Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets

Abstract

O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1-O-Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3-N-acetylglucosaminyltransferases.

Fig. 1.

Overview of the CEPC method (A) and experimental work flow (B) used in this study for the identification of O-GlcNAc proteins and modification sites. (A) Steps modified in the enrichment strategy are depicted in dashed boxes. PNGase F and calf intestine phosphatase (CIP) are added to the reaction mixture to ensure selective and complete derivatization with GalNAz (18). During the CuAAC reaction, Cu(I) is stabilized with TBTA. (B) The original and modified wash methods for the biotin-avidin enrichment step were compared using peptides from one WT mouse and one 3xTg-AD mouse (female, 1 y old). The number of O-GlcNAc sites and proteins identified from LC-MS/MS analysis of individual samples is depicted at the bottom of the figure.

Fig. 2.

Cellular component gene ontology annotation of identified O-GlcNAcylated proteins.