title: ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes.
abstract: The precise glycosyltransferase enzymes that mediate selectin-ligand biosynthesis in human leukocytes are unknown. This knowledge is important because selectin-mediated cell tethering and rolling is a critical component of both normal immune response and various vascular disorders. We evaluated the role of 3 α(2,3)sialyltransferases, ST3Gal-3, -4, and -6, which act on the type II N-Acetyllactosamine structure (Galβ1,4GlcNAc) to create sialyl Lewis-X (sLe(X)) and related sialofucosylated glycans on human leukocytes of myeloid lineage. These genes were either silenced using lentiviral short hairpin RNA (shRNA) or functionally ablated using the clustered regularly interspaced short palindromic repeat/Cas9 technology. The results show that ST3Gal-4, but not ST3Gal-3 or -6, is the major sialyltransferase regulating the biosynthesis of E-, P-, and L-selectin ligands in humans. Reduction in ST3Gal-4 activity lowered cell-surface HECA-452 epitope expression by 75% to 95%. Glycomics profiling of knockouts demonstrate an almost complete loss of the sLe(X) epitope on both leukocyte N- and O-glycans. In cell-adhesion studies, ST3Gal-4 knockdown/knockout cells displayed 90% to 100% reduction in tethering and rolling density on all selectins. ST3Gal-4 silencing in neutrophils derived from human CD34(+) hematopoietic stem cells also resulted in 80% to 90% reduction in cell adhesion to all selectins. Overall, a single sialyltransferase regulates selectin-ligand biosynthesis in human leukocytes, unlike mice where multiple enzymes contribute to this function.
authors.affiliation: Department of Chemical and Biological Engineering and The New York State Center for Excellence in Bioinformatics and Life Sciences, State University of New York, Buffalo, NY;
authors.forename: Nandini
authors.initials: N
authors.lastname: Mondal
authors.affiliation: Department of Chemical and Biological Engineering and The New York State Center for Excellence in Bioinformatics and Life Sciences, State University of New York, Buffalo, NY;
authors.forename: Alexander
authors.initials: A
authors.lastname: Buffone
authors.affiliation: Department of Chemical and Biological Engineering and The New York State Center for Excellence in Bioinformatics and Life Sciences, State University of New York, Buffalo, NY;
authors.forename: Gino
authors.initials: G
authors.lastname: Stolfa
authors.affiliation: Department of Life Sciences, Imperial College London, London, United Kingdom; and.
authors.forename: Aristotelis
authors.initials: A
authors.lastname: Antonopoulos
authors.affiliation: Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY.
authors.forename: Joseph T Y
authors.initials: JT
authors.lastname: Lau
authors.affiliation: Department of Life Sciences, Imperial College London, London, United Kingdom; and.
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Haslam
authors.affiliation: Department of Life Sciences, Imperial College London, London, United Kingdom; and.
authors.forename: Anne
authors.initials: A
authors.lastname: Dell
authors.affiliation: Department of Chemical and Biological Engineering and The New York State Center for Excellence in Bioinformatics and Life Sciences, State University of New York, Buffalo, NY;
authors.forename: Sriram
authors.initials: S
authors.lastname: Neelamegham
mesh_terms: D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D019040:E-Selectin / Q000096:biosynthesis* / Q000235:genetics; D020868:Gene Silencing; D054794:Glycomics; D018922:HL-60 Cells; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D019041:L-Selectin / Q000096:biosynthesis* / Q000235:genetics; D036904:Leukocyte Rolling / Q000502:physiology; D051379:Mice; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D019007:P-Selectin / Q000096:biosynthesis* / Q000235:genetics; D012799:Sialyltransferases / Q000096:biosynthesis* / Q000235:genetics; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
chemical_list: D019040:E-Selectin; D019007:P-Selectin; D019041:L-Selectin; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase