title: Functional contributions of N- and O-glycans to L-selectin ligands in murine and human lymphoid organs.
abstract: L-selectin initiates lymphocyte interactions with high endothelial venules (HEVs) of lymphoid organs through binding to ligands with specific glycosylation modifications. 6-Sulfo sLe(x), a sulfated carbohydrate determinant for L-selectin, is carried on core 2 and extended core 1 O-glycans of HEV-expressed glycoproteins. The MECA-79 monoclonal antibody recognizes sulfated extended core 1 O-glycans and partially blocks lymphocyte-HEV interactions in lymphoid organs. Recent evidence has identified the contribution of 6-sulfo sLe(x) carried on N-glycans to lymphocyte homing in mice. Here, we characterize CL40, a novel IgG monoclonal antibody. CL40 equaled or surpassed MECA-79 as a histochemical staining reagent for HEVs and HEV-like vessels in mouse and human. Using synthetic carbohydrates, we found that CL40 bound to 6-sulfo sLe(x) structures, on both core 2 and extended core 1 structures, with an absolute dependency on 6-O-sulfation. Using transfected CHO cells and gene-targeted mice, we observed that CL40 bound its epitope on both N-glycans and O-glycans. Consistent with its broader glycan-binding, CL40 was superior to MECA-79 in blocking lymphocyte-HEV interactions in both wild-type mice and mice deficient in forming O-glycans. This superiority was more marked in human, as CL40 completely blocked lymphocyte binding to tonsillar HEVs, whereas MECA-79 inhibited only 60%. These findings extend the evidence for the importance of N-glycans in lymphocyte homing in mouse and indicate that this dependency also applies to human lymphoid organs.
authors.affiliation: Department of Anatomy, University of California, San Francisco, California 94143-0452, USA.
authors.forename: Hanayo
authors.initials: H
authors.lastname: Arata-Kawai
authors.forename: Mark S
authors.initials: MS
authors.lastname: Singer
authors.forename: Annette
authors.initials: A
authors.lastname: Bistrup
authors.forename: Annemieke van
authors.initials: Av
authors.lastname: Zante
authors.forename: Yang-Qing
authors.initials: YQ
authors.lastname: Wang
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ito
authors.forename: Xingfeng
authors.initials: X
authors.lastname: Bao
authors.forename: Stefan
authors.initials: S
authors.lastname: Hemmerich
authors.forename: Minoru
authors.initials: M
authors.lastname: Fukuda
authors.forename: Steven D
authors.initials: SD
authors.lastname: Rosen
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D006801:Humans; D019041:L-Selectin / Q000276:immunology* / Q000378:metabolism; D008024:Ligands; D008198:Lymph Nodes / Q000276:immunology*; D008565:Membrane Proteins / Q000276:immunology*; D051379:Mice; D014066:Palatine Tonsil / Q000276:immunology*; D011134:Polysaccharides / Q000276:immunology* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D000954:Antigens, Surface; D000939:Epitopes; C069886:L-selectin counter-receptors; D008024:Ligands; D008565:Membrane Proteins; D011134:Polysaccharides; D019041:L-Selectin