Differential distribution of N- and O-Glycans and variable expression of sialyl-T antigen on HeLa cells-Revealed by direct fluorescent glycan imaging.

Cells are covered with glycans. The expression and distribution of specific glycans on the surface of a cell are important for various cellular functions. Imaging these glycans is essential to aid elucidation of their biological roles. Here, utilizing methods of direct fluorescent glycan imaging, in which fluorescent sialic acids are directly incorporated into substrate glycans via recombinant sialyltranferases, we report the differential distribution of N- and O-glycans and variable expression of sialyl-T antigen on HeLa cells. While the expression of N-glycans tends to be more peripheral at positions where cell-cell interaction occurs, O-glycan expression is more granular but relatively evenly distributed on positive cells. While N-glycans are expressed on all cells, sialyl-T antigen expression exhibits a wide spectrum of variation with some cells being strongly positive and some cells being almost completely negative. The differential distribution of N- and O-glycans on cell surface reflects their distinctive roles in cell biology.

Direct fluorescent glycan labeling with recombinant sialyltransferases.

Glycosylation is a common modification found on numerous proteins and lipids. However, direct detection of glycans on these intact biomolecules has been challenge. Here, utilizing enzymatic incorporation of fluorophore-conjugated sialic acids, dubbed as direct fluorescent glycan labeling, we report the labeling and detection of N- and O-glycans on glycoproteins. The method allows detection of specific glycans without the laborious gel blotting and chemiluminescence reactions used in Western blotting. The method can also be used with a variety of fluorescent dyes.

Probing sialoglycans on fetal bovine fetuin with azido-sugars using glycosyltransferases.

Sialic acids are negatively charged sugar residues commonly found on the terminal positions of most glycoproteins. They play important roles in the stability and solubility of these proteins. Due to their unique positioning, they also frequently act as receptors for various ligands, and therefore are involved in numerous cell-cell and cell-pathogen interactions. Here, using in vitro incorporation of clickable monosaccharides with various glycosyltransferases, we probed the sialoglycans on fetal bovine fetuin. The incorporated monosaccharides were detected with chemiluminescence via click chemistry in a format of western blotting. The results indicate that the non-reducing end Gal residues on both N- and O-glycans are fully sialylated, but the peptide-linked GalNAc residues in O-glycans are not. The presence of sialyl core-1 glycan was repeatedly confirmed by probing with α-2,3-sialyltransferases, N-acetylgalactosaminide α-2,6-sialyltransferases and a ß-1,6-N-acetylglucosaminyltransferase that is specific for core-1 glycan. The results also suggest the presence of a minute amount of sialyl Tn antigen on the protein.

Glycoprotein labeling with click chemistry (GLCC) and carbohydrate detection.

Molecular labeling and detection techniques are essential to research in life science. Here, a method for glycoprotein labeling/carbohydrate detection through glycan replacement, termed glycoprotein labeling with click chemistry (GLCC), is described. In this method, a glycoprotein is first treated with specific glycosidases to remove certain sugar residues, a procedure that creates acceptor sites for a specific glycosyltransferase. A 'clickable' monosaccharide is then installed onto these sites by the glycosyltransferase. This modified glycoprotein is then conjugated to a reporter molecule using a click chemistry reaction. For glycoproteins that already contain vacant glycosylation sites, deglycosylation is not needed before the labeling step. As a demonstration, labeling on fetal bovine fetuin, mouse immunoglobulin IgG and bacterial expressed human TNFα and TNFß are shown. Compared to traditional ways of protein labeling, labeling at glycosylation sites with GLCC is considerably more specific and less likely to have adverse effects, and, when utilized as a method for carbohydrate detection, this method is also highly specific and sensitive.

New caged neurotransmitter analogs selective for glutamate receptor sub-types based on methoxynitroindoline and nitrophenylethoxycarbonyl caging groups.

Photolysis is widely used in experimental neuroscience to isolate post-synaptic receptor activation from presynaptic processes, to determine receptor mechanisms in situ, for pharmacological dissection of signaling pathways, or for photostimulation/inhibition in neural networks. We have evaluated new caged neuroactive amino acids that use 4-methoxy-7-nitroindolinyl- (MNI) or 1-(2-nitrophenyl)ethoxycarbonyl (NPEC) photoprotecting groups to make caged ligands specific for glutamate receptor sub-types. Each was tested for interference with synaptic transmission and excitability and for receptor-specific actions in slice preparations. No adverse effects were found at glutamate receptors. At high concentration, MNI-caged, but not NPEC-caged ligands, interfered with GABA-ergic transmission. MNI-caged amino acids have sub-microsecond release times suitable for investigating mechanisms at fast synaptic receptors in situ. MNI-NMDA and MNI-kainate were synthesized and tested. MNI-NMDA showed stoichiometric release of chirally pure NMDA. Wide-field photolysis in cerebellar interneurons produced a fast-rising sustained activation of NMDA receptors, and localized laser photolysis gave a fast, transient response. Photolysis of MNI-kainate to release up to 4 µM kainate generated large inward currents at resting membrane potential in Purkinje neurons. Application of GYKI 53655 indicated that 40% of the current was due to AMPA receptor activation by kainate. Signaling via metabotropic glutamate receptors (mGluR) does not require fast release rates. NPEC cages are simpler to prepare but have slower photorelease. Photolysis of NPEC-ACPD or NPEC-DHPG in Purkinje neurons generated slow inward currents blocked by the mGluR type 1 antagonist CPCCOEt similar to the slow sEPSC seen with parallel fiber burst stimulation. NPEC-AMPA was also tested in Purkinje neurons and showed large sustained inward currents selective for AMPA receptors with little activation of kainate receptors. MNI-caged l-glutamate, NMDA and kainate inhibit GABA-A receptors with IC50 concentrations close to the maximum concentrations useful in receptor signaling experiments.