Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid β-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 ðNeu5AcÞ 1−2 HexðNeu5AcÞHexNAc-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.

SorLA Complement-type Repeat Domains Protect the Amyloid Precursor Protein against Processing

“…No other glycoforms were observed. Further glycosylation of Tyr-681 and the tryptic peptide Thr-652, Thr-659, and Thr-663 had previously been identified on sAPP in cerebrospinal fluid (52). Similar studies showed an identical glycan structure of Thr-651 using media from SorLA-DDD-transfected cells, but we were unable to detect other glycosylated peptides (data not shown).…”

Human Urinary Glycoproteomics; Attachment Site Specific Analysis of N- and O-Linked Glycosylations by CID and ECD

“…Thus, the main purpose of the ECD experiments was to determine the amino acid attachment sites of O-linked glycans. Traditionally, O-linked glycans are attached to serine or threonine residues but recently we reported a tyrosine residue to be modified by a sialylated O-linked glycan on amyloid beta peptides in human cerebrospinal fluid (59). However, our ECD…”

Enhanced Mass Spectrometric Mapping of the Human GalNAc-type O-Glycoproteome with SimpleCells

“…For example, lipolysis-stimulated lipoprotein receptor was newly identified in this study as a GalNAc-O-glycoprotein, and even though stimulated lipoprotein receptor was found in lysates of Capan-1 and COLO-205 as well as in secretomes of all three cell lines, the secretomes contributed to the identification of almost all the O-glycosites observed. In addition, peptides found exclusively in the secretomes of COLO-205 and K562 pointed to several new O-glycosites not previously identified in studies of amyloid ␤ A4 precursor (APP) (see "Discussion") (4,28,29).…”

“…In this study, we also found these peptides and identified glycoforms bearing up to five of the six available sites. This region of APP has been identified in two previous MS-based studies, as recombinant human isoform APP695 secreted from CHO cells (28), and in the form of short APP/A␤ glycopeptides immunoprecipitated from human cerebrospinal fluid (4). In neither of these studies were more than three sites within the 649 -670 segment unambiguously identified.…”

“…At the same time, this type of protein glycosylation is the least understood, especially with respect to sites of glycosylation and specific biological functions (2,3). As a prime example, the possibility of attachment of GalNAc to Tyr residues has only recently been discovered (4,5). Currently, site-specific O-glycosylation as a regulator of protein function is a dynamic area of investigation, with the most recently discovered function being co-regulation of proprotein convertase processing of proteins (6 -9).…”