HNK-1 Glyco-epitope Regulates the Stability of the Glutamate Receptor Subunit GluR2 on the Neuronal Cell Surface
2009; Elsevier BV; Volume: 284; Issue: 44 Linguagem: Inglês
10.1074/jbc.m109.024208
ISSN1083-351X
AutoresIppei Morita, Shinako Kakuda, Yusuke Takeuchi, Satsuki Itoh, Nana Kawasaki, Yasuhiko Kizuka, Toshisuke Kawasaki, Shogo Oka,
Tópico(s)Immune Cell Function and Interaction
ResumoHNK-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 (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. Expression of Concern: Ca2+/calmodulin-dependent protein kinase II promotes cell cycle progression by directly activating MEK1 and subsequently modulating p27 phosphorylation.Journal of Biological ChemistryVol. 295Issue 26PreviewVOLUME 284 (2008) PAGES 3021–3027 Full-Text PDF Open Access 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 (1Morita I. Kizuka Y. Kakuda S. Oka S. J. Biochem. 2008; 143: 719-724Crossref PubMed Scopus (46) Google Scholar). It has been reported that HNK-1 mediates the interaction of these adhesion molecules, thereby controlling their functions, including cell-to-cell adhesion (2Künemund V. Jungalwala F.B. Fischer G. Chou D.K. Keilhauer G. Schachner M. J. Cell Biol. 1988; 106: 213-223Crossref PubMed Scopus (323) Google Scholar), migration (3Bronner-Fraser M. Dev. Biol. 1987; 123: 321-331Crossref PubMed Scopus (147) Google Scholar), and neurite extension (4Martini R. Xin Y. Schmitz B. Schachner M. Eur. J. Neurosci. 1992; 4: 628-639Crossref PubMed Scopus (176) Google Scholar). 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) 3The abbreviations used are: GlcATglucuronyltransferaseAMPAα-amino-3-hydroxy-5-methylisoxazole propionateFT-ICR MSFourier transform-ion cyclotron resonance mass spectrometerLC/MS/MSliquid chromatography-tandem mass spectrometryLTPlong term potentiationPSDpostsynaptic densityCHOChinese hamster ovaryDTSSP3,3′-dithiobis(sulfosuccinimidyl propionate)Ppostnatal daySTsulfotransferase. (5Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 6093-6098Crossref PubMed Scopus (122) Google Scholar, 6Seiki T. Oka S. Terayama K. Imiya K. Kawasaki T. Biochem. Biophys. Res. Commun. 1999; 255: 182-187Crossref PubMed Scopus (70) Google Scholar) and a sulfotransferase (HNK-1ST) (7Bakker H. Friedmann I. Oka S. Kawasaki T. Nifant'ev N. Schachner M. Mantei N. J. Biol. Chem. 1997; 272: 29942-29946Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). 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 (8Rabouille C. Hui N. Hunte F. Kieckbusch R. Berger E.G. Warren G. Nilsson T. J. Cell Sci. 1995; 108: 1617-1627Crossref PubMed Google Scholar). glucuronyltransferase α-amino-3-hydroxy-5-methylisoxazole propionate Fourier transform-ion cyclotron resonance mass spectrometer liquid chromatography-tandem mass spectrometry long term potentiation postsynaptic density Chinese hamster ovary 3,3′-dithiobis(sulfosuccinimidyl propionate) postnatal day sulfotransferase. 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 (9Yamamoto S. Oka S. Inoue M. Shimuta M. Manabe T. Takahashi H. Miyamoto M. Asano M. Sakagami J. Sudo K. Iwakura Y. Ono K. Kawasaki T. J. Biol. Chem. 2002; 277: 27227-27231Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). 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 (10Strekalova T. Wotjak C.T. Schachner M. Mol. Cell. Neurosci. 2001; 17: 1102-1113Crossref PubMed Scopus (30) Google Scholar, 11Pradel G. Schachner M. Schmidt R. J. Neurobiol. 1999; 39: 197-206Crossref PubMed Scopus (104) Google Scholar, 12Saghatelyan A.K. Gorissen S. Albert M. Hertlein B. Schachner M. Dityatev A. Eur. J. Neurosci. 2000; 12: 3331-3342Crossref PubMed Scopus (105) Google Scholar). 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 (13Yoshihara T. Sugihara K. Kizuka Y. Oka S. Asano M. J. Biol. Chem. 2009; 284: 12550-12561Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). 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 (14Derkach V.A. Oh M.C. Guire E.S. Soderling T.R. Nat. Rev. Neurosci. 2007; 8: 101-113Crossref PubMed Scopus (538) Google Scholar, 15Isaac J.T. Ashby M. McBain C.J. Neuron. 2007; 54: 859-871Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). 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 (16Pasternack A. Coleman S.K. Féthière J. Madden D.R. LeCaer J.P. Rossier J. Pasternack M. Keinänen K. J. Neurochem. 2003; 84: 1184-1192Crossref PubMed Scopus (17) Google Scholar). 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 (17Hall R.A. Hansen A. Andersen P.H. Soderling T.R. J. Neurochem. 1997; 68: 625-630Crossref PubMed Scopus (62) Google Scholar, 18Standley S. Tocco G. Wagle N. Baudry M. J. Neurochem. 1998; 70: 2434-2445Crossref PubMed Scopus (43) Google Scholar), but it is generally accepted that N-glycosylation is not essential for their channel function or ligand binding (19Everts I. Villmann C. Hollmann M. Mol. Pharmacol. 1997; 52: 861-873Crossref PubMed Scopus (100) Google Scholar, 20Maruo K. Nagata T. Yamamoto S. Nagai K. Yajima Y. Maruo S. Nishizaki T. Brain Res. 2003; 977: 294-297Crossref PubMed Scopus (9) Google Scholar). 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. The following mammalian expression plasmids have been described. The 3.2-kb XhoI-XbaI GluR2 fragment derived from pKC24 (pBluescript)-GluR2, which was donated by Dr. M. Mishina (Tokyo University) (21Sakimura K. Bujo H. Kushiya E. Araki K. Yamazaki M. Yamazaki M. Meguro H. Warashina A. Numa S. Mishina M. FEBS Lett. 1990; 272: 73-80Crossref PubMed Scopus (158) Google Scholar), was cloned into pcDNA3.1/myc-HisB (Invitrogen) to yield the plasmid pcDNA3.1-GluR2. The pIRES-GlcAT-P-HNK-1ST and pEF-BOS-GlcAT-P plasmids were described previously (5Terayama K. Oka S. Seiki T. Miki Y. Nakamura A. Kozutsumi Y. Takio K. Kawasaki T. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 6093-6098Crossref PubMed Scopus (122) Google Scholar, 22Kizuka Y. Matsui T. Takematsu H. Kozutsumi Y. Kawasaki T. Oka S. J. Biol. Chem. 2006; 281: 13644-13651Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The pcDNA3.1-N-cadherin was a generous gift from Dr. A. Kinoshita (Kyoto University) (23Uemura K. Kihara T. Kuzuya A. Okawa K. Nishimoto T. Ninomiya H. Sugimoto H. Kinoshita A. Shimohama S. Neurosci. Lett. 2006; 402: 278-283Crossref PubMed Scopus (91) Google Scholar). Biochemical subcellular fractionation was performed according to the method of a previous report (24Nakamura M. Sato K. Fukaya M. Araishi K. Aiba A. Kano M. Watanabe M. Eur. J. Neurosci. 2004; 20: 2929-2944Crossref PubMed Scopus (148) Google Scholar) with minor modifications. The hippocampi of C57 BL/6 mice were homogenized with 15 strokes at 800 rpm in 9 volumes of ice-cold homogenizing buffer (10 mm Tris-HCl (pH 7.4), 0.32 m sucrose, 1 mm EDTA, and protease inhibitor mixture (Nacalai Tesque)), and the homogenate was centrifuged at 1,000 × g for 20 min to remove nuclei and large debris. The supernatant fluid (S1, postnuclear fraction) was centrifuged at 10,000 × g for 20 min to pellet a crude synaptosome fraction (P2). The supernatant above the P2 fraction was centrifuged at 125,000 × g for 1 h, and the pellet was designated as the light membrane fraction (P3). The P2 fraction was lysed hypo-osmotically and centrifuged at 25,000 × g for 30 min to obtain the synaptosomal membrane fraction (LP1). The LP1 fraction was then suspended in 0.5% Triton X-100 in homogenizing buffer for 15 min and centrifuged at 125,000 × g for 1 h to obtain the PSD fraction. All biochemical experiments were carried out on ice or at 4 °C. The protein concentration of each fraction was measured with DC protein assay kit (Bio-Rad). HNK-1 carrier glycoproteins from the P2 fraction of the hippocampi were partially purified with HNK-1 antibody-conjugated beads. The purified proteins were isolated by SDS-PAGE and stained with Coomassie Brilliant Blue. The ∼100-kDa protein band was digested with trypsin, and the peptide fragments were identified by LC/MS/MS as reported previously (25Kizuka Y. Kobayashi K. Kakuda S. Nakajima Y. Itoh S. Kawasaki N. Oka S. Glycobiology. 2008; 18: 331-338Crossref PubMed Scopus (11) Google Scholar). Proteins were identified by searching Swiss Protein Database (mouse) using the Mascot (Matrixscience) and TurboSequest search engines (Thermo Fisher Scientific). Proteins were separated by SDS-PAGE with the buffer system of Laemmli (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar) and then transferred to a nitrocellulose membrane. After blocking with 5% nonfat dried milk in phosphate-buffered saline containing 0.1% Tween 20, the membrane was incubated with primary antibodies. Horseradish peroxidase-conjugated secondary antibodies and ECL (Pierce) were used for protein detection. For immunoprecipitation under denaturing conditions, the PSD fraction was solubilized with 1 volume of 10 mm Tris-HCl (pH 7.4) buffer containing 1% SDS and boiled for 5 min, and then 5 volumes of immunoprecipitation buffer (20 mm Tris-HCl (pH 7.4), 2% Triton X-100, and 300 mm NaCl) and 4 volumes of H2O were added to the sample. For immunoprecipitation under native conditions, HEK 293 cells were lysed with extraction buffer (20 mm Tris-HCl (pH 7.4), 1% Triton X-100, 0.5% deoxycholate, 150 mm NaCl, 1 mm EDTA, and protease inhibitor mixture). Primary antibodies were used at 4 μg/ml, and 20 μl of protein G-Sepharose 4 Fast Flow (GE Healthcare) were added to precipitate the immunocomplexes for 2 h at 4 °C. The antibodies used in the immunoprecipitation and immunoblot analyses are listed as follows: HNK-1 monoclonal antibody (a hybridoma cell line was purchased from the American Type Culture Collection); anti-actin N terminus (Sigma); anti-PSD-95 (Chemicon); anti-GluR1 N terminus and anti-GluR2 N terminus (Zymed Laboratories Inc.); GluR2/3 (Upstate); anti-NR1 (Upstate); anti-NR2A (Upstate);anti-synaptophysin (FabGennix); and anti-N-cadherin (BD Biosciences). The immunoprecipitates formed with the anti-GluR1 and anti-GluR2 antibodies were denatured with 20 mm sodium phosphate buffer (pH 7.2) containing 0.5% SDS, 1% 2-mercaptoethanol, and 4 mm EDTA. To reduce the concentration of SDS, we diluted the samples with 4 volumes of 20 mm sodium phosphate buffer (pH 7.2) containing 0.5% Nonidet P-40. Two units of N-glycosidase F (Roche Applied Science) were added to the solution, and the solution was incubated for 16 h at 37 °C. Anti-GluR2 antibody was raised from a rabbit immunized with a 20-mer peptide (QNFATYKEGYNVYGIESVKI) corresponding to the C terminus of mouse GluR2 and purified with an antigen peptide affinity column. The purified anti-GluR2 antibody (3 mg) was coupled to 1.5 ml of CNBr-activated Sepharose 4B (GE healthcare), and the resin was packed into a column. The P2 fractions from 4-week-old mouse hippocampi were extracted with extraction buffer containing 1% n-dodecyl-β-d-maltopyranoside. The extracted material was slowly applied to the anti-GluR2 antibody column. After washing the column with Tris-buffered saline containing 0.1% n-dodecyl-β-d-maltopyranoside, GluR2 proteins bound to the resin were eluted with the elution buffer (50 mm diethylamine (pH 11.0), 150 mm NaCl, and 0.1% n-dodecyl-β-d-maltopyranoside). This eluate was immediately neutralized with 0.5 m NaH2PO4 (pH 4.5). The purified GluR2 was concentrated and subjected to SDS-PAGE. The bands of purified GluR2 (∼100 kDa) were excised and cut into pieces. The gel pieces were destained and dehydrated with 50% acetonitrile. The protein in the gel was reduced and carboxymethylated by incubation with dithiothreitol and sodium monoiodoacetate (27Kikuchi M. Hatano N. Yokota S. Shimozawa N. Imanaka T. Taniguchi H. J. Biol. Chem. 2004; 279: 421-428Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). N-Glycans were extracted from the gel pieces as reported (28Küster B. Wheeler S.F. Hunter A.P. Dwek R.A. Harvey D.J. Anal. Biochem. 1997; 250: 82-101Crossref PubMed Scopus (323) Google Scholar) and reduced with NaBH4. The borohydride-reduced glycans were desalted with Envi-carb (Supelco). LC/MS/MS was carried out on a quadrupole linear ion trap/Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS) (Thermo Electron Corp) connected to a NanoLC system (Michrom BioResource, Inc.). The eluents were 5 mm ammonium acetate (pH 9.6), 2% CH3CN (pump A), and 5 mm ammonium acetate (pH 9.6), 80% CH3CN (pump B). The borohydride-reduced N-linked oligosaccharides were separated on a Hypercarb (0.1 × 150 mm, 5 μm; Thermo Electron Corp.) with a linear gradient of 5–20% of pump B (0–45 min), 20–50% of pump B (45–90 min), 50–70% of pump B (90–90.1 min), and 70% of pump B (90.1–130 min). A full MS scan (m/z 700–2000) by FT-ICR MS followed by data-dependent MS/MS for the most abundant ion was performed in the positive ion mode. Primary hippocampal cultures were prepared from postnatal day 0 mouse brains. Hippocampi were trypsinized for 10 min at 37 °C. Dissociated neurons were plated on BD Biocoat Poly-d-lysine Cellware 4-well culture slide (BD Biosciences) at 1.0 × 105 cells per well coated with 2 μg/ml laminin in Neurobasal medium (Invitrogen) supplemented with 2% B-27 (Invitrogen) and 500 μm l-glutamine. Every 7 days, the cultures were fed by replacing half of the medium with feeding medium. HEK 293 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum, and CHO cells were maintained in minimum Eagle's α-medium supplemented with 10% fetal calf serum. These cells were transfected using FuGENE 6 transfection reagent (Roche Applied Science). Live hippocampal neurons were prelabeled with 10 μg/ml anti-GluR2 N terminus antibody for 15 min at 37 °C. After washing out the antibody, the neurons were incubated with or without 100 μm AMPA treatment (in the presence of 50 μm d-2-amino-5-phosphonopentanoic acid) for 20 min at 37 °C to allow endocytosis of GluR2. To stain GluR2 remaining on the cell surface, neurons were fixed in 4% paraformaldehyde, 4% sucrose for 10 min at room temperature and incubated with Alexa 488-conjugated secondary antibody (Molecular Probes) for 1 h at room temperature. The neurons were fixed with methanol at −20 °C, and Alexa 546-conjugated secondary antibody (Molecular Probes) was used for internalized GluR2 staining. Internalized and surface GluR2 fluorescence intensity was quantified using FluoView (Olympus) imaging system. Hippocampal cultured neurons and CHO cells were reacted with 1 mg/ml EZ-Link Sulfo-NHS-SS-biotin (Pierce) in phosphate-buffered saline for 30 min at 4 °C. Before surface biotinylation, neurons were incubated with or without 100 μm AMPA treatment for 20 min at 37 °C. These cells were lysed with extraction buffer, and cell surface-biotinylated proteins were pulled down with immobilized streptavidin (Pierce) and immunoblotted with anti-GluR2 antibody. Hippocampal P2 fractions and transfected HEK 293 cells were cross-linked with 2 mm 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP) (Pierce) in phosphate-buffered saline at room temperature for 30 min, and the reaction was quenched by the addition of 50 mm Tris-HCl (pH 7.4). The cross-linked P2 fractions were solubilized with extraction buffer and centrifuged at 125,000 × g for 1 h to separate into two distinct fractions (1% Triton X-100-soluble and -insoluble fractions). The Triton X-100-insoluble fraction was solubilized with 10 mm Tris-HCl (pH 7.4) buffer containing 1% SDS. Both insoluble and soluble fractions were subjected to immunoprecipitation analysis. For the immunoblot analysis, a luminoimage analyzer LAS-3000 (Fuji Film) was used for the detection of protein bands. Densitometry of these bands was performed using image analysis software ImageGauge (Fuji Film). Calibration curves of protein bands for GluR2 and N-cadherin showed good linearity (r2 = 0.97072 (N-cadherin) and 0.95566 (GluR2)). To quantify the strength of interaction between GluR2 and N-cadherin, the intensity of co-immunoprecipitated N-cadherin or GluR2 was normalized by immunoprecipitated GluR2 or N-cadherin, respectively. Results are shown as percent of value relative to the wild-type or control cells. The statistical analyses performed are listed in the individual figure legends, and values are expressed as the means ± S.E. To search for a novel HNK-1 carrier protein(s) responsible for synaptic plasticity, we first examined the expression of HNK-1 glyco-epitope in P2 (crude synaptosome) fractions obtained from mouse hippocampi at several developmental stages (Fig. 1A). The expression of HNK-1 was barely detected at embryonic day 16. After birth, it reached a peak at postnatal day 14 (P14), which coincided with the major postnatal synaptogenic period, and gradually decreased in adulthood. The expression of HNK-1 epitope was almost abolished in GlcAT-P-deficient mice (Fig. 1A), indicating that the biosynthesis of HNK-1 in brains is mainly regulated by a brain-specific glucuronyltransferase (GlcAT-P). Next, we performed biochemical fractionation of mouse hippocampi and examined the subcellular distribution of the HNK-1 epitope by immunoblot analysis. HNK-1 carrier proteins were detected in a wide range of molecular mass, from 100 to 250 kDa or higher (Fig. 1B, upper panel), and an ∼100-kDa HNK-1-positive band was highly enriched in the P2 and PSD fractions, showing a similar distribution profile to the postsynaptic marker PSD-95 (Fig. 1B, lower panel). We also examined the subcellular localization of the HNK-1 epitope in cultured hippocampal neurons at 19 days in vitro. The immunoreactivity against the HNK-1 antibody was diffusely distributed on dendritic shafts and spine heads (supplemental Fig. 1). The punctate staining pattern for HNK-1 on the spines was co-localized with PSD-95 and apposed to the presynaptic marker synaptophysin. These results indicated the presence of HNK-1 carrier protein(s) at postsynaptic sites, and we focused on the specific HNK-1 carrier molecule enriched at postsynaptic sites to examine the role of HNK-1 in synaptic plasticity. To identify the ∼100-kDa candidate glycoprotein, we purified HNK-1 carrier proteins from the P2 fractions of mouse hippocampi using HNK-1 antibody-conjugated beads. The purified proteins were isolated by SDS-PAGE and stained with Coomassie Brilliant Blue. A piece of polyacrylamide gel containing the ∼100-kDa glycoprotein was excised and digested with trypsin. Using LC/MS/MS, we identified the novel HNK-1 carrier molecule as GluR2, which is an AMPA-type glutamate receptor subunit (supplemental Fig. 2). To further confirm these results, we immunoprecipitated GluR1 and GluR2 by subunit-specific antibodies from the PSD fractions of hippocampi under denaturing conditions and then immunoblotted them with HNK-1 antibody (Fig. 2A). HNK-1 epitope was specifically detected on GluR2 but not on GluR1. Furthermore, the HNK-1 epitope on GluR2 was completely removed by N-glycosidase F treatment, indicating that it is attached to the nonreducing terminus of N-glycan(s) on GluR2. We also examined the expression of HNK-1 on N-methyl-d-aspartic acid (NMDA) receptor (composed of NR1 and NR2 subunits), which is another ionotropic glutamate receptor (Fig. 2B). Neither NR1 nor NR2 carried the HNK-1 epitope. These results suggest that the HNK-1 epitope is specifically expressed on GluR2 and may regulate the function of GluR2. To determine the glycan structure, including HNK-1 glyco-epitope on GluR2, GluR2 was immunopurified from P2 fractions and separated by SDS-PAGE (Fig. 3A). The GluR2 band (∼100 kDa) was excised and treated with N-glycosidase F. The extracted N-linked oligosaccharides were reduced with NaBH4 and subjected to LC/MS/MS. Fig. 3B shows the base peak chromatograms obtained by a full MS scan (m/z 700–2000) by FT-ICR MS. The major molecular ions detected by a full MS scan were subjected to data-dependent collision-induced dissociation-tandem mass spectrometry automatically. From all the MS/MS spectra, only those of oligosaccharides carrying HNK-1 or nonsulfated HNK-1 motifs were sorted out using HNK-1 diagnostic ion, GlcA-Gal-GlcNAc+ (m/z 542) (Fig. 3B, lower panel). The carbohydrate structure of the HNK-1 and nonsulfated HNK-1 oligosaccharides (peaks 1–6) was deduced from the m/z values of precursor ions detected by FT-ICR MS and product ions obtained by MS/MS (Fig. 3C). As a typical MS/MS spectrum, the product ion spectrum of peak 3 is shown in Fig. 3D. Peak 3 was assigned to a bisected biantennary N-glycan carrying the HNK-1 motif based on the product ions, such as GlcA-Gal-GlcNAc+ (m/z 542), SO3-GlcA-Gal-GlcNAc+ (m/z 622), and [M-SO3-GlcA-Gal-GlcNAc + H]+ (m/z 1319). Together with the immunoprecipitation experiment shown in Fig. 2, these data show that HNK-1 is actually expressed on N-glycans of GluR2 in the brain. Numerous recent studies have strongly suggested that the mechanism underlying activity-dependent synaptic plasticity is related to the redistribution of GluR2 between intracellular components and the synaptic membrane (15Isaac J.T. Ashby M. McBain C.J. Neuron. 2007; 54: 859-871Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). GluR2 is internalized by clathrin-dependent endocytosis, and two distinct types of GluR2 endocytosis are known, constitutive (under basal conditions) and regulated endocytosis (under stimulation by a reagent such as AMPA, N-methyl-d-aspartic acid, and insulin) (29Man H.Y. Lin J.W. Ju W.H. Ahmadian G. Liu L. Becker L.E. Sheng M. Wang Y.T. Neuron. 2000; 25: 649-662Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). Moreover, we revealed that HNK-1 is specifically expressed on GluR2 (Fig. 2) and that loss of HNK-1 in the brain impairs the synaptic plasticity (8Rabouille C. Hui N. Hunte F. Kieckbusch R. Berger E.G. Warren G. Nilsson T. J. Cell Sci. 1995; 108: 1617-1627Crossref PubMed Google Scholar). Taken together, we hypothesized that HNK-1 expression on GluR2 regulates its intracellular distribution. To evaluate the influence of HNK-1 epitope on GluR2 endocytosis, we performed a fluorescence-based internalization assay (30Lee S.H. Liu L. Wang Y.T. Sheng M. Neuron. 2002; 36: 661-674Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar) using hippocampal neurons derived from wild-type or GlcAT-P-deficient mice. In this assay, the surface GluR2 subunits on living neurons at 18 days in vitro were labeled with 10 μg/ml antibody against the extracellular epitope of GluR2, and then antibody-labeled neurons were incubated for 20 min at 37 °C to allow endocytosis either in the absence or presence of 100 μm AMPA to determine constitutive or regulated GluR2 endocytosis, respectively. The antibody-labeled GluR2 remaining on the cell surface and the GluR2 internalized into intracellular vesicles were independently immunostained under nonpermeabilizing and permeabilizing conditions, respectively (Fig. 4A). The total fluorescence (surface and internalized fluorescence) of all four conditions was unchanged (Fig. 4B). The "internalization index" represents the ratio of internalized fluorescence/total fluorescence and was compared with wild-type neurons under basal and AMPA-stimulated conditions (Fig. 4C). Under basal conditions, GluR2 in GlcAT-P-deficient neurons showed a higher degree of intracellular accumulation than GluR2 in wild-type neurons (wild-type, 11.7 ± 1.35; GlcAT-P−/−, 25.1 ± 1.87; **, p < 0.01) (Fig. 4, A and C). Under AMPA-stimulated conditions, the amount of intracellular GluR2 in GlcAT-P-deficient neurons was more greatly increased than that in wild-type neurons (wild type, 43.7 ± 2.09; GlcAT-P−/−, 72.5 ± 1.49; **, p < 0.01). Next we performed a cell surface biotinylation assay to test the surface levels of GluR2 in cultured hippocampal neurons. The amount of surface GluR2 in GlcAT-P-deficient neurons was slightly decreased to 82.5 ± 3.5% of wild type under the basal conditions (**, p < 0.01) (Fig. 4, D and E). After AMPA treatment, the surface expression levels of GluR2 both in wild-type and GlcAT-P-deficient neurons were decreased, but the surface expression in GlcAT-P-deficient neurons was decreased more than that in wild type (wild type, 58.1 ± 2.6%; GlcAT-P−/−, 35.5 ± 4.5; **, p < 0.01). These results from two independent experiments were consistent with each other and revealed that GluR2 in GlcAT-P-deficient neurons showed facilitated endocytosis or instability on the cell surface under both basal and stimulated conditions. A recent report has shown that the extracellular N-terminal domain of GluR2 interacts with N-cadherin and that t
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