title: Peracetylated 4-fluoro-glucosamine reduces the content and repertoire of N- and O-glycans without direct incorporation.
abstract: Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLe(X)), and related lectin ligands on effector leukocytes. Based on anti-sLe(X) antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLe(X) formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLe(X) (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLe(X) structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLe(X) on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis.
authors.affiliation: Harvard Skin Disease Research Center, Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Steven R
authors.initials: SR
authors.lastname: Barthel
authors.forename: Aristotelis
authors.initials: A
authors.lastname: Antonopoulos
authors.forename: Filiberto
authors.initials: F
authors.lastname: Cedeno-Laurent
authors.forename: Lana
authors.initials: L
authors.lastname: Schaffer
authors.forename: Gilberto
authors.initials: G
authors.lastname: Hernandez
authors.forename: Shilpa A
authors.initials: SA
authors.lastname: Patil
authors.forename: Simon J
authors.initials: SJ
authors.lastname: North
authors.forename: Anne
authors.initials: A
authors.lastname: Dell
authors.forename: Khushi L
authors.initials: KL
authors.lastname: Matta
authors.forename: Sriram
authors.initials: S
authors.lastname: Neelamegham
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Haslam
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Dimitroff
mesh_terms: D000107:Acetylation; D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000737:chemistry; D000606:Amino Sugars / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007249:Inflammation; D037102:Lectins / Q000737:chemistry; D007962:Leukocytes / Q000378:metabolism; D008024:Ligands; D009844:Oligosaccharides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D000080506:Sialyl Lewis X Antigen; D001616:beta-Galactosidase / Q000737:chemistry
chemical_list: D000606:Amino Sugars; D037102:Lectins; D008024:Ligands; D009844:Oligosaccharides; D011134:Polysaccharides; D000080506:Sialyl Lewis X Antigen; C000458:N-acetyllactosamine; C022140:N-fluoroacetyl-D-glucosamine; D001616:beta-Galactosidase; D000117:Acetylglucosamine