Molecular basis of leukocyte rolling on PSGL-1. Predominant role of core-2 O-glycans and of tyrosine sulfate residue 51

J Biol Chem. 2003 Jan 3;278(1):37-47. doi: 10.1074/jbc.M204360200. Epub 2002 Oct 25.

Abstract

Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes. In this study, we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and fucosylated O-glycans for L-selectin binding. The functional importance of these modifications was determined by analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at N-terminal Thr or Tyr residues. Simultaneous expression of core-2 beta1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and P-selectin interactions with PSGL-1 further supported these observations. Additional experiments showed that core-2 O-glycans attached to Thr-57 were also of critical importance in regulating the velocity and stability of leukocyte rolling. These observations pinpoint the structural characteristics of PSGL-1 that are required for optimal interactions with L-selectin and may be responsible for the specific kinetic and mechanical bond properties of the L-selectin-PSGL-1 adhesion receptor-counterreceptor pair.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / metabolism
  • CHO Cells
  • Cell Adhesion / physiology
  • Cricetinae
  • Flow Cytometry
  • Humans
  • L-Selectin / metabolism
  • Leukocyte Rolling / physiology*
  • Ligands
  • Membrane Glycoproteins / chemistry
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Molecular Sequence Data
  • Molecular Structure
  • Mucins / chemistry
  • Mucins / genetics
  • Mucins / metabolism
  • P-Selectin / metabolism
  • Point Mutation
  • Polysaccharides / chemistry
  • Polysaccharides / metabolism*
  • Protein Binding
  • Protein Processing, Post-Translational
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Tyrosine / analogs & derivatives
  • Tyrosine / chemistry
  • Tyrosine / metabolism*

Substances

  • Antibodies, Monoclonal
  • Ligands
  • Membrane Glycoproteins
  • Mucins
  • P-Selectin
  • P-selectin ligand protein
  • Polysaccharides
  • Recombinant Proteins
  • L-Selectin
  • tyrosine O-sulfate
  • Tyrosine