Identification of Essential Residues in 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase

Lee, John; Gravel, Michel; Gao, Enoch; O'Neill, Ryan C.; Braun, Peter E.

doi:10.1074/jbc.m009434200

Cited by 37 publications

(31 citation statements)

References 62 publications

(65 reference statements)

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“…Extinction was measured at 405 nm on a Benchmark microplate reader (Bio-Rad laboratories, Veenendaal, the Netherlands). 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphodiesterase (CNPase) activity, as a marker for oligodendrocyte functioning, was measured in spheroid homogenates as described (Lee et al, 2001). In short, spheroid homogenate was added to a reaction mixture (100 mM 4-morpholine-ethanesulfonic acid pH 6, 30 mM D-glucose-6-phosphate, 2.5 mM cNADP, and 5 lg glucose-6-phosphated-dehydrogenase) in a 96-wells UV plate (Corning, Schiphol-Rijk, the Netherlands).…”

Section: Biochemical Assaysmentioning

confidence: 99%

An in vitro model for de‐ and remyelination using lysophosphatidyl choline in rodent whole brain spheroid cultures

Vereyken

Fluitsma

Bolijn

et al. 2009

Glia

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The process of demyelination occurring in diseases as multiple sclerosis is usually investigated in animal models. A major drawback of animal models is that only one condition can be tested per animal, necessitating many animals and systemic effects are factors to be considered. The aim of the study was to develop a reproducible in vitro model for de- and remyelination using whole brain spheroid cultures and lysophosphatidyl choline (LPC). In spheroid cultures, single cell suspensions of embryonic day 15 rodent brain cells reaggregate under constant rotation. Three-dimensional contacts form between the central nervous system cell types present. Multilayered myelin is maximal in four-week old cultures. A week of repeated exposure to LPC led to 30% loss of MBP protein concentration and 2',3'-cyclic nucleotide 3'-phosphodiesterase activity measurements in both rat and mouse spheroids and 56% loss in the number of myelin sheets, with partial remyelination after a week of recovery. The number of dividing cells was increased after LPC exposure and oligodendrocytes were shown to be among the dividing cells. Microglia and astrocytes were not affected and neurons were relatively spared. This suggests that LPC toxicity is specific for myelin and oligodendrocytes. LPC toxicity could be decreased using cholesterol and simvastatin, suggesting that LPC works through altering membrane composition. Thus, in different rodent species and using different read-outs, we could reproducibly show de- and remyelination in spheroid cultures after LPC exposure. This model for demyelination with potential for remyelination offers possibilities for testing novel therapies and studying mechanisms of remyelination.

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Section: Biochemical Assaysmentioning

confidence: 99%

An in vitro model for de‐ and remyelination using lysophosphatidyl choline in rodent whole brain spheroid cultures

Vereyken

Fluitsma

Bolijn

et al. 2009

Glia

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“…terminal, a number of phosphorylation sites [Ser-7; -12; -56; -110; -115; -151; -165 and Thr-34; 98] [131][132][133][134][135][136][137], two carbohydrate acceptor sites [Thr-95; -98] of Nacetylgalactosamine (GalNAc), a methylated Arg residue which occurs in both the mono-and di-methylated form, two likely deamidation sites [Gln-103; 147] and a possible Cterminal modification (i.e., loss of Arg). It also, contains an unusual tri-proline region [residues [99][100][101], which has been highlighted as an important structural feature similar to that contained in immunoglobulin G (IgG), and sequences homologous to cholera toxin A and B subunits [residues 102-118 and 67-77 in human MBP], which may possibly be involved in GM 1 ganglioside and GTP binding, as well as ADP ribosylation of MBP.…”

Section: C) Myelin Oligodendrocytic Basic Protein (Mobp)mentioning

confidence: 99%

Structure and Function of the Myelin Proteins: Current Status and Perspectives in Relation to Multiple Sclerosis

Tzakos¹,

Kursula²,

Troganis³

et al. 2005

CMC

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Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by demyelination and loss of neurological function, local macrophage infiltrate and neuroantigenspecific CD4 + T cells. MS arises from complex interactions between genetic, immunological, infective and biochemical mechanisms. Although the circumstances of MS etiology remain hypothetical, one persistent theme involves immune system recognition of myelin-specific antigens derived from myelin basic protein, the most abundant extrinsic myelin membrane protein, and/or another equally suitable myelin protein or lipid. Knowledge of the biochemical and physico-chemical properties of myelin proteins and lipids, particularly their composition, organization, structure and accessibility with respect to the compacted myelin multilayers, becomes central to understanding how and why myelin-specific antigens become selected during the development of MS. This review focuses on the current understanding of the molecular basis of MS with emphasis: (i) on the physical-chemical properties, organization, morphology, and accessibility of the proteins and lipids within the myelin multilayers; (ii) on the structure-function relationships and characterization of the myelin proteins relevant to the manifestation and evolution of MS; (iii) on conformational relationships between myelin epitopes which might become selected during the development of MS; (iv) on the structure of MHC/HLA in complex with MBP peptides as well as with TCR, which is crucial to the understanding of the pathogenesis of MS with the ultimate goal of designed antigen-specific treatments.

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“…The authenticity of the substitutions and the absence of any undesired mutations were confirmed by sequence analysis. The CNP-CF histidine mutants (H230L and H309L) were generated as previously described (6).…”

Section: Methodsmentioning

confidence: 99%

“…1-3). More recently, RICH, a neuronally associated homolog of CNP, has been discovered in fish (4,5), and the catalytic active site of CNP has been investigated (6).…”

mentioning

confidence: 99%

Structural Evidence That Brain Cyclic Nucleotide Phosphodiesterase Is a Member of the 2H Phosphodiesterase Superfamily

Kozlov

Lee

Elias

et al. 2003

Journal of Biological Chemistry

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2,3-Cyclic-nucleotide 3-phosphodiesterase (CNP) is an enzyme abundantly present in the central nervous system of mammals and some vertebrates. In vitro, CNP specifically catalyzes the hydrolysis of 2,3-cyclic nucleotides to produce 2-nucleotides, but the physiologically relevant in vivo substrate remains obscure. Here, we report the medium resolution NMR structure of the catalytic domain of rat CNP with phosphate bound and describe its binding to CNP inhibitors. The structure has a bilobal arrangement of two modules, each consisting of a four-stranded ␤-sheet and two ␣-helices. The ␤-sheets form a large cavity containing a number of positively charged and aromatic residues. The structure is similar to those of the cyclic phosphodiesterase from Arabidopsis thaliana and the 2-5 RNA ligase from Thermus thermophilus, placing CNP in the superfamily of 2H phosphodiesterases that contain two tetrapeptide HX(T/S)X motifs. NMR titrations of the CNP catalytic domain with inhibitors and kinetic studies of site-directed mutants reveal a protein conformational change that occurs upon binding.The abundance of the enzyme 2Ј,3Ј-cyclic nucleotide 3Ј-phosphodiesterase (CNP 1 ; EC 3.1.4.37) in the central nervous system of all mammals and some other vertebrates such as amphibians and birds has long been an enigma. This derives from the continuing failure to identify a physiological substrate for this enzyme. CNP has an apparent specificity for nucleoside 2Ј,3Ј-cyclic phosphate, which it cleaves to 2Ј-nucleotide end products, none of which (with the exception of NADP/NADPH) are found in metabolite pools. The last 4 decades of research have failed to attribute a function to this protein, although many possibilities have been considered (extensively reviewed in Refs. 1-3). More recently, RICH, a neuronally associated homolog of CNP, has been discovered in fish (4, 5), and the catalytic active site of CNP has been investigated (6).CNP and RICH share catalytic features with three other groups of enzymes: fungal/plant RNA ligases involved in tRNA splicing (7,8), bacterial and archaeal RNA ligases (9) that ligate tRNA half-molecules containing 2Ј,3Ј-cyclic phosphate and 5Ј-hydroxyl termini, and plant and yeast cyclic phosphodiesterases (CPDases) that hydrolyze ADP-ribose 1Љ,2Љ-cyclic phosphate to yield ADP-ribose 1Ј-phosphate (at least one of these latter enzymes also hydrolyzes nucleoside 2Ј,3Ј-cyclic phosphates) (10, 11). These enzymes are thought to play a role in the tRNA-splicing pathways. The x-ray structures of a CPDase from Arabidopsis thaliana (12-14) and, most recently, 2Ј-5Ј RNA ligase from Thermus thermophilus (15) have been determined.Members of this enzyme superfamily occur across a vast range of organisms ranging from bacteria to mammals. It has been suggested (16) that all four classes of enzymes originated from a common ancestor because they all have two similarly spaced histidine-containing tetrapeptides; their catalytic domains have a similar size of ϳ200 residues with similar pattern of predicted secondary structural ...

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Identification of Essential Residues in 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase

Cited by 37 publications

References 62 publications

An in vitro model for de‐ and remyelination using lysophosphatidyl choline in rodent whole brain spheroid cultures

An in vitro model for de‐ and remyelination using lysophosphatidyl choline in rodent whole brain spheroid cultures

Structure and Function of the Myelin Proteins: Current Status and Perspectives in Relation to Multiple Sclerosis

Structural Evidence That Brain Cyclic Nucleotide Phosphodiesterase Is a Member of the 2H Phosphodiesterase Superfamily

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