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.
Highlights
Sickle cell disease (SCD) is caused by a mutation in the b-globin gene that replaces glutamic acid with valine
P-selectin expression was analyzed in platelets and HEL cells in detail in a previous study [30], phorbol myristate acetate, a tumor promoter [31], was used to investigate the mechanisms for P-selectin expression
To verify that the 140 kDa band is a glycosylated form, cell lysates prepared from human umbilical vein endothelial cells (HUVECs) treated with thrombin (1 U/mL) and from untreated platelets were immunprecipiated with P-selectin antibody
Summary
Sickle cell disease (SCD) is caused by a mutation in the b-globin gene that replaces glutamic acid with valine. Fetal hemoglobin inhibits sickle hemoglobin polymerization in vitro [4] and is an important protein ameliorating disease severity [5], as evidenced by the fact that SCD patients who express high levels of fetal hemoglobin have a milder clinical course [6]. Our intravital microscopic studies found that anti-P-selectin aptamer, with its high affinity to P-selectin and inhibition of P-selectin function, enables SCD model mice to survive hypoxic stress [21]. This is consistent with the work by Embury and colleagues which revealed an important role for Pselectin in sRBC adhesion to ECs [13,22]. A recent phase II clinical trial of pentosan polysulfate sodium (PPS), an orally available heparin compound, improved microvascular flow and reduced serum VCAM-1 levels in SCD patients, but did not reduce daily pain scores [23], prompting us to search for novel P-selectin inhibitors
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