Competition between core-2 GlcNAc-transferase and ST6GalNAc-transferase regulates the synthesis of the leukocyte selectin ligand on human P-selectin glycoprotein ligand-1

J Biol Chem. 2013 May 17;288(20):13974-13987. doi: 10.1074/jbc.M113.463653. Epub 2013 Apr 2.


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.

Keywords: Carbohydrate-binding Protein; Cell Adhesion; Glycobiology; Glycoprotein; Glycosyltransferases; Inflammation; Mass Spectrometry (MS); Mucins.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Cell Adhesion
  • Cricetinae
  • Gene Expression Regulation*
  • Glycoproteins / chemistry
  • Glycosylation
  • Glycosyltransferases / chemistry
  • HEK293 Cells
  • HL-60 Cells
  • Humans
  • L Cells
  • Leukocyte Rolling
  • Leukocytes / cytology
  • Lewis X Antigen / chemistry
  • Mass Spectrometry
  • Membrane Glycoproteins / chemistry*
  • Mice
  • Mucins / chemistry
  • N-Acetylglucosaminyltransferases / chemistry*
  • Protein Binding
  • Receptors, Cell Surface / chemistry
  • Shear Strength
  • Sialyl Lewis X Antigen
  • Sialyltransferases / chemistry*
  • Stress, Mechanical


  • Glycoproteins
  • Lewis X Antigen
  • Membrane Glycoproteins
  • Mucins
  • P-selectin ligand protein
  • Receptors, Cell Surface
  • Sialyl Lewis X Antigen
  • saccharide-binding proteins
  • Glycosyltransferases
  • N-Acetylglucosaminyltransferases
  • Sialyltransferases
  • beta-D-galactoside alpha 2-6-sialyltransferase