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Abstract
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 (HSO(3)-3GlcAβ1-3Galβ1-4GlcNAc-), is biosynthesized by the successive actions of β-1,4-galactosyltransferase (β4GalT), glucuronyltransferase (GlcAT-P and GlcAT-S), and sulfotransferase (HNK-1ST). A previous study showed that mice lacking β4GalT-II, one of seven β4GalTs, exhibited a dramatic loss of HNK-1 expression in the brain, although β4GalT-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 β4GalT-II is not likely due to a general loss of the β1,4-galactose residue as an acceptor for GlcAT-P. Instead, we demonstrated by co-immunoprecipitation and endoplasmic reticulum-retention analyses using Neuro2a (N2a) cells that β4GalT-II physically and specifically associates with GlcAT-P. In addition, we revealed by pulldown assay that Golgi luminal domains of β4GalT-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 β4GalT-II was increased about 2.5-fold compared with that in the absence of β4GalT-II. Finally, we showed that co-expression of β4GalT-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 β4GalT-II with GlcAT-P plays an important role in the biosynthesis of HNK-1 carbohydrate.
Highlights
Glycosylation is a major post-translational modification, especially for cell surface and extracellular proteins, and plays important roles in cellular functions such as adhesion, endocytosis, and receptor signaling [1, 2]
Unaltered Expression of N-Acetyllactosamine on a Major Human natural killer-1 (HNK-1) Carrier in 4GalT-II-deficient Mice—Recently, we reported that HNK-1 expression is substantially reduced in 4GalT-II-deficient mice, an earlier study revealed that 4GalT-I-deficient mice expressed normal levels of SEPTEMBER 9, 2011
We recently reported that 4GalT-II-deficient mice showed a marked reduction in levels of HNK-1 carbohydrate and impaired spatial memory, and the phenotypes are similar to those of GlcAT-P-deficient mice [19]
Summary
Glycosylation is a major post-translational modification, especially for cell surface and extracellular proteins, and plays important roles in cellular functions such as adhesion, endocytosis, and receptor signaling [1, 2]. These results indicate that the specific enzyme complex of 4GalT-II with GlcAT-P plays an important role in the biosynthesis of HNK-1 carbohydrate.
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