title: Discordant expression of selectin ligands and sialyl Lewis x-related epitopes on murine myeloid cells.
abstract: Murine leukocytes are thought to express alpha2-3-sialylated and alpha1-3-fucosylated selectin ligands such as sialyl Lewis x (sLe(x)), although monoclonal antibodies (mAbs) to sLe(x) or Le(x) reportedly do not bind to murine leukocytes. We observed that P- and E-selectin bound to pronase-sensitive ligands on murine monocytic WEHI-3 cells and murine neutrophils, indicating that the ligands for both selectins are glycoproteins. CSLEX-1, HECA-452, and other widely used mAbs to sLe(x) and Le(x) did not bind to WEHI-3 cells and bound at very low levels to murine neutrophils. Only the anti-sLe(x) mAbs 2H5 and KM93, which also recognize nonfucosylated glycans, bound to WEHI-3 cells. 2H5 and KM93 bound to pronase-resistant structures, indicating that the mAbs did not identify selectin ligands. Treatment of WEHI-3 cells with glycosidases or chlorate demonstrated that sialic acid modifications, alpha1-3-galactosylation, or sulfation did not mask epitopes for mAbs to sLe(x) or Le(x). Compared to human promyelocytic HL-60 cells, WEHI-3 cells and murine neutrophils expressed low alpha1-3-fucosyltransferase activities. Consistent with very low endogenous fucosylation, forced fucosylation of intact WEHI-3 cells or murine neutrophils by exogenous alpha1-3-fucosyltransferase FTVI and GDP-fucose created many new epitopes for anti-sLe(x) mAbs such as HECA-452 and CSLEX-1. Nevertheless, forced fucosylation of intact cells did not significantly augment their ability to bind to fluid-phase P- or E-selectin or to roll on immobilized P- or E-selectin under flow. These data suggest that murine myeloid leukocytes fucosylate only a few specific glycans, which interact preferentially with P- and E-selectin.
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104, USA.
authors.forename: Marcin M A
authors.initials: MM
authors.lastname: Kobzdej
authors.forename: Anne
authors.initials: A
authors.lastname: Leppänen
authors.forename: Vishwanath
authors.initials: V
authors.lastname: Ramachandran
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cummings
authors.forename: Rodger P
authors.initials: RP
authors.lastname: McEver
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D019040:E-Selectin / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism*; D005643:Fucose / Q000276:immunology; D005647:Fucosyltransferases / Q000096:biosynthesis / Q000494:pharmacology; D005690:Galactose / Q000276:immunology; D018922:HL-60 Cells / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D022423:Myeloid Cells / Q000276:immunology / Q000378:metabolism*; D009439:Neuraminidase / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism; D009844:Oligosaccharides / Q000276:immunology / Q000378:metabolism*; D019007:P-Selectin / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D011402:Pronase / Q000494:pharmacology; D011485:Protein Binding; D000080506:Sialyl Lewis X Antigen; D013045:Species Specificity; D013379:Substrate Specificity; D013431:Sulfates / Q000276:immunology; D000519:alpha-Galactosidase / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D019040:E-Selectin; D000939:Epitopes; D008024:Ligands; D009844:Oligosaccharides; D019007:P-Selectin; D011134:Polysaccharides; D000080506:Sialyl Lewis X Antigen; D013431:Sulfates; D005643:Fucose; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D009439:Neuraminidase; D000519:alpha-Galactosidase; D011402:Pronase; D005690:Galactose