title: Glycosphingolipids on Human Myeloid Cells Stabilize E-Selectin-Dependent Rolling in the Multistep Leukocyte Adhesion Cascade.
abstract: OBJECTIVE
  Recent studies suggest that the E-selectin ligands expressed on human leukocytes may differ from those in other species, particularly mice. To elaborate on this, we evaluated the impact of glycosphingolipids expressed on human myeloid cells in regulating E-selectin-mediated cell adhesion.
  
  
  RESULTS
  A series of modified human cell lines and primary neutrophils were created by targeting UDP-Glucose Ceramide Glucosyltransferase using either lentivirus-delivered shRNA or CRISPR-Cas9-based genome editing. Enzymology and mass spectrometry confirm that the modified cells had reduced or abolished glucosylceramide biosynthesis. Glycomics profiling showed that UDP-Glucose Ceramide Glucosyltransferase disruption also increased prevalence of bisecting N-glycans and reduced overall sialoglycan expression on leukocyte N- and O-glycans. Microfluidics-based flow chamber studies demonstrated that both the UDP-Glucose Ceramide Glucosyltransferase knockouts and knockdowns display ≈60% reduction in leukocyte rolling and firm adhesion on E-selectin bearing stimulated endothelial cells, without altering cell adhesion to P-selectin. Consistent with the concept that the glycosphingolipids support slow rolling and the transition to firm arrest, inhibiting UDP-Glucose Ceramide Glucosyltransferase activity resulted in frequent leukocyte detachment events, skipping motion, and reduced diapedesis across the endothelium. Cells bearing truncated O- and N-glycans also sustained cell rolling on E-selectin, although their ability to be recruited from free fluid flow was diminished.
  
  
  CONCLUSIONS
  Glycosphingolipids likely contribute to human myeloid cell adhesion to E-selectin under fluid shear, particularly the transition of rolling cells to firm arrest.
authors: From the Department of Chemical and Biological Engineering (N.M., G.S., Y.Z., S.-S.W., A.B., S.N.), Department of Chemistry (G.E.A.-G.), and The NY State Center for Excellence in Bioinformatics and Life Sciences (S.N.), State University of New York, Buffalo; and Department of Life Sciences, Imperial College London, London, UK (A.A., S.M.H., A.D.).
authors: Nandini
authors: N
authors: Mondal
authors: From the Department of Chemical and Biological Engineering (N.M., G.S., Y.Z., S.-S.W., A.B., S.N.), Department of Chemistry (G.E.A.-G.), and The NY State Center for Excellence in Bioinformatics and Life Sciences (S.N.), State University of New York, Buffalo; and Department of Life Sciences, Imperial College London, London, UK (A.A., S.M.H., A.D.).
authors: Gino
authors: G
authors: Stolfa
authors: From the Department of Chemical and Biological Engineering (N.M., G.S., Y.Z., S.-S.W., A.B., S.N.), Department of Chemistry (G.E.A.-G.), and The NY State Center for Excellence in Bioinformatics and Life Sciences (S.N.), State University of New York, Buffalo; and Department of Life Sciences, Imperial College London, London, UK (A.A., S.M.H., A.D.).
authors: Aristotelis
authors: A
authors: Antonopoulos
authors: From the Department of Chemical and Biological Engineering (N.M., G.S., Y.Z., S.-S.W., A.B., S.N.), Department of Chemistry (G.E.A.-G.), and The NY State Center for Excellence in Bioinformatics and Life Sciences (S.N.), State University of New York, Buffalo; and Department of Life Sciences, Imperial College London, London, UK (A.A., S.M.H., A.D.).
authors: Yuqi
authors: Y
authors: Zhu
authors: From the Department of Chemical and Biological Engineering (N.M., G.S., Y.Z., S.-S.W., A.B., S.N.), Department of Chemistry (G.E.A.-G.), and The NY State Center for Excellence in Bioinformatics and Life Sciences (S.N.), State University of New York, Buffalo; and Department of Life Sciences, Imperial College London, London, UK (A.A., S.M.H., A.D.).
authors: Shuen-Shiuan
authors: SS
authors: Wang
authors: From the Department of Chemical and Biological Engineering (N.M., G.S., Y.Z., S.-S.W., A.B., S.N.), Department of Chemistry (G.E.A.-G.), and The NY State Center for Excellence in Bioinformatics and Life Sciences (S.N.), State University of New York, Buffalo; and Department of Life Sciences, Imperial College London, London, UK (A.A., S.M.H., A.D.).
authors: Alexander
authors: A
authors: Buffone
authors: From the Department of Chemical and Biological Engineering (N.M., G.S., Y.Z., S.-S.W., A.B., S.N.), Department of Chemistry (G.E.A.-G.), and The NY State Center for Excellence in Bioinformatics and Life Sciences (S.N.), State University of New York, Buffalo; and Department of Life Sciences, Imperial College London, London, UK (A.A., S.M.H., A.D.).
authors: G Ekin
authors: GE
authors: Atilla-Gokcumen
authors: From the Department of Chemical and Biological Engineering (N.M., G.S., Y.Z., S.-S.W., A.B., S.N.), Department of Chemistry (G.E.A.-G.), and The NY State Center for Excellence in Bioinformatics and Life Sciences (S.N.), State University of New York, Buffalo; and Department of Life Sciences, Imperial College London, London, UK (A.A., S.M.H., A.D.).
authors: Stuart M
authors: SM
authors: Haslam
authors: From the Department of Chemical and Biological Engineering (N.M., G.S., Y.Z., S.-S.W., A.B., S.N.), Department of Chemistry (G.E.A.-G.), and The NY State Center for Excellence in Bioinformatics and Life Sciences (S.N.), State University of New York, Buffalo; and Department of Life Sciences, Imperial College London, London, UK (A.A., S.M.H., A.D.).
authors: Anne
authors: A
authors: Dell
authors: From the Department of Chemical and Biological Engineering (N.M., G.S., Y.Z., S.-S.W., A.B., S.N.), Department of Chemistry (G.E.A.-G.), and The NY State Center for Excellence in Bioinformatics and Life Sciences (S.N.), State University of New York, Buffalo; and Department of Life Sciences, Imperial College London, London, UK (A.A., S.M.H., A.D.). neel@buffalo.edu.
authors: Sriram
authors: S
authors: Neelamegham
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002448:Cell Adhesion; D019040:E-Selectin / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism*; D005260:Female; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism; D054794:Glycomics / Q000379:methods; D006028:Glycosphingolipids / Q000378:metabolism*; D057809:HEK293 Cells; D018922:HL-60 Cells; D006801:Humans; D036904:Leukocyte Rolling; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D046210:Microfluidic Analytical Techniques; D009504:Neutrophils / Q000378:metabolism*; D061251:Primary Cell Culture; D011402:Pronase / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction; D013045:Species Specificity; D013997:Time Factors; D057705:Transendothelial and Transepithelial Migration; D014162:Transfection
chemical_list: D019040:E-Selectin; D006028:Glycosphingolipids; C546936:SELE protein, human; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; D011402:Pronase
keywords: cell adhesion molecule; endothelial cell; flow shear stress; inflammation; leukocyte