HNK-1 Glyco-epitope Regulates the Stability of the Glutamate Receptor Subunit GluR2 on the Neuronal Cell Surface

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 α-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 (HSO₃-3GlcAβ1–3Galβ1–4GlcNAc) 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 (Galβ1–4GlcNAc) 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 β4-galactosyltransferase-2 synthesizes the glycan backbone structure of HNK-1, Galβ1–4GlcNAc. The mice lacking β4-galactosyltransferase-2 showed decreased HNK-1 expression in their brains and also exhibited impaired learning and memory (13). Overall, these studies suggest that HNK-1 plays unique functional roles in some types of neuronal plasticity, but the molecular mechanisms of HNK-1 remain unclear.AMPA-type glutamate receptors mediate most of the fast excitatory synaptic transmissions in the mammalian brain and control synaptic strength. The regulated trafficking of AMPA receptors to the postsynaptic membrane is thought to be a major mechanism contributing to long lasting changes in synaptic strength, including LTP and long term depression (14, 15). AMPA receptors are mainly heterotetrameric channels assembled from the subunits GluR1 to GluR4, and all subunits have 4–6 potential N-glycosylation sites in their extracellular domains (16). Few studies have focused on the function of N-glycosylation in AMPA receptors. Some investigations showed that AMPA receptor subunits expressed at both the cell surface and synaptic sites possess the mature glycosylated form (17, 18), but it is generally accepted that N-glycosylation is not essential for their channel function or ligand binding (19, 20).In this study, we searched for a candidate molecule(s) responsible for the defects in synaptic plasticity seen in GlcAT-P-deficient mice. We found that the HNK-1 epitope is mainly expressed on a specific molecule in the hippocampal postsynaptic density (PSD) fraction. We focused on the molecule and identified a subunit of AMPA-type glutamate receptors, GluR2, as a novel HNK-1 carrier protein. Furthermore, we showed that the loss of HNK-1 epitope on GluR2 greatly increases both constitutive and regulated endocytosis of GluR2, resulting in a decrease in the amount of surface GluR2 in cultured hippocampal neurons and CHO cells. This is the first report demonstrating that the N-glycan on GluR2 regulates its protein function, and our results suggest that HNK-1 epitope on GluR2 is an important factor for synaptic plasticity.