TY - JOUR
T1 - Structural and biochemical characterization of O-mannose-linked human natural killer-1 glycan expressed on phosphacan in developing mouse brains
AU - Morise, Jyoji
AU - Kizuka, Yasuhiko
AU - Yabuno, Keiko
AU - Tonoyama, Yasuhiro
AU - Hashii, Noritaka
AU - Kawasaki, Nana
AU - Manya, Hiroshi
AU - Miyagoe-Suzuki, Yuko
AU - Takeda, Shin'Ichi
AU - Endo, Tamao
AU - Maeda, Nobuaki
AU - Takematsu, Hiromu
AU - Oka, Shogo
N1 - Funding Information:
This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (grant number 23110006 to S.O. and 23110002 to H.M.) from MEXT of Japan, a Grant-in-Aid for Scientific Research (B) (grant number 25293016) from the Japan Society for the Promotion of Science (to T.E.) and Intramural Research Grant (23-5) for Neurological and Psychiatric Disorders of NCNP (to T.E.).
PY - 2014/3
Y1 - 2014/3
N2 - 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.
AB - 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.
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U2 - 10.1093/glycob/cwt116
DO - 10.1093/glycob/cwt116
M3 - Article
C2 - 24352591
AN - SCOPUS:84894270094
SN - 0959-6658
VL - 24
SP - 314
EP - 324
JO - Glycobiology
JF - Glycobiology
IS - 3
ER -