title: Selective Exo-Enzymatic Labeling Detects Increased Cell Surface Sialoglycoprotein Expression upon Megakaryocytic Differentiation.
abstract: Selective exo-enzymatic labeling (or SEEL) uses recombinant glycosyltransferases and nucleotide-sugar analogues to allow efficient labeling of cell surface glycans. SEEL can circumvent many of the possible issues associated with metabolic labeling, including low incorporation of sugar precursors, and allows for sugars to be added selectively to different types of glycans by virtue of the inherent specificity of the glycosyltransferases. Here we compare the labeling of sialoglycoproteins in undifferentiated and differentiated human erythroleukemia cells (HEL) using SEEL using the sialyltransferases ST6Gal1 and ST3Gal1, which label N- and O-glycans, respectively. Our results show that the profile of glycoproteins detected varies between undifferentiated HEL cells and those differentiated to megakaryocytes, with a shift to more N-linked sialoglycoproteins in the differentiated cells. The efficiency of SEEL for both sialyltransferases in HEL cells was greatly increased with prior neuraminidase treatment highlighting the necessity for the presence of available acceptors with this labeling method. Following metabolic labeling or SEEL, tagged glycoproteins were enriched by immunoprecipitation and identified using mass spectrometry. The proteomic findings demonstrated that the detection of many glycoproteins is markedly improved by SEEL labeling, and that unique glycoproteins can be identified using either ST6Gal1 or ST3Gal1. Furthermore, this analysis enabled the identification of increased surface expression of several sialylated cell adhesion molecules, including the known megakaryocytic markers integrinβ3 and CD44, upon differentiation of HEL cells to adherent megakaryocytes.
authors.affiliation: From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602.
authors.forename: Seok-Ho
authors.initials: SH
authors.lastname: Yu
authors.affiliation: From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhao
authors.affiliation: From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602.
authors.forename: Tiantian
authors.initials: T
authors.lastname: Sun
authors.affiliation: From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602.
authors.forename: Zhongwei
authors.initials: Z
authors.lastname: Gao
authors.affiliation: From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602.
authors.forename: Kelley W
authors.initials: KW
authors.lastname: Moremen
authors.affiliation: From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602.
authors.forename: Geert-Jan
authors.initials: GJ
authors.lastname: Boons
authors.affiliation: From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602 lwell@ccrc.uga.edu.
authors.forename: Lance
authors.initials: L
authors.lastname: Wells
authors.affiliation: From the Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602 rsteet@ccrc.uga.edu.
authors.forename: Richard
authors.initials: R
authors.lastname: Steet
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis; D039661:Integrin beta3 / Q000096:biosynthesis; D007553:Isotope Labeling / Q000379:methods*; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism*; D012795:Sialoglycoproteins / Q000096:biosynthesis*; D012799:Sialyltransferases / Q000378:metabolism; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D015703:Antigens, CD; D018960:Hyaluronan Receptors; D039661:Integrin beta3; D012795:Sialoglycoproteins; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase; C497483:CD44 protein, human; C093493:ST6GAL1 protein, human; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
keywords: cell differentiation; click chemistry; glycosyltransferase; proteomics; sialic acid; β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1)