Cyclic Nucleotide-3 -Phosphodiesterase
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Juxtanodin: An oligodendroglial protein that promotes cellular arborization and 2,3-cyclic nucleotide-3-phosphodiesterase trafficking Bin Zhang*, Qiong Cao*, Anchen Guo*, Haiying Chu*, Yee Gek Chan*, Jan Paul Buschdorf†, Boon Chuan Low†, Eng Ang Ling*, and Fengyi Liang*‡ *Department of Anatomy, Faculty of Medicine, National University of Singapore, Singapore 117597; and †Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 117543 Edited by Edward G. Jones, University of California, Davis, CA, and approved June 16, 2005 (received for review February 4, 2005) In the process of screening cell-type-specific genes, we identified databases revealed another cDNA sequence encoding a trun- juxtanodin (JN), an oligodendroglial protein featuring a putative cated JN of 268 residues (JN268). C-terminal actin-binding domain. At the cellular level, JN in the rat CNS colocalized with 2,3-cyclic nucleotide-3-phosphodiesterase Generation of Fusion Proteins and JN Antibody. The DNA fragments (CNPase), a cytoskeleton-related oligodendroglial protein. In the encoding residues 1–170 and 1–282 of deduced JN were cloned myelin sheath, JN was found mainly in the abaxon and the lateral into pET41a (Novagen) to produce GST-JN170 and His-tagged few terminal loops. Its apposition to the myelinated axon, through JN. GST and GST-JN170 were purified by using Bulk and the latter, defined an axonal subregion, herewith termed juxta- RediPack GST Purification modules (Amersham Pharmacia, node, at the Ranvier node–paranode junction. During forebrain Piscataway, NJ). Polyclonal antibody against GST-JN170 was ontogenesis, JN expression paralleled that of MBPs but lagged developed in New Zealand White rabbits and affinity purified behind CNPase. Juxtanodin transfection promoted arborization of with GST-JN170-Sepharose 4B. cultured OLN-93 cells and augmented endogenous CNPase expres- sion and transport to the process arbors of cultured primary Mammalian Expression Plasmids and Transfection of Cell Culture. The oligodendrocyte precursors. These results reveal JN as a cytoskel- spontaneously transformed, microfilament-containing OLN-93 eton-related oligodendroglial protein that delineates the juxtan- cells (5) were cultured at 37°C in DMEM supplemented with ode and might serve oligodendrocyte motility, differentiation, or 10% FCS. Primary oligodendrocyte precursor (OLP) cells were myelin–axon signaling. Functionally, JN may be involved in CNS prepared from the optic nerves of 5- to 7-day-old rats (6, 7) in myelination and͞or specialization of the node of Ranvier. the facilities at the Department of Clinical Research, Singapore General Hospital (Singapore). myelin–axon interaction ͉ oligodendrocyte ͉ node of Ranvier ͉ cytoskeleton To elucidate the functional roles of JN and its putative C-terminal actin-binding domain (as reported by National Cen- ligodendroglia are highly specialized myelin-forming cells ter for Biotechnology Information reverse-position-specific Oof the CNS. Adequate myelination serves to insulate and BLAST), the nucleotide sequences encoding JN (282 amino acid accelerate the propagation of action potentials. Abnormalities in residues), JN268, and JN residues 1–247, 1–141, and 101–282 were Ј myelin formation͞maintenance may underlie diverse neurolog- subcloned into pXJ40 (8) with a FLAG tag at the 5 end of the ical disorders, ranging from multiple sclerosis to schizophrenia inserts, generating the constructs for FLAG-tagged JN, JN268, (1, 2). Matched with highly specialized functions and unique JN247,JN141, and JNc, respectively. Empty pXJ40 and pXJ40- architecture, various oligodendrocyte͞myelin-selective mole- BNIP-S served as controls (9). Cultured cells were transfected cules, such as myelin basic protein (MBP), myelin-associated with the recombinant plasmids by using GIBCO Lipofectamine glycoprotein, and 2Ј,3Ј-cyclic nucleotide-3Ј-phosphodiesterase 2000 (Invitrogen). Forty-eight hours later, the cells were solu- (CNPase), have been isolated and characterized (1, 3). It is likely bilized for Western blotting or fixed with 3% paraformaldehyde that many more are yet to be revealed. The identification and for immunocytochemistry. characterization of such molecules will probably help elucidate molecular mechanisms of myelination and dys- or demyelinating Immunocytochemistry, Electron Microscopy, and Multiple Labeling. Adult rats (n ϭ 18, body weight Ϸ200 g) were deeply anesthe- diseases. ͞ The unique architecture of the myelin sheath entails special- tized with Nembutal (100 mg kg of body weight, i.p.) and NEUROSCIENCE ized, yet elusive, cytoskeletal mechanisms of myelin-forming transcardially perfused with saline, followed by 3% paraformal- cells. Here, we report the molecular features, cellular expression, dehyde (plus 0.1% glutaraldehyde for immunoelectron micros- and functional roles of juxtanodin (JN), a previously unidentified copy) in 0.1 M phosphate buffer (pH 7.4). The brain and spinal cytoskeleton-related oligodendrocyte-specific protein. cord were dissected, postfixed, and sectioned with a cryostat (for light microscopy) or a vibratome (for electron microscopy). All Materials and Methods procedures involving experimental animals were approved by Identification and Characterization of the mRNA. Cell-type-specific the Ethics Committee at the National University of Singapore. CNS genes were sought out by in situ hybridization histochem- istry (ISH). Briefly, cDNA clones (n ϭ 1,500) from a rat brain This paper was submitted directly (Track II) to the PNAS office. cDNA library (Invitrogen) were sequenced and compared by Abbreviations: CNPase, 2Ј,3Ј-cyclic nucleotide-3Ј-phosphodiesterase; ERM, ezrin–radixin– BLAST searches against National Center for Biotechnology In- moesin; FERM, band four-point-one ERM homology; ISH, in situ hybridization histochem- formation nucleotide and protein nonredundant databases (4). istry; JN, juxtanodin; MBP, myelin basic protein; OLP, primary oligodendrocyte precursor; For the unannotated cDNA sequences (n ϭ 274), expression of PD, postnatal day. the mRNAs in the CNS was mapped by ISH using digoxigenin- Data deposition: The sequence reported in this paper has been deposited in the GenBank labeled riboprobes. A 3.6-kb cDNA clone with a predicted ORF database (accession no. DQ119821). encoding 282 amino acid residues was thereby identified, and the ‡To whom correspondence should be addressed. E-mail: [email protected]. gene was subsequently named juxtanodin (JN). A search of EST © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500952102 PNAS ͉ August 9, 2005 ͉ vol. 102 ͉ no. 32 ͉ 11527–11532 Downloaded by guest on September 29, 2021 The following antibodies were used (mouse monoclonal an- 282). BLAST searches against National Center for Biotechnology tibody from Sigma, unless otherwise noted): anti-JN (1:100, Information nonredundant nucleotide and protein databases rabbit polyclonal antibody, in-house produced), anti-CNPase yielded no positive hits, except for an unnamed mRNA sequence (1:500, Chemicon), anti-FLAG (1:200), anti-glial fibrillary acidic predicted by automated genome computation (GenBank acces- protein (1:1,000, Chemicon), anti-MBP (1:1,000, goat polyclonal sion no. NM001008311, 100% identity with JN), a deduced antibody, Santa Cruz Biotechnology), anti-neurofilament 200 human KIAA1189 protein (284 amino acid residues, 58% amino (1:1,000), anti-OX42 (1:50, Harlan Sera-lab, Sussex, U.K.), acid identity with JN), and an unnamed mouse protein inferred anti-pan sodium channel (NavP, 1:200), and anti-potassium from a cDNA sequence (accession no. NP084248, 235 amino channel Kv1.2 (1:300, Upstate Biotechnology, Lake Placid, NY). acid residues, 64% amino acid identity with JN). EST databases In vitro transcription of digoxigenin-labeled riboprobes, ISH presented a cDNA clone encoding a truncated JN consisting of (probe concentration, 0.2 g͞ml), immunofluorescence (IF), 268 amino acid residues (JN268; Fig. 1A). immunoperoxidase (avidin-biotinylated peroxidase complex In comparison with characterized proteins, the JN C terminus method), and immunoelectron microscopy followed protocols showed homology to the actin-binding domain of the ezrin- described in refs. 10 and 11. For simultaneous IF double͞triple radixin-moesin (ERM) proteins. Fig. 1A compares JN and JN268 labeling, bound primary antibodies were revealed by appropriate with moesin (NCBI Protein Data Bank accession no. secondary antibodies conjugated to either Alexa Fluor 568 or AAB61666). The boxed residues denote the homologous region Alexa Fluor 488 (1:400, Invitrogen). For sequential double- among the three. Unlike the ERM proteins, however, JN labeling, IF signals for the first antigen were documented before exhibited no detectable plasma-membrane-binding FERM immunoperoxidase for the second antigen and Luxol fast blue (band four-point-one ERM homology) domain. Instead, a scan counterstaining (0.1% in 95% ethanol, overnight at 50°C) for the of the Prosite database of protein families and domain patterns visualization of immunonegative cell arbors. Unexpected cross (12) reported four potential N-myristoylation sites near the JN reactivity in double͞triple labeling could be ruled out, based on N terminus (at residues 10–15, 22–27, 43–48, and 61–66; un- control experiments in which one of the primary antibodies was derlined by dots in Fig. 1A). omitted. On Western blots of adult rat brain lysate, anti-JN antibody, but not preimmune or GST-JN170 preabsorbed