U.S. Department of Energy

Pacific Northwest National Laboratory

Mapping and Quantifying Protein O-GlcNAcylation in Human Brain for Studies of Alzheimer’s Disease

Introduction 

O-GlcNAcylation, in which Ser/Thr residues are modified with a single N-acetylglucosamine, is a wide spread dynamic protein modification. Recently, the link between O-GlcNAc and Alzheimer’s disease (AD), the most common form of dementia, is emerging as an important topic. Besides dementia, AD is confirmed post-mortem by the presence of extracellular amyloid-beta (Aβ) plaques and intracellular neurofibrillary tangles (NFTs) made up with hyper-phosphorylated protein tau. It has been found that both amyloid precursor protein (APP) and protein tau were O-GlcNAcylated. O-GlcNAcylation may mediate many pathways from disrupted brain metabolism to AD. Thus, comprehensive understanding of brain protein O-GlcNAcylation and its dysregulation in AD may be crucial to better understanding the molecular mechanisms leading to sporadic AD.

 

Methods 

In this report, we characterized O-GlcNAcylation in post-mortem brains with (10 samples) and without AD (10 samples) using isobaric tandem mass tagging (TMT, 6-plex), chemoenzymatic photocleavage (CEPC) enrichment, and liquid chromatography coupled to tandem MS (LC-MS/MS). In each TMT experiment, the same amount of peptides from a reference sample (pooled from all 20 samples) and five individual samples were each labelled with a different TMT tag, combined and fractionated with basic pH RPLC, and enriched for O-GlcNAc peptides. Consecutive electron transfer dissociation (ETD) and higher-energy collisional dissociation (HCD) techniques were used in the LC-MS/MS analysis of these samples where ETD provides site determination information and HCD provides TMT reporter ion intensity and CEPC reporter ion intensity information.

 

Preliminary data

In total, 2,049 O-GlcNAc peptides from 588 proteins in the human brain were identified. Gene ontology (GO) analysis of these proteins supported the involvement of O-GlcNAc in various cellular functions in the brain, including cytoskeleton organization, axonogenesis, cell adhesion, regeneration, etc. KEGG pathway analysis indicated the involvement of O-GlcNAc in extracellular matrix (ECM)-receptor interaction (p = 3.10E-04), focal adhesion (p = 6.73E-03), MAPK signaling pathway (p = 7.98E-04), prion diseases (p = 4.74E-03), long-term potentiation (p = 5.73E-03), Endocytosis (p =5.78E-3), etc. Interestingly, ten secreted proteins related to ECM as well as some membrane proteins, including NOTCH, were found to be O-GlcNAcylated. These proteins’ O-GlcNAcylation may be the result of the action of the EGF domain specific O-GlcNAc transferase (EOGT), which is located in the endoplasmic reticulum and is responsible for O-GlcNAcylation of extracellular proteins. 1,091 O-GlcNAcylation sites were determined (Ascore >13, with ≥95% confidence) and 578 (53%) of these O-GlcNAc sites we identified in the present study may have reciprocal or proximal (within ±10 aa) relationship with phosphorylation sites.

 

A total of 1,621 O-GlcNAc peptides from 521 proteins have quantitative information relative to the reference sample. 623 O-GlcNAc peptides had >50% occurrence in the samples and were thus used to study the dysregulation of O-GlcNAc in AD. Among these 623 peptides, 131 peptides showed significant change in abundance in AD (q<0.05). Interestingly, among these 131 O-GlcNAc peptides, only 12 peptides from two proteins (SYNPO and ANK3) decreased in AD, while the majority (119 peptides) increased in AD. The implications of these dysregulated O-GlcNAcylation events are of great interest for future study in Alzheimer’s disease.

 

Novel aspect

This comprehensive and quantitative human brain O-GlcNAc data provides a unique opportunity for understanding its role in Alzheimer’s disease.

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