No abstract

Selectins play a major role in the inflammatory reaction by initiating neutrophil attachment to activated vascular endothelium. Some heparin preparations can interact with L-and P-selectin; however, the determinants required for inhibiting selectin-mediated cell adhesion have not yet been characterized. We now report that carboxyl-reduced and sulfated heparin (prepared by chemical modifications of porcine intestinal mucosal heparin leading to the replacement of carboxylates by O-sulfate groups) and trestatin A sulfate (obtained by sulfation of trestatin A, a non-uronic pseudo-nonasaccharide extracted from Streptomyces dimorphogenes) exhibit strong anti-P-selectin and anti-L-selectin activity while lacking antithrombin-mediated anticoagulant activity. In vitro experiments revealed that both compounds inhibited P-selectin-and L-selectin-mediated cell adhesion under laminar flow conditions. Moreover, carboxyl-reduced and sulfated heparin and trestatin A sulfate were also active in vivo, as assessed by experiments showing 1) that microinfusion of trestatin A sulfate reduced by 96% leukocyte rolling along rat mesenteric postcapillary venules and 2) that both compounds inhibited (by 58 -81%) neutrophil migration into thioglycollate-inflamed peritoneum of BALB/c mice. These results indicate that nonanticoagulant sulfated saccharides targeted at P-selectin and L-selectin may have therapeutic potential in inflammatory disorders.Leukocyte migration in inflammatory lesions is a reaction that is sequentially regulated by adhesion receptors and inflammation products. Selectins play a major role in initiating neutrophil attachment to cytokine-activated endothelium. Lselectin is expressed by most circulating leukocytes; E-selectin expression is induced after several hours of endothelial cell activation by interleukin-1, tumor necrosis factor-␣, or endotoxin; P-selectin is rapidly expressed by endothelial cells or platelets exposed to thrombin or histamine (1-6).Due to different kinetics of expression, the various selectins function at different, although overlapping, phases of the inflammatory reaction. The earliest phase (Ͻ20 min) of neutrophil spontaneous rolling in rat postcapillary venules is mainly dependent on interactions between P-selectin and its major ligand P-selectin glycoprotein-1 (PSGL-1), 1 whereas L-selectin involvement is observed during a later phase (7-12). Several observations indicated that PSGL-1 is a common ligand for L-, P-, and E-selectin (13-16). This mucin-like glycoprotein is expressed by most leukocytes and interacts with L-selectin and P-selectin through a N-terminal tyrosine sulfation consensus and sialylated, fucosylated core-2 branched O-glycans (4,14,(17)(18)(19)(20)(21)(22)(23)(24). Importantly, interactions between L-selectin and PSGL-1 mediate the attachment of circulating neutrophils to neutrophils already adherent to the endothelium, a process that may increase neutrophil recruitment in inflamed tissues (25,26).Selectins share a common primary structure with a N-terminal lectin domain tha...

Previous studies have shown that yeast glycosylphosphatidylinositol-anchored proteins (GPI-APs) and other secretory proteins are preferentially incorporated into distinct coat protein II (COPII) vesicle populations for their transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and that incorporation of yeast GPI-APs into COPII vesicles requires specific lipid interactions. We compared the ER exit mechanism and segregation of GPI-APs from other secretory proteins in mammalian and yeast cells. We find that, unlike yeast, ER-to-Golgi transport of GPI-APs in mammalian cells does not depend on sphingolipid synthesis. Whereas ER exit of GPI-APs is tightly dependent on Sar1 in mammalian cells, it is much less so in yeast. Furthermore, in mammalian cells, GPI-APs and other secretory proteins are not segregated upon COPII vesicle formation, in contrast to the remarkable segregation seen in yeast. These findings suggest that GPI-APs use different mechanisms to concentrate in COPII vesicles in the two organisms, and the difference might explain their propensity to segregate from other secretory proteins upon ER exit.