Structural and biochemical characterization of O-mannose-linked human natural killer-1 glycan expressed on phosphacan in developing mouse brains

Jyoji Morise, Yasuhiko Kizuka, Keiko Yabuno, Yasuhiro Tonoyama, Noritaka Hashii, Nana Kawasaki, Hiroshi Manya, Yuko Miyagoe-Suzuki, Shin'Ichi Takeda, Tamao Endo, Nobuaki Maeda, Hiromu Takematsu, Shogo Oka

Research output: Contribution to journalArticlepeer-review

30 Citations (Scopus)


The human natural killer-1 (HNK-1) carbohydrate comprising a sulfated trisaccharide (HSO3-3GlcA1-3Gal1-4GlcNAc-) 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 1,2-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.

Original languageEnglish
Pages (from-to)314-324
Number of pages11
Issue number3
Publication statusPublished - 03-2014
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry


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