Structural characterization of N-glycans on human transthyretin and their impact on amyloid-β binding.

Transthyretin (TTR), a plasma and cerebrospinal fluid protein, binds amyloid-β (Aβ) peptides, inhibiting their aggregation and amyloid fibril formation-key processes implicated in Alzheimer's disease pathogenesis. Despite its critical functions, the structural characterization of TTR remains incomplete. In this study, liquid chromatography-tandem mass spectrometry was employed to identify and characterize the N-glycans on human TTR and evaluate their role in Aβ binding. A total of 18 N-glycan structures were identified, comprising high-mannose (2.1 %), hybrid (0.4 %), and complex (97.5 %) types, with a total N-glycan quantity of 0.22 pmol/pmol TTR. The N-glycan modifications included galactosylation (96.0 %), fucosylation (26.8 %), mono-sialylation (30.1 %), and bisected GlcNAc (5.5 %). Proteolytic peptide analysis revealed partial N-glycosylation at Asn-98 with an occupancy of 16.5 %. Functional assays showed that desialylation and complete deglycosylation resulted in reduced Aβ binding, as assessed by ELISA, and reduced Aβ-aggregation inhibition by thioflavin T assay, compared to intact TTR. These results indicate that, despite its low occupancy (4.9 %, with 30.1 % sialylation at the 16.5 % occupied site) at Asn-98, negatively charged sialylation plays an important role in the Aβ binding capacity of TTR. This study provides the first detailed characterization of human TTR N-glycans and highlights their essential role in the binding of TTR to Aβ.