16 May 2007. A new technique for detecting proteins modified by β-N-acetyl-D-glucosamine (i.e., O-GlcNAc glycosylation) reveals that the process is reversible and dynamic in neurons and may even play a role in memory. Linda Hsieh-Wilson, California Institute of Technology, Pasadena, and colleagues have developed a quantitative isotopic and chemoenzymatic tagging system (QUIC-Tag) to detect glycosylated proteins inside neurons. Unlike some other methodologies, QUIC-Tag does not require metabolic incorporation of isotopes or rounds of cell division. Cellular levels of O-GlcNAc-proteins are also unperturbed. The technique, which involves protection of the O-GlcNAc moieties by biotinylation followed by avidin-based purification and then mass spectrometry detection, is particularly suited to non-dividing cells, such as neurons.
The technique is explained in detail in the May 13 Nature Chemical Biology online. First author Nelly Khidekel and colleagues also describe how they used the QUIC-Tag method to detect kainic acid-stimulated glycosylation in rat brain. The glutamate receptor agonist reproducibly elevated thirteen of 83 O-GlcNAc-derived peptides detected in the cerebral cortices. One of the peptides derives from the transcription factor early growth response-1 (EGR-1), which has been linked to long-term memory formation (see Jones et al., 2001).
Khidekel and colleagues also found that glycosylation is reversible, suggesting that it may serve a regulatory function akin to phosphorylation and other post-translational modifications. On that note, they report that the axonal guidance protein CRMP-2 is glycosylated near sites that spur incorporation of the protein into neurofibrillary tangles when phosphorylated. Tau, and amyloid-β precursor protein, are also known to be modified by glycosylation. “Deciphering the mechanisms that regulate the interplay of glycosylation and phosphorylation for these and other proteins may have important ramifications for the study of neuronal signaling and neurodegenerative disorders,” write the authors.—Tom Fagan.
Khidekel N, Ficarro SB, Clark PM, Bryan MC, Swaney DL, Rexach JE, Sun YE, Coon JJ, Peters EC, Hsieh-Wilson LC. Probing the dynamics of O-GlcNAc glycoslyation in the brain using quantitative proteomics. Nature Chemical Biology. 2007 May 13. Advanced online publication. Abstract