title: Competition between core-2 GlcNAc-transferase and ST6GalNAc-transferase regulates the synthesis of the leukocyte selectin ligand on human P-selectin glycoprotein ligand-1.
abstract: The binding of selectins to carbohydrate ligands expressed on leukocytes regulates immunity and inflammation. Among the human selectin ligands, the O-linked glycans at the N-terminus of the leukocyte cell-surface molecule P-selectin glycoprotein ligand-1 (PSGL-1, CD162) are important because they bind all selectins (L-, E-, and P-selectin) with high affinity under hydrodynamic shear conditions. Analysis of glycan microheterogeneity at this site is complicated by the presence of 72 additional potential O-linked glycosylation sites on this mucinous protein. To overcome this limitation, truncated forms of PSGL-1, called "PSGL-1 peptide probes," were developed. Ultra-high sensitivity mass spectrometry analysis of glycans released from such probes along with glycoproteomic analysis demonstrate the presence of both the sialyl Lewis-X (sLe(X)) and the di-sialylated T-antigen (NeuAcα2,3Galβ1,3(NeuAcα2,6)GalNAc) at the PSGL-1 N-terminus. Overexpression of glycoprotein-specific ST6GalNAc-transferases (ST6GalNAc1, -2, or -4) in human promyelocytic HL-60 cells altered glycan structures and cell adhesion properties. In particular, ST6GalNAc2 overexpression abrogated cell surface HECA-452/CLA expression, reduced the number of rolling leukocytes on P- and L-selectin-bearing substrates by ~85%, and increased median rolling velocity of remaining cells by 80-150%. Cell rolling on E-selectin was unaltered although the number of adherent cells was reduced by 60%. ST6GalNAc2 partially co-localizes in the Golgi with the core-2 β(1,6)GlcNAc-transferase C2GnT-1. Overall, the data describe the glycan microheterogeneity at the PSGL-1 N-terminus. They suggest that a competition between ST6GalNAc2 and C2GnT-1 for the core-1/Galβ1,3GalNAc glycan may regulate leukocyte adhesion under fluid shear.
authors.affiliation: Department of Chemical and Biological Engineering, The State University of New York, Buffalo, New York 14260.
authors.forename: Chi Y
authors.initials: CY
authors.lastname: Lo
authors.affiliation: Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, United Kingdom.
authors.forename: Aristotelis
authors.initials: A
authors.lastname: Antonopoulos
authors.affiliation: Department of Chemical and Biological Engineering, The State University of New York, Buffalo, New York 14260.
authors.forename: Rohitesh
authors.initials: R
authors.lastname: Gupta
authors.affiliation: Pharmaceutical Sciences, The State University of New York, Buffalo, New York 14260; The New York State Center for Excellence in Bioinformatics and Life Sciences, The State University of New York, Buffalo, New York 14260.
authors.forename: Jun
authors.initials: J
authors.lastname: Qu
authors.affiliation: Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, United Kingdom.
authors.forename: Anne
authors.initials: A
authors.lastname: Dell
authors.affiliation: Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, United Kingdom.
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Haslam
authors.affiliation: Department of Chemical and Biological Engineering, The State University of New York, Buffalo, New York 14260; The New York State Center for Excellence in Bioinformatics and Life Sciences, The State University of New York, Buffalo, New York 14260. Electronic address: neel@buffalo.edu.
authors.forename: Sriram
authors.initials: S
authors.lastname: Neelamegham
mesh_terms: D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion; D006224:Cricetinae; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D016695:Glycosyltransferases / Q000737:chemistry; D057809:HEK293 Cells; D018922:HL-60 Cells; D006801:Humans; D007739:L Cells; D036904:Leukocyte Rolling; D007962:Leukocytes / Q000166:cytology; D016256:Lewis X Antigen / Q000737:chemistry; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry*; D051379:Mice; D009077:Mucins / Q000737:chemistry; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry; D033081:Shear Strength; D000080506:Sialyl Lewis X Antigen; D012799:Sialyltransferases / Q000737:chemistry*; D013314:Stress, Mechanical; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D006023:Glycoproteins; D016256:Lewis X Antigen; D008562:Membrane Glycoproteins; D009077:Mucins; C084882:P-selectin ligand protein; D011956:Receptors, Cell Surface; D000080506:Sialyl Lewis X Antigen; C055213:saccharide-binding proteins; D016695:Glycosyltransferases; D017351:N-Acetylglucosaminyltransferases; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
keywords: Carbohydrate-binding Protein; Cell Adhesion; Glycobiology; Glycoprotein; Glycosyltransferases; Inflammation; Mass Spectrometry (MS); Mucins
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