title: The monoclonal antibody CHO-131 binds to a core 2 O-glycan terminated with sialyl-Lewis x, which is a functional glycan ligand for P-selectin.
abstract: Core 2 O-glycans terminated with sialyl-Lewis x (sLe(X)) are functionally important oligosaccharides that endow particular macromolecules with high-affinity glycan ligands for the selectin family. To date, antibodies that recognize these structures on leukocytes have not been described. We characterize such a monoclonal antibody (mAb) here (CHO-131). The binding specificity of CHO-131 was directly examined by means of synthetic glycopeptides containing precise O-glycan structures. CHO-131 bound to sLe(X) extended from a core 2 branch (C2-O-sLe(X)), but CHO-131 demonstrated no reactivity if this oligosaccharide lacked fucose or if sLe(X) was extended from a core 1 branch. Using transfected cell lines, we found that CHO-131 binding required the functional activity of the glycosyltransferases alpha2,3-sialyltransferase, alpha1,3-fucosyltransferase-VII, and core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT). The C2-O-sLe(X) motif occurs primarily on sialomucins and has been directly shown to contribute to high-affinity P-selectin glycoprotein ligand-1 binding by P-selectin. Indeed, CHO-131 staining of neutrophils was diminished following sialomucin removal by O-glycoprotease, and its reactivity with transfected hematopoietic cell lines correlated with the expression of P-selectin ligands. CHO-131 also stained a small population of lymphocytes that were primarily CD3(+), CD4(+), and CD45RO(+) and represented a subset (37.8% +/- 18.3%) of cutaneous lymphocyte-associated antigen (CLA) T cells, distinguished by the mAb HECA-452, which detects sLe(X)-related glycans. Unlike anti-sLe(X) mAbs, CHO-131 binding also indicates C2GnT activity and demonstrates that CLA T cells are heterogeneous based on the glycan structures they synthesize. These findings support evidence that differential C2GnT activity results in T-cell subsets that express ligands for E-selectin, P-selectin, or both.
authors: Department of Veterinary PathoBiology, University Minnesota Academic Health Center, University of Minnesota, St Paul 55108, USA. walch003@umn.edu
authors: Bruce
authors: B
authors: Walcheck
authors: Anne
authors: A
authors: Leppanen
authors: Richard D
authors: RD
authors: Cummings
authors: Randall N
authors: RN
authors: Knibbs
authors: Lloyd M
authors: LM
authors: Stoolman
authors: Shelia R
authors: SR
authors: Alexander
authors: Polly E
authors: PE
authors: Mattila
authors: Rodger P
authors: RP
authors: McEver
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000276:immunology; D006020:Glycopeptides / Q000737:chemistry / Q000276:immunology; D006602:Hexosyltransferases / Q000276:immunology; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000276:immunology; D009844:Oligosaccharides / Q000276:immunology*; D019007:P-Selectin / Q000737:chemistry / Q000276:immunology*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D000080506:Sialyl Lewis X Antigen; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D006020:Glycopeptides; D008024:Ligands; D009844:Oligosaccharides; D019007:P-Selectin; D011994:Recombinant Proteins; D000080506:Sialyl Lewis X Antigen; D006602:Hexosyltransferases; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase