title: Structural and biochemical characterization of O-mannose-linked human natural killer-1 glycan expressed on phosphacan in developing mouse brains.
abstract: The human natural killer-1 (HNK-1) carbohydrate comprising a sulfated trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-) is expressed on N-linked and O-mannose-linked glycans in the nervous system and involved in learning and memory functions. Although whole/core glycan structures and carrier glycoproteins for the N-linked HNK-1 epitope have been studied, carrier glycoproteins and the biosynthetic pathway of the O-mannose-linked HNK-1 epitope have not been fully characterized. Here, using mass spectrometric analyses, we identified the major carrier glycoprotein of the O-linked HNK-1 as phosphacan in developing mouse brains and determined the major O-glycan structures having the terminal HNK-1 epitope from partially purified phosphacan. The O-linked HNK-1 epitope on phosphacan almost disappeared due to the knockout of protein O-mannose β1,2-N-acetylglucosaminyltransferase 1, an N-acetylglucosaminyltransferase essential for O-mannose-linked glycan synthesis, indicating that the reducing terminal of the O-linked HNK-1 is mannose. We also showed that glucuronyltransferase-P (GlcAT-P) was involved in the biosynthesis of O-mannose-linked HNK-1 using the gene-deficient mice of GlcAT-P, one of the glucuronyltransferases for HNK-1 synthesis. Consistent with this result, we revealed that GlcAT-P specifically synthesized O-linked HNK-1 onto phosphacan using cultured cells. Furthermore, we characterized the as-yet-unknown epitope of the 6B4 monoclonal antibody (mAb), which was thought to recognize a unique phosphacan glycoform. The reactivity of the 6B4 mAb almost completely disappeared in GlcAT-P-deficient mice, and exogenously expressed phosphacan was selectively recognized by the 6B4 mAb when co-expressed with GlcAT-P, suggesting that the 6B4 mAb preferentially recognizes O-mannose-linked HNK-1 on phosphacan. This is the first study to show that 6B4 mAb-reactive O-mannose-linked HNK-1 in the brain is mainly carried by phosphacan.
authors.affiliation: Department of Biological Chemistry, Human Health Sciences, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Jyoji
authors.initials: J
authors.lastname: Morise
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Kizuka
authors.forename: Keiko
authors.initials: K
authors.lastname: Yabuno
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Tonoyama
authors.forename: Noritaka
authors.initials: N
authors.lastname: Hashii
authors.forename: Nana
authors.initials: N
authors.lastname: Kawasaki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Manya
authors.forename: Yuko
authors.initials: Y
authors.lastname: Miyagoe-Suzuki
authors.forename: Shin'ichi
authors.initials: S
authors.lastname: Takeda
authors.forename: Tamao
authors.initials: T
authors.lastname: Endo
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Maeda
authors.forename: Hiromu
authors.initials: H
authors.lastname: Takematsu
authors.forename: Shogo
authors.initials: S
authors.lastname: Oka
mesh_terms: D000818:Animals; D001921:Brain / Q000254:growth & development / Q000378:metabolism*; D018959:CD57 Antigens / Q000737:chemistry / Q000378:metabolism*; D019556:COS Cells; D002236:Carbohydrate Conformation; D002522:Chlorocebus aethiops; D014453:Glucuronosyltransferase / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008358:Mannose / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D018959:CD57 Antigens; D017351:N-Acetylglucosaminyltransferases; C443106:protein O-mannose beta-1,2-N-acetylglucosaminyltransferase; C111364:galactosylgalactoylxylosylprotein 3-beta-glucuronosyltransferase; D014453:Glucuronosyltransferase; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5; D008358:Mannose
keywords: HNK-1; O-mannose; glucuronyltransferase-P; phosphacan