ABSTRACT
The mammalian cell surface is rich with carbohydrate polymers involved in a diversity of biological recognition events. Dynamic alterations of surface glycans mediate cell-cell communication in the immune system and host specificity of bacterial and viral pathogens. In addition, altered surface glycosylation has been implicated in disease progression of many cancers and may serve as important new targets for therapeutics. Despite the importance of glycosylation, the systematic analysis of sugars, i.e., glycomics, has lagged behind the well-studied disciplines of genomics and proteomics. This deficiency is due in part to the unique analytical challenges presented by glycans and the overwhelming diversity of sugars in nature. New microarray technologies have provided a high-throughput methods with which to probe the glycome. Our laboratory has pioneered a shown ratiometric two-color lectin microarray method that rapidly evaluates differences in the glycosylation of mammalian cells. Herein, we present a detailed protocol of our lectin microarray methodology for the differential analysis of mammalian glycomes.
Subject(s)
Cell Membrane/chemistry , Glycomics/methods , Lectins/chemistry , Microarray Analysis/methods , Polysaccharides/analysis , Animals , Carbohydrate Sequence , Cell Line , Glycosylation , Humans , Lectins/metabolism , Microscopy, Fluorescence/methods , Molecular Sequence Data , Polysaccharides/isolation & purification , Polysaccharides/metabolismABSTRACT
beta-O-N-Acetyl-d-glucosamine (O-GlcNAc) is a dynamic carbohydrate modification that is involved in cell signaling and has been implicated in a variety of disease states, including Alzheimer's and type-II diabetes. Despite the importance of this modification, little is known about the spatial and temporal localization of O-GlcNAc during signaling. This is due to the lack of methods for the study of O-GlcNAc in living cell systems. Herein we report the first genetically encoded FRET-based sensor for the detection of O-GlcNAc dynamics in live mammalian cells.
Subject(s)
Acetylglucosamine/analysis , Carbohydrates/chemical synthesis , Fluorescence Resonance Energy Transfer/instrumentation , Signal Transduction , Blotting, Western , HeLa Cells , HumansSubject(s)
Genetic Techniques , Glycoproteins/analysis , Lectins/genetics , Protein Array Analysis/methods , Carbohydrates/chemistry , Glycolipids/chemistry , Glycoproteins/chemistry , Glycoproteins/genetics , Glycosylation , Lectins/chemistry , Microscopy, Fluorescence , Oligonucleotide Array Sequence AnalysisABSTRACT
Leukemia-associated RhoGEF (LARG) is a multidomain protein that relays signals from Galpha(12/13)-coupled heptahelical receptors to GTPases that regulate the cytoskeleton. To understand the molecular basis of LARG-mediated signal transduction, structural analysis of its DH/PH domains has been initiated. The LARG DH/PH domains have been overexpressed in Escherichia coli as a TEV protease-cleavable fusion protein containing maltose-binding protein and a hexahistidine tag at the N- and C-termini, respectively. Crystals of the DH/PH domains were obtained (space group C2; unit-cell parameters a = 195.5, b = 46.0, c = 75.1 A, beta = 105.0 degrees ) and xenon and NaBr derivatives were generated which should allow the structure to be determined by MIRAS.
