Brain Research 966 (2003) 194205www.elsevier.com/ locate /brainres
L ocalization of the O-GlcNAc transferase and O-GlcNAc-modifiedproteins in rat cerebellar cortex
a b b aYoshihiro Akimoto , Frank I. Comer , Robert N. Cole , Akihiko Kudo ,a a,c b ,*Hayato Kawakami , Hiroshi Hirano , Gerald W. Hart
aDepartment of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, JapanbDepartment of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MA 21205, USA
cNittai Jusei Medical College for Judo Therapeutics, Yoga, Setagaya, Tokyo 158-0097, JapanAccepted 26 November 2002
O-linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous nucleocytoplasmic protein modification that has a complex interplay withphosphorylation on cytoskeletal proteins, signaling proteins and transcription factors. O-GlcNAc is essential for life at the single celllevel, and much indirect evidence suggests it plays an important role in nerve cell biology and neurodegenerative disease. Here we showthe localization of O-GlcNAc Transferase (OGTase) mRNA, OGTase protein, and O-GlcNAc-modified proteins in the rat cerebellarcortex. The sites of OGTase mRNA expression were determined by in situ hybridization histochemistry. Intense hybridization signalswere present in neurons, especially in the Purkinje cells. Fluorescent-tagged antibody against OGTase stained almost all of the neuronswith especially intense reactivity in Purkinje cells, within which the nucleus, perikaryon, and dendrites were most intensely stained. Usingimmuno-electron microscopic labeling, OGTase was seen to be enriched in euchromatin, in the cytoplasmic matrix, at the nerve terminal,and around microtubules in dendrites. In nerve terminals, immuno-gold labeling was observed around synaptic vesicles, with the enzymemore densely localized in the presynaptic terminals than in the postsynaptic ones. Using an antibody to O-GlcNAc, we found the sugarlocalizations reflected results seen for OGTase. Collectively, these data support hypothesized roles for O-GlcNAc in key processes ofbrain cells, including the regulation of transcription, synaptic vesicle secretion, transport, and signal transduction. Thus, by modulating thephosphorylation or protein associations of key regulatory and cytoskeletal proteins, O-GlcNAc is likely important to many functions ofthe cerebellum. 2002 Elsevier Science B.V. All rights reserved.
Theme: Neurotransmitters, modulators, transporter, and receptors
Topic: Signal transduction
Keywords: O-GlcNAc transferase; O-GlcNAcylation; Purkinje cell; Synapse; Post-translational modification; Cerebellum; Phosphorylation; Signaling
1 . Introduction cytoplasmic proteins [31,66]. Phosphorylation and O-GlcNAc modification are often reciprocal, occurring at the
Phosphorylation of nerve terminal proteins and post- same or adjacent hydroxyl moieties [10,41]. O-GlcNAcsynaptic proteins plays an important role in neuronal modification, which is termed O-GlcNAcylation, is afunction, where it is involved in such processes as growth regulatory modification that has a complex dynamic inter-cone migration, synapse formation, neurotransmission, and play with O-phosphorylation (Fig. 1) [15,72].synaptic plasticity [39,65,67]. O-linked N-acetylglucos- O-GlcNAc transferase (OGTase: EC 2-4-1) is a uniqueamine moieties (O-GlcNAc) are also dynamically attached nuclear and cytosolic glycosyltransferase that containsto serine or threonine residues of many nuclear and multiple tetratricopeptide repeats [30,42]. The liver en-
zyme contains two immunologically related subunits of Mr110 kDa (a-subunit) and 78 kDa (b-subunit). Other
*Corresponding author. Tel.: 11-410-614-5993; fax: 11-410-614-tissues, such as brain, contain only the a-subunit, which8804.
E-mail address: firstname.lastname@example.org (G.W. Hart). contains the active site. The a-subunit forms homo and
0006-8993/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.doi:10.1016/S0006-8993(02)04158-6
195Y. Akimoto et al. / Brain Research 966 (2003) 194205
cortex. These studies show that OGTase and O-GlcNAc-modified proteins are abundant in all neurons, but areparticularly enriched in the nucleus, perikaryon, dendrite,and synapses of the Purkinje cells. The high concentrationof O-GlcNAc-modified proteins in neurons may reflect animportant functional plasticity of neurons in the cere-bellum.
2 . Materials and methods
2 .1. Rat cerebellum
Cerebellum was extirpated from Wistar rats (male, 911weeks, n55). All experimental procedures using labora-tory animals were approved by the Animal Care and UseCommittee of Kyorin University School of Medicine. Allefforts were made to minimize their suffering.
2 .2. AntibodiesFig. 1. Complex and dynamic interplay between O-GlcNAc and phos-
Rabbit polyclonal anti-OGTase antibody (AL-25,phate modifications. This model depicts some of the possible combina-purified IgG) was raised against a purified recombinanttions of O-GlcNAc and phosphate in the simple examples of a protein
with two modification sites. Most of the O-GlcNAc-modified proteins 110-kDa subunit of OGTase expressed in Escherichia coliidentified to date have multiple sites of O-GlcNAc and phosphate either at . AL-25 recognizes both 110-kDa and 78-kDa subunitsthe same site or at adjacent sites. The potential complexity of the
of OGTase. Moreover, OGTase enzymatic activity isinterplay between O-GlcNAc and phosphate may exert multiple levels ofprecipitated by AL-25 . The generation of the mousecontrol.monoclonal anti-O-GlcNAc antibody (Mab CTD 110.6,
heterotrimers that appear to have different binding af- isotype: IgM) was previously described . CTD 110.6finities for UDP-GlcNAc over the entire physiological specifically recognizes O-GlcNAc in b-O-glycosidic link-range . The cDNA encoding the OGTase a-subunit has age to either serine or threonine and has no cross-reactivitybeen cloned from rat, Caenorhabditis elegans, and humans toward similar carbohydrate antigens or toward peptide[42,49]. OGT is highly conserved from C. elegans to determinants . Cy3-conjugated donkey anti-rabbit IgGhumans, and maps to the locus for X-linked Parkinsonia antibody and FITC-conjugated donkey anti-mouse IgG1dystonia . OGTase is unlike any glycosyltransferase IgM were obtained from Jackson Immunoresearch (Westpreviously described [35,57]. The brain has among the Grove, PA). Secondary antibodies (ultra small gold-conju-highest mRNA and protein levels of OGTase [42,58]. gated F(ab9) fragments of goat anti-rabbit IgG and goat2O-GlcNAc is particularly abundant in the nerve terminal, anti-mouse IgG1IgM), acetylated bovine serum albuminand several of the hundreds of O-GlcNAc-modified pro- (BSA-c), coldwater fish skin gelatin (CWFS gelatin),teins in neurons have been recently identified, e.g., neuro- Enhancement Conditioning Solution (ECS), and R-gentfilaments, tau, synapsins, collapsin response mediator SE-EM electron microscopy grade silver enhancementprotein-2 (CRMP-2), b-synuclein, and ubiquitin-carboxyl reagent were purchased from Aurion (Wageningen, Thehydrolase-L1, (UCH-L1), b-amyloid precursor protein; Netherlands).synapse-specific clathrin assembly protein (AP-3), andankyrin G [5,13,14,18,19,28,52,80]. All of these proteins 2 .3. Western blot analysisare also phosphoproteins. Addition of O-GlcNAc is re-quired for life at the single cell level and for development Crude protein extracts were prepared from the dissected. Collectively, the data strongly suggest that O- cerebellum of Wistar rats (males, 911 weeks). CerebellarGlcNAc is important to numerous neuronal functions. tissue was homogenized in a homogenization buffer (20
Here, we have extended these biochemical studies to the mM TrisHCl, pH 7.4, containing 5 mM EDTA, 5 mMmorphological level, using DIG-labeled anti-sense cRNA EGTA, 1 mM DTT, 2 mM phenylmethylsulfonyl fluorideprobe, anti-OGTase and anti-O-GlcNAc antibodies for in [PMSF], and protease inhibitor cocktail [Pic]-1 and -2situ hybridization analysis, immunofluorescence and im- [1 /1000 dilution]; ). The proteins were separated bymunoelectron microscopic experiments to localize OGTase 7.5% sodium dodecyl sulfate-polyacrylamide gel electro-mRNA, OGTase protein, and O-GlcNAc-modified pro- phoresis (SDS-PAGE) and transferred to a polyvinylideneteins, respectively, in sections of intact fixed rat cerebellar difluoride (PVDF) membrane. Purified rabbit polyclonal
Y. Akimoto et al. / Brain Research 966 (2003) 194205196
IgG AL-25 (dilution, 1:5000) or mouse monoclonal IgM with 10 mM PBS, pH 7.4, and then with 4% form-CTD 110.6 (dilution, 1:5000) was used as the primary aldehyde-PBS at room temperature for 20 min. Theantibody; and goat anti-rabbit IgG or goat anti-mouse IgM cerebellum was immediately dissected out and placed incoupled to horseradish peroxidase (Amersham-Pharmacia 4% formaldehyde-PBS at 4 8C for 2 h. After having beenBiotech, Piscotaway, NJ), as the secondary antibody washed with PBS, it was cut into sections (40 mm in(dilution, 1:20 000). For carbohydrate competition experi- thickness) with a microslicer DTD-1500 (DSK; Kyoto,ments 10 mM N-acetylglucosamine was included during Japan). After quenching of excess formaldehyde for 10the incubation with the primary antibody (CTD 110.6). min with 10 mM glycine in PBS, the sections were washedDetection of the horseradish peroxidase activity was three times with PBS and treated with 5% BSA-PBS for 1enhanced by using chemiluminescence (ECL), as described h. They were then incubated with AL25 (1:250) and CTDby the manufacturer (Amersham-Pharmacia Biotech). (1:500) in 0.1% BSA-PBS for 24 h at 4 8C, washed three
times for 20 min each with PBS, and then incubated with2 .4. Preparation of cRNA probes Cy3-conjug