Tyrosine sulfation enhances but is not required for PSGL-1 rolling adhesion on P-selectin.
P-selectin glycoprotein ligand-1 (PSGL-1) is a large (240 kDa) glycoprotein found on the surface of nearly all leukocytes. The mature molecule is decorated with multiple N- and O-linked glycans and displays copies of the tetrasaccharide sialyl-Lewis(x) (sLe(X)), as well as a cluster of three tyrosine sulfate (tyr-SO(3)) groups near the N-terminus of the processed protein. Previous studies have suggested that PSGL-1 needs to be tyrosine-sulfated, in addition to glycosylated with sLe(X), to successfully interact with P-selectin. To better understand how biochemical features of the PSGL-1 ligand are related to its adhesion phenotype, we have measured the dynamics of adhesion under flow of a series of well-defined PSGL-1 variants that differ in their biochemical modification, to both P- and E-selectin-coated substrates. These variants are distinct PSGL-1 peptides: one that possesses sLe(X) in conjunction with three N-terminal tyr-SO(3) groups (SGP3), one that possesses sLe(X) without tyrosine sulfation (GP1), and one that lacks sLe(X) but has three N-terminal tyr-SO(3) groups (SP3). Although all peptides expressing sLe(X), tyr-SO(3), or both supported some form of rolling adhesion on P-selectin, only peptides expressing sLe(X) groups showed rolling adhesion on E-selectin. On P-selectin, the PSGL-1 peptides demonstrated a decreasing strength of adhesion in the following order: SGP3 > GP1 > SP3. Robust, rolling adhesion on P-selectin was mediated by the GP1 peptide, despite its lack of tyrosine sulfation. However, the addition of tyrosine sulfation to glycosylated peptides (SGP3) creates a super ligand for P-selectin that supports slower rolling adhesion at all shear rates and supports rolling adhesion at much higher shear rates. Tyrosine sulfation has no similar effect on PSGL-1 rolling on E-selectin. Such functional distinctions in rolling dynamics are uniquely realized with a cell-free system, which permits precise, unambiguous identification of the functional activity of adhesive ligands. These findings are consistent with structural and functional characterizations of the interactions between these peptides and E- and P-selectin published recently.
Rodgers
S D
SD
Department of Bioengineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Camphausen
R T
RT
Hammer
D A
DA
D019943:Amino Acid Substitution / Q000235:genetics; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D019040:E-Selectin / Q000378:metabolism; D005434:Flow Cytometry; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry* / Q000494:pharmacology; D019007:P-Selectin / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000080506:Sialyl Lewis X Antigen; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology
D019040:E-Selectin; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D019007:P-Selectin; C084882:P-selectin ligand protein; D010455:Peptides; D000080506:Sialyl Lewis X Antigen; C005662:tyrosine O-sulfate; D014443:Tyrosine