Publication Date:
2014-02-11
Description:
The human natural killer-1 (HNK-1) carbohydrate comprising a sulfated trisaccharide (HSO 3 -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.
Print ISSN:
0959-6658
Electronic ISSN:
1460-2423
Topics:
Biology
,
Medicine
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