Critical Parameters and Troubleshooting

Extraction of proteins from cells

The O-GlcNAc modification can be removed from proteins either by cytosolic O-GlcNAcase or lysosomal hexosaminidases. The inclusion of inhibitors during the extraction and purification process will preserve the levels of O-GlcNAc on proteins. Commonly used inhibitors (Dong and Hart, 1994) include 1-amino-GlcNAc (1 mM), GlcNAc (100 mM), and PUGNAc (5 |M). Note that these may have to be removed, as they will act as inhibitors in other methods.

Product characterization

Product characterization is a critical step showing that a protein is modified by O-GlcNAc, and not other glycans. While many proteins modified by O-GlcNAc have been identified, there is evidence based on metabolic labeling (Medina et al., 1998) and lectin labeling studies (Hart et al., 1989) that indicate that O-GlcNAc is not the only intracellular carbohydrate post-translational modification. In addition, at least one peptide mimic of O-GlcNAc has been identified in cytokeratins (Shikhman et al., 1994).

Moreover, many techniques used for breaking open cells also release proteins that are modified by complex N- and O-linked sugars, which may contain terminal GlcNAc. Many of the techniques described in this unit will recognize any GlcNAc residue, and it is important to perform the described controls such as PNGase F digestion to show specificity.

Product analysis is critical for metabolic labeling with glucosamine. While UDP-GlcNAc is the major product, glucosamine can enter other biosynthetic pathways, such as those used for amino acid synthesis. This issue was highlighted by studies of the SV40 large T-antigen. Some researchers have found that the SV40 large T-antigen labels with a number of different tritiated carbohydrates. However, O-GlcNAc is the only carbohydrate post-trans-lational modification of the SV40 large T-anti-gen. The incorporation of glucosamine into amino acid biosynthetic pathways could be reduced by growing cells in the presence of excess nonessential amino acids (Medina et al., 1998).

Lastly, while galactosyltransferase is specific for terminal GlcNAc residues, researchers (Elling et al., 1999) have shown that galacto-syltransferase will modify GlcNAc linked in either the a- or P-anomeric conformation. The authors of this unit have shown that proteins modified by a-O-GlcNAc will be labeled using the procedure described (N. Zachara, unpub. observ.). While a-O-GlcNAc has not been identified in complex eukaryotes, it is a common modification of cell surface proteins of simple eukaryotes such as trypanosomes and Dictyostelium. Product analysis, such as HPAEC of the sugar alditols, will resolve many of the issues discussed.

Time Considerations

Detection of O-GlcNAc proteins using antibodies (see Basic Protocol 2) and lectins (see Basic Protocol 3) will take approximately 2 to

3 days after the extraction of the proteins from cells. Samples and controls must be treated with PNGase F and/or hexosaminidase (1 hr to overnight), before SDS-PAGE and blotting. In either case, overnight incubation at 4°C provides the best signal-to-noise ratio.

WGA affinity chromatography of low-copy-number proteins will take 2 to 3 days. The ITT and WGA affinity chromatography can be completed in 1 day; subsequent analysis of the product by SDS-PAGE will take 1 to 2 days depending on the label used and the amount of label incorporated into proteins eluting from the WGA-agarose.

Autogalactosylation of the galactosyltrans-ferase and subsequent analysis of the activity will take 1 to 2 days. As the enzyme is stable for 6 to 12 months at -20°C, autogalactosyla-tion does not need to be repeated for each analysis. Labeling of the proteins can take several hours to overnight, though optimization of the conditions may take a few days. The subsequent analysis, as well as desalting (dependent on the technique used), PNGase F digestion (1 hr to overnight), precipitation of protein (3 hr to overnight), SDS-PAGE, and autoradiography (1 to 10 days), can take up to 2 weeks. The length of time allotted to product analysis is dependent on the methods chosen, but will almost certainly require 7 to 10 days. Further analysis, including digestion of labeled proteins and subsequent purification of peptides, will take at least 3 days.

Literature Cited

Amano, J. and Kobata, A. 1990. Quantitative conversion of mucin-type sugar chains to radioactive oligosaccharides. Methods Enzymol. 179:261-270.

Batteiger, B., Newhall, W.J., and Jones, R.B. 1982. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J. Immunol. Methods 55:297-307.

Brew, K., Vanaman, T.C., and Hill, R.L. 1968. The role of alpha-lactalbumin and the A protein in lactose synthetase: A unique mechanism for the control of a biological reaction. Proc. Natl. Acad. Sci. U.S.A. 59:491-497.

Comer, F.I. and Hart, G.W. 2000. O-Glycosylation of nuclear and cytosolic proteins: Dynamic interplay between O-GlcNAc and O-phosphate. J. Biol. Chem. 275:29179-29182.

Comer, F.I., Vosseller, K., Wells, L., Accavitti, M.A., and Hart, G.W. 2001. Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine. Anal. Biochem. 293:169-177.

Post-Translational

Modification:

Glycosylation

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