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Intracellular glycosylation: A QUICtag for GlcNAc

Differential isotope labeling of GlcNAcylated proteins. For a figure having a higher resolution, please click here.

O-GlcNAcylation — linking of N-acetylglucosamine to polypeptide hydroxy groups — is a dynamic protein modification that often alternates with phosphorylation to modify protein function. GlcNAcylation is abundant in the mammalian brain and the GlcNAc transferase is essential for neuronal function and embryonic viability. However, GlcNAc analysis is still a challenge due to the small size of the sugar and the sparseness of many functionally important intracellular proteins. The sites and dynamics of GlcNAcylation are difficult to analyze with current immunodetection methods and tandem mass spectrometry. In Nature Chemical Biology, Khidekel et al. now describe a dramatically improved way of analyzing GlcNAcylation.

The authors expanded on their previously described QUIC-Tag method that couples galactose bearing a keto group to GlcNAc and links biotin to the keto function. The biotinylated proteins are then purified and analyzed by tandem mass spectrometry, which shows characteristic masses for GlcNAcylated peptide fragments. In the current study, Khidekel et al. coupled QUIC-tagging with differential isotope labeling and tracked changes in GlcNAcylation in response to neuronal activity. Lysine and N-terminal amino groups of extracted proteins from two cell samples were dimethylated – in one sample the methyl groups carried hydrogen (yielding "light" peptides) and in the other they were deuterated (resulting in "heavy" peptides). QUIC-tagged and modified proteins from both samples were mixed and analyzed by mass spectrometry. Light and heavy isotope-labeled QUIC-tagged peptides led to two distinct spectrometric signals, and the relative intensity ratio of these signals revealed the change in GlcNAcylation between the two cell states.

The use of tandem mass spectrometry for identifying GlcNAc sites often leads to a loss of amino acid side chains and post-translational modifications. However, electron-transfer dissociation (ETD) of peptide chains as described by Khidekel et al. does not alter peptide structure in this way. In ETD, peptides are fragmented by small-molecule radical anions that deliver electrons to peptides, leading to a series of distinctive mass fragments that reveal individual GlcNAcylated amino acid residues.

Khidekel et al. analyzed mouse brain GlcNAcylation and identified 22 nuclear and 11 cytosolic peptides that exhibited enhanced GlcNAcylation upon inhibition of cellular GlcNAc glycosidase. Furthermore, GlcNAcylation of the translation factor eIF4G and the transcription factor early growth response-1 (EGR-1) was increased upon neuron stimulation. A role for GlcNAcylation in transcription was supported by the finding that the histone-binding transcriptional repressor p66 not only undergoes sumoylation but also dynamic GlcNAcylation. Finally, GlcNAcylation and phosphorylation of Rab3 GEP and CRMP-2 — proteins involved in neurotransmitter release and axonal guidance — underscored the relevance of GlcNAcylation in neuronal processes.

The new approach of Khidekel et al. suggests that GlcNAcylation has a fundamental role in cellular protein production and neuronal activity. As CRMP-2 is involved in the genesis of Alzheimer disease, further studies into the details of intracellular GlcNAcylation may lead to clinically relevant insights.

Author: Mirko von Elstermann