Abstract The cell surface glycoprotein CD34 is widely recognized as a marker of hematopoietic stem/progenitor cells (HSPCs), but its physiological role on HSPCs remains elusive. Although CD34 is well recognized for mediating homing of L-selectin+ naïve T cells to high endothelial venules of secondary lymphoid organs, the natural ligand for HSPC specific-CD34 is unknown. Studies in both humans and mice indicate that vascular selectins (E-selectin and P-selectin) are constitutively expressed on marrow endothelial cells and intravital studies revealed that HSPC migration to marrow occurs at specialized microvascular beds expressing E-selectin. Given that the identity of these glycoprotein E-selectin ligands (E-selL) on human HSPCs is limited, we used mass spectrometry (MS)-based proteomics to fully characterize all these potential ligands expressed on primary human CD34+ HSPCs and on the CD34+ KG1a cell line (human acute myelogenous leukemia that serves as a model for human HSPCs). We obtained a very rich resource for further investigation and chose to highlight CD34 as an E-selL candidate. The physiological binding of CD34 to E-selectin was addressed at both the molecular level and cellular level using novel binding assays. At the molecular level we developed a novel-binding assay to capture endogenous candidate glycoprotein ligands from whole cell lysates prepared from human HSPCs onto a surface plasmon resonance (SPR) chip and characterized its binding kinetics to recombinant E-sel. At the cellular level we used the blot rolling assay to monitor the ability of cells stably transfected with E-selectin (CHO-E) to roll on CD34 immunoprecipitated from HSPCs to demonstrate that CD34 binds to E-selectin with affinity comparable to well-described E-selectin ligands. Further biochemical analysis demonstrated that E-selectin binding was restricted to a specific glycoform that expresses sialyl Lewis X (sLex). This interaction was dependent on calcium, sialylation and O-glycans as removal of these abolished binding to E-selectin. CD34 binding to the other vascular selectin, P-selectin was dependent on O-glycan and tyrosine sulfation making CD34 the first selectin ligand since P-selectin glycoprotein ligand-1 (PSGL-1) reported to bind all three selectins. Among the stem cell specific processes such as self-renewal and differentiation that CD34 has been implicated to play a significant role in, these data are the first to define a role for human CD34 in mediating the direct migration of HSPCs to the bone marrow and suggests that a natural ligand for CD34 on human HSPCs are vascular selectins. Disclosures No relevant conflicts of interest to declare.
ASJC Scopus subject areas
- Cell Biology