursulfase beta for lysosomal targeting in Hunter syndrome therapy.

Mucopolysaccharidosis type II (Hunter syndrome) is a pediatric lysosomal storage disorder caused by mutations in the iduronate-2-sulfatase (I2S) gene, leading to impaired degradation and pathological accumulation of glycosaminoglycans. Mannose-6-phosphate (M6P)-containing N-glycans are critical for lysosomal targeting through mannose-6-phosphate receptors (MPRs). Idursulfase beta (IDS, Hunterase®), a recombinant human I2S produced in CHO cells, is used clinically in enzyme replacement therapy (ERT). This study aimed to characterize the site-specific N-glycosylation in trypsin/chymotrypsin-treated IDS using liquid chromatography-tandem mass spectrometry. A total of 62 N-glycopeptides were identified, encompassing 39 N-glycan structures, including four mono-M6P (P1), three bis-M6P (P2), and 22 sialylated N-glycans. Phosphorylated N-glycans exhibited site-specifically distribution: Asn221 (one P1, three P2) and Asn255 (three P1, three P2) were fully phosphorylated; Asn90 (one P1) and Asn512 (two P1) showed partial phosphorylation. In contrast, Asn6, Asn119, Asn300, and Asn488 were predominantly modified with sialylated non-phosphorylated N-glycans. Surface plasmon resonance analysis demonstrated high-affinity binding of IDS to the cation-independent MPR (KD = 27.2 nM), indicating effective M6P-mediated recognition. Furthermore, cellular uptake studies using fluorescent-labeled IDS in human fibroblasts confirmed efficient endocytosis and lysosomal delivery. This study provides the first site-specific characterization of M6P-containing N-glycans in IDS, supporting its therapeutic efficacy in ERT for Hunter syndrome.