Molecular Characterization of the Interactions between Vascular Selectins and Glycoprotein Ligands on Human Hematopoietic Stem/Progenitor Cells

Handle URI:
http://hdl.handle.net/10754/621910
Title:
Molecular Characterization of the Interactions between Vascular Selectins and Glycoprotein Ligands on Human Hematopoietic Stem/Progenitor Cells
Authors:
Abusamra, Dina ( 0000-0002-0974-5362 )
Abstract:
The human bone marrow vasculature constitutively expresses both E-selectin and P-selectin where they interact with the cell-surface glycan moiety, sialyl Lewis x, on circulating hematopoietic stem/progenitor cells (HSPCs) to mediate the essential tethering/rolling step. Although several E-selectin glycoprotein ligands (E-selLs) have been identified, the importance of each E-selL on human HSPCs is debatable and requires additional methodologies to advance their specific involvement. The first objective was to fill the knowledge gap in the in vitro characterization of the mechanisms used by selectins to mediate the initial step in the HSPCs homing by developing a real time immunoprecipitation-based assay on a surface plasmon resonance chip. This novel assay bypass the difficulties of purifying ligands, enables the use of natively glycosylated forms of selectin ligands from any model cell of interest and study its binding affinities under flow. We provide the first comprehensive quantitative binding kinetics of two well-documented ligands, CD44 and PSGL-1, with E-selectin. Both ligands bind monomeric E-selectin transiently with fast on- and off-rates while they bind dimeric E-selectin with remarkably slow on- and off-rates with the on-rate, but not the off-rate, is dependent on salt concentration. Thus, suggest a mechanism through which monomeric selectins mediate initial fast-on and -off binding to capture the circulating cells out of shear-flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to slow rolling significantly. The second objective is to fully identify and characterize E/P-selectin ligand candidates expressed on CD34+ HSPCs which cause enhanced migration after intravenous transplantation compared to their CD34- counterparts. CD34 is widely recognized marker of human HSPCs but its natural ligand and function on these cells remain elusive. Proteomics identified CD34 as an E-selL candidate on human HSPCs, whose binding to E-selectin was confirmed using some static and flow-based assays. E-selectin binds to CD34 with an affinity comparable to the well-described E-selLs CD44/HCELL and PSGL-1. CD34 knockdown resulted in faster-rolling velocities compared to control cells especially at and above three dyne/cm2. CD34 is the first selectin ligand since PSGL-1 reported to bind E-/P-/L-selectins and likely plays a key role in directing the migration of human HSPCs to the bone marrow.
Advisors:
Merzaban, Jasmeen ( 0000-0002-7276-2907 )
Committee Member:
Eppinger, Jörg ( 0000-0001-7886-7059 ) ; Habuchi, Satoshi ( 0000-0002-6663-2807 ) ; Dimitroff, Charles
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division
Program:
Bioscience
Issue Date:
Dec-2016
Type:
Dissertation
Appears in Collections:
Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.advisorMerzaban, Jasmeenen
dc.contributor.authorAbusamra, Dinaen
dc.date.accessioned2016-12-01T07:15:53Z-
dc.date.available2016-12-01T07:15:53Z-
dc.date.issued2016-12-
dc.identifier.urihttp://hdl.handle.net/10754/621910-
dc.description.abstractThe human bone marrow vasculature constitutively expresses both E-selectin and P-selectin where they interact with the cell-surface glycan moiety, sialyl Lewis x, on circulating hematopoietic stem/progenitor cells (HSPCs) to mediate the essential tethering/rolling step. Although several E-selectin glycoprotein ligands (E-selLs) have been identified, the importance of each E-selL on human HSPCs is debatable and requires additional methodologies to advance their specific involvement. The first objective was to fill the knowledge gap in the in vitro characterization of the mechanisms used by selectins to mediate the initial step in the HSPCs homing by developing a real time immunoprecipitation-based assay on a surface plasmon resonance chip. This novel assay bypass the difficulties of purifying ligands, enables the use of natively glycosylated forms of selectin ligands from any model cell of interest and study its binding affinities under flow. We provide the first comprehensive quantitative binding kinetics of two well-documented ligands, CD44 and PSGL-1, with E-selectin. Both ligands bind monomeric E-selectin transiently with fast on- and off-rates while they bind dimeric E-selectin with remarkably slow on- and off-rates with the on-rate, but not the off-rate, is dependent on salt concentration. Thus, suggest a mechanism through which monomeric selectins mediate initial fast-on and -off binding to capture the circulating cells out of shear-flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to slow rolling significantly. The second objective is to fully identify and characterize E/P-selectin ligand candidates expressed on CD34+ HSPCs which cause enhanced migration after intravenous transplantation compared to their CD34- counterparts. CD34 is widely recognized marker of human HSPCs but its natural ligand and function on these cells remain elusive. Proteomics identified CD34 as an E-selL candidate on human HSPCs, whose binding to E-selectin was confirmed using some static and flow-based assays. E-selectin binds to CD34 with an affinity comparable to the well-described E-selLs CD44/HCELL and PSGL-1. CD34 knockdown resulted in faster-rolling velocities compared to control cells especially at and above three dyne/cm2. CD34 is the first selectin ligand since PSGL-1 reported to bind E-/P-/L-selectins and likely plays a key role in directing the migration of human HSPCs to the bone marrow.en
dc.language.isoenen
dc.subjectHematopoietic Stem/Pogenitor Cell (HSPCS)en
dc.subjectCD34en
dc.subjectBone Marrowen
dc.subjectVascular Selectionen
dc.subjectSurface Plasmon Resonanceen
dc.subjectCD44 / HCellen
dc.titleMolecular Characterization of the Interactions between Vascular Selectins and Glycoprotein Ligands on Human Hematopoietic Stem/Progenitor Cellsen
dc.typeDissertationen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
thesis.degree.grantorKing Abdullah University of Science and Technologyen_GB
dc.contributor.committeememberEppinger, Jörgen
dc.contributor.committeememberHabuchi, Satoshien
dc.contributor.committeememberDimitroff, Charlesen
thesis.degree.disciplineBioscienceen
thesis.degree.nameDoctor of Philosophyen
dc.person.id101939en
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