HNK-1 (human natural killer-1) glyco-epitope, a sulfated glucuronic acid attached to N-acetyllactosamine on the nonreducing termini of glycans, is highly expressed in the nervous system. Our previous report showed that mice lacking a glucuronyltransferase (GlcAT-P), a key enzyme for biosynthesis of the HNK-1 epitope, showed reduced long term potentiation at hippocampal CA1 synapses. In this study, we identified an ␣-amino-3-hydroxy-5-methylisoxazole propionate (AMPA)-type glutamate receptor subunit, GluR2, which directly contributes to excitatory synaptic transmission and synaptic plasticity, as a novel HNK-1 carrier molecule. We demonstrated that the HNK-1 epitope is specifically expressed on the N-linked glycan(s) on GluR2 among the glutamate receptors tested, and the glycan structure, including HNK-1 on GluR2, was determined using liquid chromatography-tandem mass spectrometry. As for the function of HNK-1 on GluR2, we found that the GluR2 not carrying HNK-1 was dramatically endocytosed and expressed less on the cell surface compared with GluR2 carrying HNK-1 in both cultured hippocampal neurons and heterologous cells. These results suggest that HNK-1 stabilizes GluR2 on neuronal surface membranes and regulates the number of surface AMPA receptors. Moreover, we showed that the expression of the HNK-1 epitope enhanced the interaction between GluR2 and N-cadherin, which has important roles in AMPA receptor trafficking. Our findings suggest that the HNK-1 epitope on GluR2 regulates cell surface stability of GluR2 by modulating the interaction with N-cadherin.HNK-1 glyco-epitope (HSO 3 -3GlcA␤1-3Gal␤1-4GlcNAc) is characteristically expressed on some cell adhesion molecules (NCAM, L1, and MAG, etc.) and extracellular matrix molecules (tenascin-R and phosphacan, etc.) in the nervous system (1). It has been reported that HNK-1 mediates the interaction of these adhesion molecules, thereby controlling their functions, including cell-to-cell adhesion (2), migration (3), and neurite extension (4). The unique structural feature of the HNK-1 epitope is the sulfated glucuronic acid, because sialic acids are usually attached to the terminal galactose residue of the inner N-acetyllactosamine structure (Gal␤1-4GlcNAc) on various glycoproteins. HNK-1 is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) 3 (5, 6) and a sulfotransferase (HNK-1ST) (7). These enzymes are thought to localize and function in the Golgi apparatus, especially the trans-Golgi to trans-Golgi network, like most sialyltransferases and galactosyltransferases (8).We previously demonstrated that mice deficient in GlcAT-P showed an almost complete loss of HNK-1 expression in the brain and exhibited reduced LTP in hippocampal CA1 synapses (9). Similarly, HNK-1ST-deficient mice also exhibited a reduction of LTP, and several other studies also revealed that HNK-1 is associated with neural plasticity (10 -12). A recent study showed that ␤4-galactosyltransferase-2 synthesizes the glycan backbone structure of HNK-1, Gal␤1-4GlcNAc. ...

Glycosylation is a major post-translational protein modification, especially for cell surface proteins, which play important roles in a variety of cellular functions, including recognition and adhesion. Among them, we have been interested in HNK-1 (human natural killer-1) carbohydrate, which is characteristically expressed on a series of cell adhesion molecules in the nervous system. The HNK-1 carbohydrate has a unique structural feature, i.e. a sulfated glucuronic acid is attached to the non-reducing terminal of an N-acetyllactosamine residue (HSO(3)-3GlcAbeta1-3Galbeta1-4GlcNAc-). We have cloned and characterized the biosynthetic enzymes (two glucuronyltransferases and a sulfotransferase), and also obtained evidence that the HNK-1 carbohydrate is involved in synaptic plasticity and memory formation. In this review, we describe recent findings regarding the expression mechanism and functional roles of this carbohydrate.

The AMPA-type glutamate receptor (AMPAR), which is a tetrameric complex composed of four subunits (GluA1-4) with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc), human natural killer-1 (HNK-1) carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413) within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER) in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.

The human natural killer-1 (HNK-1) glyco-epitope possesses a unique structural feature, a sulfated glucuronic acid attached to lactosamine on the non-reducing termini of glycans. The expression of HNK-1 is temporally and spatially regulated by glucuronyltransferase (GlcAT-P) in the brain. Our previous report showed that mice lacking GlcAT-P almost completely lost HNK-1 expression in the brain and exhibited reduced long-term potentiation (LTP) at hippocampal CA1 synapses. GlcAT-P-deficient mice also showed impaired hippocampus-dependent spatial learning. Although HNK-1 plays an essential role in synaptic plasticity and memory formation, it remains unclear how HNK-1 regulates these functions. In this study, we showed that loss of the HNK-1 epitope resulted in an increase of filopodium-like immature spines and a decrease of mushroom-like mature spines in both the early postnatal mouse hippocampus and cultured hippocampal neurons. However, HNK-1 had no influence on spine density or filopodium formation. Immunofluorescence staining revealed that loss of HNK-1 altered the distribution of postsynaptic proteins such as alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)-type glutamate receptor subunit GluR2 and PSD-95 from spine heads onto dendritic shafts without affecting synapse formation, resulting in an increase of shaft synapses in cultured GlcAT-P-deficient neurons. GluR2, a major HNK-1 carrier glycoprotein in postsynaptic density, has the ability to promote spine morphogenesis. Overexpression of GluR2 promoted spine growth in both wild-type and GlcAT-P-deficient neurons, but the increase in GlcAT-P-deficient neurons was lower than that in wild-type neurons. This is the first evidence that HNK-1 is a key factor for normal dendritic spine maturation and is involved in the distribution of postsynaptic proteins.

The HNK-1 carbohydrate epitope is found in various neural cell adhesion molecules. Two glucuronyltransferases (GlcAT-P and GlcAT-S) are involved in the biosynthesis of HNK-1 carbohydrate. Our previous study on the crystal structure of GlcAT-P revealed the reaction and substrate recognition mechanisms of this enzyme. Comparative analyses of the enzymatic activities of GlcAT-S and GlcAT-P showed that there are notable differences in the acceptor substrate specificities of these enzymes. To elucidate differences between their specificities, we now solved the crystal structure of GlcAT-S. Residues interacting with UDP molecule, which is a part of the donor substrate, are highly conserved between GlcAT-P and GlcAT-S. On the other hand, there are some differences between these proteins in the manner they recognize their respective acceptor substrates. Phe245, one of the most important GlcAT-P residues for the recognition of acceptors, is a tryptophan in GlcAT-S. In addition, Val320, which is located on the C-terminal long loop of the neighboring molecule in the dimer and critical in the recognition of the acceptor sugar molecule by the GlcAT-P dimer, is an alanine in GlcAT-S. These differences play key roles in establishing the distinct specificity for the acceptor substrate by GlcAT-S, which is further supported by site-directed mutagenesis of GlcAT-S and a computer-aided model building of GlcAT-S/substrate complexes.

Hyperinsulinemia is widely thought to be a compensatory response to insulin resistance, whereas its potentially causal role in the progression of insulin resistance remains to be established. Here, we aimed to examine whether hyperinsulinemia could affect the progression of insulin resistance in Zucker fatty diabetic (ZDF) rats. Male ZDF rats at 8 wk of age were fed a diet ad libitum (AL) or dietary restriction (DR) of either 15 or 30% from AL feeding over 6 wk. Insulin sensitivity was determined by hyperinsulinemic euglycemic clamp. ZDF rats in the AL group progressively developed hyperglycemia and hyperinsulinemia by 10 wk of age, and then plasma insulin rapidly declined to nearly normal levels by 12 wk of age. Compared with AL group, DR groups showed delayed onset of hyperglycemia and persistent hyperinsulinemia, leading to weight gain and raised plasma triglycerides and free fatty acids by 14 wk of age. Notably, insulin sensitivity was significantly reduced in the DR group rather than the AL group and inversely correlated with plasma levels of insulin and triglyceride but not glucose. Moreover, enhanced lipid deposition and upregulation of genes involved in lipogenesis were detected in liver, skeletal muscle, and adipose tissues of the DR group rather than the AL group. Alternatively, continuous hyperinsulinemia induced by insulin pellet implantation produced a decrease in insulin sensitivity in ZDF rats. These results suggest that chronic hyperinsulinemia may lead to the progression of insulin resistance under DR conditions in association with altered lipid metabolism in peripheral tissues in ZDF rats.

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Background Integrin αvβ3, which are expressed by activated hepatic stellate cells in non-alcoholic steatohepatitis (NASH), play an important role in the fibrosis. Recently, we reported that an RGD peptide positron emission tomography (PET) probe is useful as a predictor of hepatic fibrosis. Kinetic analysis of the RGD PET probe has been performed in tumours, but not in hepatic fibrosis. Therefore, we aimed to quantify hepatic integrin αvβ3 in a model of NASH by kinetic analysis using 18F-FPP-RGD2, an integrin αvβ3 PET probe. Methods 18F-FPP-RGD2 PET/CT scans were performed in control and NASH rats. Tissue kinetic analyses were performed using a one-tissue, two-compartment (1T2C) and a two-tissue, three-compartment (2T3C) model using an image-derived input function (IDIF) for the left ventricle. We then conducted correlation analysis between standard uptake values (SUVs) or volume of distribution (VT), evaluated using compartment kinetic analysis and integrin αv or β3 protein expression. Results Biochemical and histological evaluation confirmed the development of NASH rats. Integrin αvβ3 protein expression and hepatic SUV were higher in NASH- than normal rats. The hepatic activity of 18F-FPP-RGD2 peaked rapidly after administration and then gradually decreased, whereas left ventricular activity rapidly disappeared. The 2T3C model was found to be preferable for 18F-FPP-RGD2 kinetic analysis in the liver. The VT (IDIF) for 18F-FPP-RGD2, calculated using the 2T3C model, was significantly higher in NASH- than normal rats and correlated strongly with hepatic integrin αv and β3 protein expression. The strengths of these correlations were similar to those between SUV60–90 min and hepatic integrin αv or β3 protein expression. Conclusions We have demonstrated that the VT (IDIF) of 18F-FPP-RGD2, calculated using kinetic modelling, positively correlates with integrin αv and β3 protein in the liver of NASH rats. These findings suggest that hepatic VT (IDIF) provides a quantitative assessment of integrin αvβ3 protein in liver.