title: Analysis of glycoprotein E-selectin ligands on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells.
abstract: Although well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using E-selectin-Ig chimera (E-Ig) shows that human marrow cells enriched for HSPCs (CD34(+) cells) display greater E-selectin binding than those obtained from mouse (lin(-)/Sca-1(+)/c-kit(+) [LSK] cells). To define the relevant glycoprotein E-selectin ligands, lysates from human CD34(+) and KG1a cells and from mouse LSK cells were immunoprecipitated using E-Ig and resolved by Western blot using E-Ig. In both human and mouse cells, E-selectin ligand reactivity was observed at ~ 120- to 130-kDa region, which contained two E-selectin ligands, the P-selectin glycoprotein ligand-1 glycoform "CLA," and CD43. Human, but not mouse, cells displayed a prominent ~ 100-kDa band, exclusively comprising the CD44 glycoform "HCELL." E-Ig reactivity was most prominent on CLA in mouse cells and on HCELL in human cells. To further assess HCELL's contribution to E-selectin adherence, complementary studies were performed to silence (via CD44 siRNA) or enforce its expression (via exoglycosylation). Under physiologic shear conditions, CD44/HCELL-silenced human cells showed striking decreases (> 50%) in E-selectin binding. Conversely, enforced HCELL expression of LSK cells profoundly increased E-selectin adherence, yielding > 3-fold more marrow homing in vivo. These data define the key glycoprotein E-selectin ligands of human and mouse HSPCs, unveiling critical species-intrinsic differences in both the identity and activity of these structures.
authors.affiliation: Department of Dermatology, Brigham and Women's Hospital, Boston, MA, USA.
authors.forename: Jasmeen S
authors.initials: JS
authors.lastname: Merzaban
authors.forename: Monica M
authors.initials: MM
authors.lastname: Burdick
authors.forename: S Zeineb
authors.initials: SZ
authors.lastname: Gadhoum
authors.forename: Nilesh M
authors.initials: NM
authors.lastname: Dagia
authors.forename: Julia T
authors.initials: JT
authors.lastname: Chu
authors.forename: Robert C
authors.initials: RC
authors.lastname: Fuhlbrigge
authors.forename: Robert
authors.initials: R
authors.lastname: Sackstein
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D019040:E-Selectin / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism; D051917:Leukosialin / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008810:Mice, Inbred C57BL; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D018952:Antigens, CD34; D019040:E-Selectin; D018960:Hyaluronan Receptors; D051917:Leukosialin; D008024:Ligands; D011134:Polysaccharides; D034741:RNA, Small Interfering; C497483:CD44 protein, human