We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3+2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct ingel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of OGlcNAc.Understanding posttranslational modifications to proteins is critical for elucidating the functional roles of proteins within the dynamic environment of cells. O-Linked β-Nacetylglucosamine (O-GlcNAc) glycosylation has emerged as important for the regulation of diverse cellular processes, including transcription, cell division, and glucose homeostasis. 1 While new chemical tools have provided rapid, sensitive methods for detecting the modification and enabled better control over the activity of O-GlcNAc enzymes, 1a,2 significant challenges remain with regard to elucidating the functions of O-GlcNAc in cells. For instance, a robust method for the direct fluorescence detection of O-GlcNAc proteins in gels would permit monitoring of changes in glycosylation levels in response to cellular stimuli and greatly extend the reach of existing technologies. Furthermore, new tools for imaging OGlcNAc-glycosylated proteins would enable the expression and dynamics of the modification to be monitored in cells and tissues. Here, we report an advanced chemoenzymatic labeling strategy that addresses these important needs. HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptPrevious studies have shown that an engineered β-1,4-galactosyltransferase enzyme (Y289L GalT) efficiently transfers a ketogalactose moiety from an unnatural UDP substrate selectively onto O-GlcNAc-modified proteins. 2a However, treatment of cell lysates with an aminooxy fluorescein derivative resulted in some nonspecific labeling of proteins. We therefore investigated whether Y289L GalT would accept the UDP-azidogalactose substrate 1 (UDPGalNAz), which would allow for labeling of O-GlcNAc proteins using [3+2] azide-alkyne cycloaddition chemistry ( Figure 1A). 3 In addition to providing alternative dyes to potentially reduce nonspecific interactions, this Cu(I)-catalyzed cycloaddition reaction would have the advantage of being performed more rapidly and at physiological pH.We tested the approach using α-crystallin, a known O-GlcNAc-modified protein with a low extent (~10%) of glycosylation. α-Crystallin was treated with 1 and Y289L GalT, followed by reaction with CuSO 4 , sodium ascor...

The 5-bromo-2'-deoxyuridine (BrdU) labeling of cells followed by antibody staining has been the standard method for direct measurement of cells in the S-phase. Described is an improved method for the detection of S-phase cell cycle progression based upon the application of click chemistry, the copper(I)-catalyzed variant of the Huisgen [3+2] cycloaddition between a terminal alkyne and an azide. 5-ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that is incorporated into DNA during active DNA synthesis, just like BrdU. While the BrdU assay requires harsh chemical or enzymatic disruption of helical DNA structure to allow for direct measurement of cells in the S-phase by the anti-BrdU antibody, the EdU method does not. Elimination of this requirement results in the preservation of helical DNA structure and other cell surface epitopes, decreased assay time, and increased reproducibility.

S100(3 is a calcium-binding protein that is expressed at high levels in brain primarily by astrocytes. Addition of the disulfide-bonded dimeric form of S100(3 to primary neuronal and glial cultures and established cell lines induces axonal extension and alterations in astrocyte proliferation and phenotype, but evidence that S100(3 exerts the same effects in vivo has not been presented. An 8.9-kb murine SlOOb genomic clone was used to produce two lines of transgenic mice in which S100(3 RNA is increased in a dose-related manner to 2-fold and 7-fold above normal. These lines show concomitant increased S10013 protein throughout the brain. Expression in both lines is cell type-and tissue-appropriate, and expression levels are correlated with the transgene copy number, demonstrating that sequences necessary for normal regulation of the gene are included within the cloned segment. In the hippocampus of adult transgenic mice, Western blotting detects elevated levels of glial fibrillary acidic protein and several markers of axonal sprouting, including neurofilament L, phosphorylated epitopes of neurofilament H and M, and (-tubulin. Immunocytochemistry demonstrates alterations in astrocyte morphology and axonal sprouting, especially in the dentate gyrus.Thus, both astrocytosis and neurite proliferation occur in transgenic mice expressing elevated levels of S100g3. These transgenic mice provide a useful model for studies of the role of S100(3 in glial-neuronal interactions in normal development and function of the brain and for analyzing the significance of elevated levels of S10013 in Down syndrome and Alzheimer disease.