Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 286, Issue 36, Pages 31337-31346Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M111.233353
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Funding
- Ministry of Education, Culture, Sports, Science and Technology [21370053]
- Ministry of Health, Labor, and Welfare of Japan Health and Labour Sciences [H21-012]
- Grants-in-Aid for Scientific Research [23700509, 22300142, 21370053, 23650232, 23659153, 23500493] Funding Source: KAKEN
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Human natural killer-1 (HNK-1) carbohydrate is highly expressed in the nervous system and is involved in synaptic plasticity and dendritic spine maturation. This unique carbohydrate, consisting of a sulfated trisaccharide (HSO3-3GlcA beta 1-3Gal beta 1-4GlcNAc-), is biosynthesized by the successive actions of beta-1,4-galactosyltransferase (beta 4GalT), glucuronyltransferase (GlcAT-P and GlcAT-S), and sulfotransferase (HNK-1ST). A previous study showed that mice lacking beta 4GalT-II, one of seven beta 4GalTs, exhibited a dramatic loss of HNK-1 expression in the brain, although beta 4GalT-I-deficient mice did not. Here, we investigated the underlying molecular mechanism of the regulation of HNK-1 expression. First, focusing on a major HNK-1 carrier, neural cell adhesion molecule, we found that reduced expression of an N-linked HNK-1 carbohydrate caused by a deficiency of beta 4GalT-II is not likely due to a general loss of the beta 1,4-galactose residue as an acceptor for GlcAT-P. Instead, we demonstrated by co-immunoprecipitation and endoplasmic reticulum-retention analyses using Neuro2a (N2a) cells that beta 4GalT-II physically and specifically associates with GlcAT-P. In addition, we revealed by pull-down assay that Golgi luminal domains of beta 4GalT-II and GlcAT-P are sufficient for the complex to form. With an in vitro assay system, we produced the evidence that the kinetic efficiency k(cat)/K-m of GlcAT-P in the presence of beta 4GalT-II was increased about 2.5-fold compared with that in the absence of beta 4GalT-II. Finally, we showed that co-expression of beta 4GalT-II and GlcAT-P increased HNK-1 expression on various glycoproteins in N2a cells, including neural cell adhesion molecule. These results indicate that the specific enzyme complex of beta 4GalT-II with GlcAT-P plays an important role in the biosynthesis of HNK-1 carbohydrate.
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