Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid β-peptides in human cerebrospinal fluid

The proteolytic processing of human amyloid precursor protein (APP) into shorter aggregating amyloid beta (A beta)-peptides, e. g., A beta 1-42, is considered a critical step in the pathogenesis of Alzheimer's disease (AD). Although APP is a well-known membrane glycoprotein carrying both N-and O-glycans, nothing is known about the occurrence of released APP/A beta glycopeptides in cerebrospinal fluid (CSF). We used the 6E10 antibody and immunopurified A beta peptides and glycopeptides from CSF samples and then liquid chromatography-tandem mass spectrometry for structural analysis using collision-induced dissociation and electron capture dissociation. In addition to 33 unglycosylated APP/A beta peptides, we identified 37 APP/A beta glycopeptides with sialylated core 1 like O-glycans attached to Thr(-39, -21, -20, and -13), in a series of APP/A beta X-15 glycopeptides, where X was -63, -57, -52, and -45, in relation to Asp1 of the A beta sequence. Unexpectedly, we also identified a series of 27 glycopeptides, the A beta 1-X series, where X was 20 (DAEFRHDSGYEVHHQKLVFF), 19, 18, 17, 16, and 15, which were all uniquely glycosylated on Tyr10. The Tyr10 linked O-glycans were (Neu5Ac)(1-2)Hex(Neu5Ac)HexNAc-O- structures with the disialylated terminals occasionally O-acetylated or lactonized, indicating a terminal Neu5Aca2,8Neu5Ac linkage. We could not detect any glycosylation of the A beta 1-38/40/42 isoforms. We observed an increase of up to 2.5 times of Tyr10 glycosylated A beta peptides in CSF in six AD patients compared to seven non-AD patients. APP/A beta sialylated O-glycans, including that of a Tyr residue, the first in a mammalian protein, may modulate APP processing, inhibiting the amyloidogenic pathway associated with AD.