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

Abstract

The proteolytic processing of human amyloid precursor protein (APP) into shorter aggregating amyloid β (Aβ)-peptides, e.g., Aβ1-42, 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/Aβ glycopeptides in cerebrospinal fluid (CSF). We used the 6E10 antibody and immunopurified Aβ 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/Aβ peptides, we identified 37 APP/Aβ glycopeptides with sialylated core 1 like O-glycans attached to Thr(-39, -21, -20, and -13), in a series of APP/AβX-15 glycopeptides, where X was -63, -57, -52, and -45, in relation to Asp1 of the Aβ sequence. Unexpectedly, we also identified a series of 27 glycopeptides, the Aβ1-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 Neu5Acα2,8Neu5Ac linkage. We could not detect any glycosylation of the Aβ1-38/40/42 isoforms. We observed an increase of up to 2.5 times of Tyr10 glycosylated Aβ peptides in CSF in six AD patients compared to seven non-AD patients. APP/Aβ 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.

Fig. 1.

Glycosylated and sialylated Aβ1-15 peptides from human CSF. CID MS² spectra of (A) SA₂-Aβ1-15 and (B) SA₃-Aβ1-15. (C) O-AcSA₃-Aβ1-15 (Left) with MS³ fragmentation at m/z 694.4 (Right), and (D) lactonized SA₃-Aβ1-15. (E) Structure of α2,8-linked disialic acid terminal and its lactonized form. The Neu5Ac oxonium ion and its loss of H₂O are present at m/z 292 and 274, respectively. Neu5Ac, N-acetyl-5-neuraminic acid; Hex, hexose; HexNAc, N-acetylhexosamine; O-Ac, O-acetyl.

Fig. 2.

ECD MS² fragmentation spectra of Aβ1-15 and Aβ1-17 glycopeptides. ECD spectra of (A) SA₂-Aβ1-15, including an m/z expansion showing the z6, c8, and c9 fragments. (B) SA₂-Aβ1-17, with an m/z expansion showing the z8 and z9 fragments. The isotopic peaks of the z8 fragment are resolved in the Inset. (C) HexHexNAc-Aβ1-15. (D) HexHexNAc-Aβ1-17 including an m/z expansion showing the z8 and z9 fragments. Parent ions (M) and the charged-reduced forms were base peaks and were cropped. Noise peaks are denoted as a. Fully m/z annotated ECD spectra and lists of c- and z-ions for all observed Tyr10 glycosylated Aβ1-X peptides are shown in SI Appendix, Fig. S4 A–K.

Fig. 3.

Heatmap for the relative signal intensities of individual Aβ1-X peptides and glycopeptides for Alzheimer (AD1-6) and non-Alzheimer (N1-7) patients. The samples are arranged according to their Aβ1-42 concentrations. An increase of Tyr10 glycosylated Aβ peptides for AD patients is seen as a red shift.

Fig. 4.

MS¹ and CID MS² of APP/AβX-15 glycopeptides. (A) Amino acid sequence of APP/AβX-15, where X = -63, −57, −51, and −25. Underlined residues show Thr(−39, −21, −20, and −13) glycosylation sites. Either Ser(−5) or Thr(−9) was glycosylated (dashed underlines). The b9, b15, b21, and y57 fragmentation site is indicated. (B) CID MS² of triglycosylated APP/Aβ−25-15 showing neutral loss of Neu5Ac from the glycopeptide. (C) Triglycosylated APP/Aβ−51-15 with diagnostic b9 (m/z 987.4) and glycosylated y57 fragment ions. (D) Triglycosylated APP/Aβ−57-15 with diagnostic b15 (m/z 835.0) and glycosylated y57 fragments. (E) Tetraglycosylated APP/Aβ−63-15 with a diagnostic b21 fragment (m/z 1,151.5).