Isolated P-selectin Glycoprotein Ligand-1 Dynamic Adhesion to P- and E-selectin

Abstract

Leukocyte adhesion to vascular endothelium under flow involves an adhesion cascade consisting of multiple receptor pairs that may function in an overlapping fashion. P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin have been implicated in neutrophil adhesion to P- and E-selectin under flow conditions. To study, in isolation, the interaction of PSGL-1 with P-and E-selectin under flow, we developed an in vitro model in which various recombinant regions of extracellular PSGL-1 were coupled to 10-μm-diameter microspheres. In a parallel plate chamber with well defined flow conditions, live time video microscopy analyses revealed that microspheres coated with PSGL-1 attached and rolled on 4-h tumor necrosis factor-α–activated endothelial cell monolayers, which express high levels of E-selectin, and CHO monolayers stably expressing E-or P-selectin. Further studies using CHO-E and -P monolayers demonstrate that the first 19 amino acids of PSGL-1 are sufficient for attachment and rolling on both E- and P-selectin and suggest that a sialyl Lewis x–containing glycan at Threonine-16 is critical for this sequence of amino acids to mediate attachment to E- and P-selectin. The data also demonstrate that a sulfated, anionic polypeptide segment within the amino terminus of PSGL-1 is necessary for PSGL-1–mediated attachment to P- but not to E-selectin. In addition, the results suggest that PSGL-1 has more than one binding site for E-selectin: one site located within the first 19 amino acids of PSGL-1 and one or more sites located between amino acids 19 through 148.

Distinct kinetic and mechanical properties govern selectin-leukocyte interactions

“…Moreover, rupture-force histograms, representing the most probable rupture force at a given reproach velocity, always developed a single peak as opposed to regularly spaced force distributions, which would be indicative of the unbinding of oligomeric adhesions (Benoit et al, 2000). The observance of a single significant force histogram for E-selectin/PMN binding is particularly noteworthy, considering the evidence supporting E-selectin binding to glycosphingolipids on the leukocyte cell surface (Alon et al, 1995b;Burdick et al, 2001) as well as multiple regions on PSGL-1 (Goetz et al, 1997). Several distinct E-selectin ligands or simultaneous multiple binding regions might be revealed as a broadened histogram distribution or histograms with multiple peaks (Benoit et al, 2000).…”

“…PSGL-1 serves as the primary counter-receptor for P-and L-selectin, in that it selectively binds with relatively high affinity and removal or absence of PSGL-1 essentially abrogates P-or L-selectin-mediated rolling (Varki, 1997). E-selectin also binds effectively to PSGL-1 (Goetz et al, 1997;Li et al, 1996), but accumulating evidence supports the concept that glycosphingolipids might also be physiologically relevant Eselectin ligands (Alon et al, 1995a;Bochner et al, 1994;Burdick et al, 2001;Kobzdej et al, 2002). Dynamic rolling assays conducted in the presence of a blocking anti-PSGL-1 mAb (KPL-1) indicated that PSGL-1-coated microspheres are still capable of tethering on E-but not P-selectin substrates (Goetz et al, 1997), supporting the notion that PSGL-1 has binding sites for E-selectin other than the crucial 19 amino acid sequence recognized by KPL-1.…”

“…E-selectin also binds effectively to PSGL-1 (Goetz et al, 1997;Li et al, 1996), but accumulating evidence supports the concept that glycosphingolipids might also be physiologically relevant Eselectin ligands (Alon et al, 1995a;Bochner et al, 1994;Burdick et al, 2001;Kobzdej et al, 2002). Dynamic rolling assays conducted in the presence of a blocking anti-PSGL-1 mAb (KPL-1) indicated that PSGL-1-coated microspheres are still capable of tethering on E-but not P-selectin substrates (Goetz et al, 1997), supporting the notion that PSGL-1 has binding sites for E-selectin other than the crucial 19 amino acid sequence recognized by KPL-1. Along these lines, E-selectin appears to be capable of binding to N-linked oligosaccharides, including those found on PSGL-1 (Aeed et al, 2001;Patel et al, 1994;Vestweber, 1996).…”

Partial characterization of the N-linked oligosaccharide structures on P-selectin glycoprotein ligand-1 (PSGL-1)

“…Previous reports have described the N-glycans on PSGL-1 as dispensable for binding [28] and a considerable body of evidence suggest that the selectins bind to Olinked oligosaccharide structures on PSGL-1 [7][8][9][10], [14]. A specific threonine residue has been identified in the NH2-terminal portion of PSGL-1 that is believed to carry the (O-linked) ligand oligosac-charide recognized by P-selectin [29][30][31]. Moreover, results from recent investigations clearly demonstrate that a significant portion of the O-linked structures on PSGL-1 are fucosylated [18], [19], thereby lending further support the involvement of O-linked oligosaccharides in selectin binding.…”

L- and P-Selectins Collaborate to Support Leukocyte Rolling in Vivo When High-Affinity P-Selectin-P-Selectin Glycoprotein Ligand-1 Interaction Is Inhibited

“…One strategy for studying the contributions of individual components of that surface is to coat artificial microspheres with purified or recombinant versions of molecules of interest. We and others have used this strategy in the past to investigate the capacity of PSGL-1 and other adhesion molecules to mediate rolling in vitro [27][28][29][30] and in vivo. 20,22,31 Ideally, the properties (size, ligand density, deformabilty, and so forth) of microspheres used for such investigations will closely match those of leukocytes, allowing direct comparisons to be made.…”