Our results emphasize the importance of epigenetic control i

Our results emphasize the importance of epigenetic control in the 188968-51-6 regulation of N-glycosylation but also suggest the stability of complex biosynthetic pathways responsible for the establishment of glycan profiles in human cells in culture. To define more accurately the origin of glycan fraction isolated from the embedded cells, we observed cells following their embedding into the acrylamide gels by confocal scanning microscopy. Staining with Ricinus Communis Agglutinin I confirmed the preserved integrity of the cell membrane during the embedding process , thus arguing in favor of glycans originating mostly from the cell membrane glycoproteins. However, since we could not exclude the 1624602-30-7 possibility of leakage during the subsequent steps, we analyzed glycans from embedded cells that were not treated with PNGase F, in order to release glycans from glycoproteins. Interestingly, we obtained certain glycan peaks and attributed those to oligomannose structures, due to an existing efflux of oligomannose glycans . We validated this hypothesis by mannosidase treatment, which resulted in almost complete disappearance of the corresponding chromatographic peaks , thus confirming the contribution of free oligomannose glycans to the pool of glycans released by PNGase F from glycoproteins associated with the cell membrane. In addition, a tiny fraction of remaining peaks could as well predominantly represent glycan structures transported to the cell surface unattached to their protein counterparts. Based on the presented experiments, we conclude that there was no significant glycan leakage from embedded cells, since the chromatogram obtained from the analysis of untreated cells did not contain structures other than the oligomannose. Each of the two glycan release methods was repeated six times to estimate their reproducibility. For nearly all glycan peaks, we observed higher coefficients of variation when glycome was analyzed from cell lysates . Especially high experimental variation was observed in highly branched glycan structures , which are important for the regulation of membrane half-life of many receptors . This increased variation of complex structures from experiment to experiment probably reflects their small contribution to the total glycom