Cell surface N-glycans remodeled in vitro using commercial glycosidases.

PAULA MAGNELLI; ALEX LUEBBERS; ALEJANDRO CAGNONI; LUCIANO MOROSI; CUTINE, ANABELA; AMBERLYN WANDS; JENNIFER KOHLER; KARINA MARIÑO

San Francisco

Congreso; 2015 Annual Meeting - Society for Glycobiology; 2015

Society for Glycobiology

Glycan-protein interactions are involved in different biologicalprocesses including development, immunity, inflammation andcancer. As a result, manipulation of cell surface glycans is anessential tool for understanding the mechanisms by which glycansgovern cell fate. Traditional approaches using gene KOs, genesilencing, or metabolic blockers are not always adequate, aspleiotropic effects are common when metabolic inhibitors blockglycosylation pathways. Targeted approaches like site-directedmutagenesis or gene silencing are not available for every cell typeor organism, limiting research on this area.Specific glycosidases can effectively remove selected glycanresidues and/or epitopes at the cell surface; these enzymes couldbe a viable alternative to remodel the cell surface glycome whenother methods are not suitable. Although this approach has beenreported in the literature, there has never been a systematicstudy (comparing different cell lines, enzymes, and reactionconditions) where the extent and efficacy of glycan removal wasevaluated along with a measurement of cell integrity and viability.Here, we present optimized conditions (buffer-media-enzymecombinations) for an efficient in vitro cell surface glycanremodeling in different cell types, while monitoring cultureviability. After enzymatic treatment, modifications in the glycanstructure were analyzed by flow cytometry using specific lectins