title: 13C-sialic acid labeling of glycans on glycoproteins using ST6Gal-I.
abstract: Glycans that are either N-linked to asparagine or O-linked to serine or threonine are the hallmark of glycoproteins, a class of protein that dominates the mammalian proteome. These glycans perform important functions in cells and in some cases are required for protein activity. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for studying glycan structure and interactions, particularly in a form that exploits heteronuclei such as 13C. Here an approach is presented that that uses alpha-2,6-sialyltransferase (ST6Gal-I) to enzymatically add 13C-N-acetylneuraminic acid (NeuAc or sialic acid) to glycoproteins after their preparation using nonbacterial hosts. ST6Gal-I is itself a glycoprotein, and in this initial application, labeling of its own glycans and observation of these glycans by NMR are illustrated. The catalytic domain from rat ST6Gal-I was expressed in mammalian HEK293 cells. The glycans from the two glycosylation sites were analyzed with mass spectrometry and found to contain sialylated biantennary structures. The isotopic labeling approach involved removal of the native NeuAc residues from ST6Gal-I with neuraminidase, separation of the neuramindase with a lectin affinity column, and addition of synthesized 13C-CMP-NeuAc to the desialylated ST6Gal-I. Chemical shift dispersion due to the various 13C-NeuAc adducts on ST6Gal-I was observed in a 3D experiment correlating 1H-13C3-13C2 atoms of the sugar ring.
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, USA.
authors.forename: Megan A
authors.initials: MA
authors.lastname: Macnaughtan
authors.forename: Fang
authors.initials: F
authors.lastname: Tian
authors.forename: Shan
authors.initials: S
authors.lastname: Liu
authors.forename: Lu
authors.initials: L
authors.lastname: Meng
authors.forename: Seongha
authors.initials: S
authors.lastname: Park
authors.forename: Parastoo
authors.initials: P
authors.lastname: Azadi
authors.forename: Kelley W
authors.initials: KW
authors.lastname: Moremen
authors.forename: James H
authors.initials: JH
authors.lastname: Prestegard
mesh_terms: D002247:Carbon Isotopes; D006023:Glycoproteins / Q000138:chemical synthesis / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011134:Polysaccharides / Q000138:chemical synthesis / Q000737:chemistry*; D012799:Sialyltransferases / Q000737:chemistry*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D002247:Carbon Isotopes; D006023:Glycoproteins; D011134:Polysaccharides; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase