title: Glycosylation inhibitors efficiently inhibit P-selectin-mediated cell adhesion to endothelial cells.
abstract: Adhesion molecules play a critical role in the adhesive interactions of multiple cell types in sickle cell disease (SCD). We previously showed that anti-P-selectin aptamer efficiently inhibits cell adhesion to endothelial cells (ECs) and permits SCD mice to survive hypoxic stress. In an effort to discover new mechanisms with which to inhibit P-selectin, we examined the role of glycosylation. P-selectin is a 90 kDa protein but was found to migrate as 90 and 140 kDa bands on gel electrophoresis. When P-selectin isolated from ECs was digested with peptide N-glycosidase F, but not O-glycosidase, the 140 kDa band was lost and the 90 kDa band was enhanced. Treatment of ECs with tunicamycin, an N-glycosylation inhibitor, suppressed CD62P (P-selectin) expression on the cell surface as well as the 140 kDa form in the cytoplasm. These results indicate that the 140 kDa band is N-glycosylated and glycosylation is critical for cell surface expression of P-selectin in ECs. Thrombin, which stimulates P-selectin expression on ECs, induced AKT phosphorylation, whereas tunicamycin inhibited AKT phosphorylation, suggesting that AKT signaling is involved in the tunicamycin-mediated inhibition of P-selectin expression. Importantly, the adhesion of sickle red blood cells (sRBCs) and leukocytes to ECs induced by thrombin or hypoxia was markedly inhibited by two structurally distinct glycosylation inhibitors; the levels of which were comparable to that of a P-selectin monoclonal antibody which most strongly inhibited cell adhesion in vivo. Knockdown studies of P-selectin using short-hairpin RNAs in ECs suppressed sRBC adhesion, indicating a legitimate role for P-selectin in sRBC adhesion. Together, these results demonstrate that P-selectin expression on ECs is regulated in part by glycosylation mechanisms and that glycosylation inhibitors efficiently reduce the adhesion of sRBCs and leukocytes to ECs. Glycosylation inhibitors may lead to a novel therapy which inhibits cell adhesion in SCD.
authors.affiliation: Department of Anesthesiology and Perioperative Medicine, Medical College of Georgia, Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: Pushpankur
authors.initials: P
authors.lastname: Ghoshal
authors.affiliation: Department of Anesthesiology and Perioperative Medicine, Medical College of Georgia, Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: Mythilypriya
authors.initials: M
authors.lastname: Rajendran
authors.affiliation: Department of Anesthesiology and Perioperative Medicine, Medical College of Georgia, Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: Nadine
authors.initials: N
authors.lastname: Odo
authors.affiliation: Department of Anesthesiology and Perioperative Medicine, Medical College of Georgia, Georgia Regents University, Augusta, Georgia, United States of America.
authors.forename: Tohru
authors.initials: T
authors.lastname: Ikuta
mesh_terms: D000755:Anemia, Sickle Cell / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects / Q000235:genetics*; D015687:Cell Hypoxia / Q000235:genetics; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D006031:Glycosylation; D007962:Leukocytes / Q000378:metabolism / Q000473:pathology; D051379:Mice; D019007:P-Selectin / Q000096:biosynthesis* / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014415:Tunicamycin / Q000008:administration & dosage
chemical_list: D019007:P-Selectin; D014415:Tunicamycin; D013917:Thrombin