title: Molecular basis of leukocyte rolling on PSGL-1. Predominant role of core-2 O-glycans and of tyrosine sulfate residue 51.
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.
authors.affiliation: Division and Central Laboratory of Hematology, Centre Hospitalier Universitaire Vaudois, Bugnon 46, 1011 Lausanne, Switzerland.
authors.forename: Michael Pierre
authors.initials: MP
authors.lastname: Bernimoulin
authors.forename: Xian-Lu
authors.initials: XL
authors.lastname: Zeng
authors.forename: Claire
authors.initials: C
authors.lastname: Abbal
authors.forename: Sylvain
authors.initials: S
authors.lastname: Giraud
authors.forename: Manuel
authors.initials: M
authors.lastname: Martinez
authors.forename: Olivier
authors.initials: O
authors.lastname: Michielin
authors.forename: Marc
authors.initials: M
authors.lastname: Schapira
authors.forename: Olivier
authors.initials: O
authors.lastname: Spertini
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology; D006224:Cricetinae; D005434:Flow Cytometry; D006801:Humans; D019041:L-Selectin / Q000378:metabolism; D036904:Leukocyte Rolling / Q000502:physiology*; D008024:Ligands; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009077:Mucins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism; D017354:Point Mutation; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D008024:Ligands; D008562:Membrane Glycoproteins; D009077:Mucins; D019007:P-Selectin; C084882:P-selectin ligand protein; D011134:Polysaccharides; D011994:Recombinant Proteins; D019041:L-Selectin; C005662:tyrosine O-sulfate; D014443:Tyrosine