Selective synthesis of 2',3'-cyclic nucleotide 3'-phosphodiesterase isoform 2 and identification of specifically phosphorylated serine residues

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is a protein found abundantly in the cytoplasmic compartments of CNS myelin. Two isoforms of this protein, CNP1 and CNP2, are detectable. They differ by a 20-amino acid extension exclusive to CNP2. Additionally, CNP2 is essentially the only isoform to be phosphorylated in vivo. In this study, we examine the phosphorylation of CNP2 in transfected cells. CNP2 was selectively expressed ectopically in 293T cells and labeled with 32P. Immunoprecipitation of labeled CNP2 and tryptic phosphopeptide mapping analyses identified serines 9 and 22 as the major sites of phosphorylation. Only serine 22 was phosphorylated initially in oligodendrocyte-enriched cultures of neonatal rat brain glial cells. However, 4beta-phorbol 12,13-dibutyrate (PDB) induced the phosphorylation of serine 9, thereby producing the same pattern seen in 293T cells. These results suggest that serine 9 is phosphorylated by a PDB-sensitive kinase, likely protein kinase C, and that serine 22 appears to be constitutively phosphorylated.     

Age-dependent myelin degeneration and proteolysis of oligodendrocyte proteins is associated with the activation of calpain-1 in the rhesus monkey

Myelin provides important insulating properties to axons allowing for propagation of action potentials over large distances at high velocity. Disruption of the myelin sheath could therefore contribute to cognitive impairment, such as that observed during the normal aging process. In the present study, age-related changes in myelin, myelin proteins and oligodendrocyte proteins were assessed in relationship to calpain-1 expression and cognition in the rhesus monkey. Isolation of myelin fractions from brain white matter revealed that as the content of the intact myelin fraction decreased with age, there was a corresponding increase in the floating or degraded myelin fraction, suggesting an increased breakdown of intact myelin with age. Of the myelin proteins examined, only the myelin-associated glycoprotein decreased with age. Levels of the oligodendrocyte-specific proteins 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and myelin/oligodendrocyte-specific protein (MOSP) increased dramatically in white matter homogenates and myelin with age. Age-related increases in degraded CNPase also were demonstrable in white matter in association with increases in activated calpain-1. Degraded CNPase was also detectable in myelin fractions, with only the floating fraction containing activated calpain-1. The increases in the activated enzyme in white matter were much greater than those found in myelin fractions suggesting a source other than the myelin membrane for the marked overexpression of activated calpain-1 with age. In addition, CNPase was demonstrated to be a substrate for calpain in vitro. In summary, changes in myelin and oligodendrocyte proteins occur with age, and they appear to have a significant relationship to cognitive impairment. The overexpression of CNPase and MOSP suggests new formation of myelin by oligodendrocytes, which may occur in response to myelin degradation and injury caused by proteolytic enzymes such as calpain.     

Cloning and characterization of zRICH, a 2',3'-cyclic-nucleotide 3'-phosphodiesterase induced during zebrafish optic nerve regeneration

We previously reported cloning of cDNAs encoding both components of a protein doublet induced during goldfish optic nerve regeneration. The predicted protein sequences showed significant homology with the mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases (CNPases). CNPases are well-established markers of mammalian myelin; hence, the cDNAs were designated gRICH68 and gRICH70 (for goldfish Regeneration-Induced CNPase Homologues of 68 and 70 kDa). Homologous cDNAs have now been isolated from zebrafish encoding a highly related protein, which we have termed zRICH. RNase protection assays show that zRICH mRNA is induced significantly (fivefold) in optic nerve regenerating zebrafish retinas 7 days following nerve crush. Western blots show a single band in zebrafish brain and retina extracts, with immunoreactivity increasing three-fold in regenerating retinas 21 days postcrush. Immunohistochemical analysis indicated that this increase in zRICH protein expression is localized to the retinal ganglion cell layer in regenerating retina. We have characterized and evaluated the relevance of a conserved beta-ketoacyl synthase motif in zRICH to CNPase activity by means of site-directed mutagenesis. Two residues within the motif, H334 and T336, are critical for enzymatic activity. A cysteine residue within the motif, which corresponds to a critical residue for beta-ketoacyl synthase, does not appear to participate in the phosphodiesterase activity.     

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase Binds to Actin-Based Cytoskeletal Elements in an Isoprenylation-Independent Manner

Abstract: 2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is an isoprenylated protein enriched in myelin and oligodendrocytes but also present in several other tissues at low levels. CNP binds avidly to membranes and in addition possesses several characteristics of cytoskeletal proteins. The role of isoprenylation in the association of CNP with the cytoskeleton was analyzed by ectopic expression in L cells of epitope-tagged CNP1 and a non-isoprenylated mutant CNP1. Using nonionic detergent extraction, drug-mediated cytoskeletal disruption, and coimmunoprecipitation with an anti-actin antibody, we show that CNP1 is associated with actin-based cytoskeletal elements independently of its isoprenylation status. A control protein, p21c-H-ras, which is also modified by isoprenylation at its carboxyl-terminus, does not bind to cytoskeletal structures as judged by the same criteria. We present a model that accounts for the association of CNP1 with membranes and the cytoskeleton.     

Cellular and Subcellular Distribution of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase and Its mRNA in the Rat Central Nervous System

The 2',3'-cyclic nucleotide 3'-phosphodiesterases (CNPs) are closely related oligodendrocyte proteins whose in vivo function is unknown. To identify subcellular sites of CNP function, the distribution of CNP and CNP mRNA was determined in tissue sections from rats of various developmental ages. Our results indicate that CNP gene products were expressed exclusively by oligodendrocytes in the CNS. CNP mRNA was concentrated around oligodendrocyte perinuclear regions during all stages of myelination. Developmentally, initial detection of CNP mRNA closely paralleled initial detection of its translation products. In electron micrographs of immunostained ultrathin cryosections, CNP was associated with oligodendrocyte membranes during the earliest phase of axonal ensheathment. In more mature fibers, immunocytochemistry established that the CNPs are not major components of compact myelin but are concentrated within specific regions of the oligodendrocyte and myelin internode. These include (a) the plasma membrane of oligodendrocytes and their processes, (b) the periaxonal membrane and inner mesaxon, (c) the outer tongue process, (d) the paranodal myelin loops, and (e) the "incisure-like" membranes found in many larger CNS myelin sheaths. A cytoplasmic pool of CNP was also detected in oligodendrocyte perikarya and larger oligodendrocyte processes. CNP was also enriched in similar locations in myelinated fibers of the PNS.     

An investigation of 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37, CNP) in peripheral blood elements and CNS myelin

Activity of 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNP) of human erythrocyte membranes was determined in the presence of various brain CNP inhibitory compounds. Also, the hydrolysis of 2':3'-cAMP and 2':3'-cCMP by CNP of human platelets and lymphocytes was confirmed by thin layer chromatography and CNP activity was measured in lymphocytes, platelets, erythrocytes and CNS myelin. Human erythrocyte CNP activity was reduced 75 percent by the organomercurial p-chloromercuriphenyl sulfonate (1 X 10(-4) M), 46 percent by thimerosal (1 X 10(-4) M) and 35 percent by cupric chloride (1 X 10(-3) M). The 2'-AMP or 2'-CMP isomer was produced, exclusively, by the hydrolysis of 2':3'-cAMP or 2':3'-cCMP, respectively, by CNP of human lymphocytes and platelets and indicates a CNP-like activity is not only present in erythrocytes and the central and peripheral nervous systems, but also platelets and lymphocytes. CNP activities of human erythrocytes, human human and rat lymphocytes and human platelets were less than 4 percent of the activity of human and bovine CNS myelin.     

Differential Ultrastructural Localization of Myelin Basic Protein, Myelin/Oligodendroglial Glycoprotein, and 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase in the CNS of Adult Rats

In a light and electron microscopic immunocytochemical study we have examined the distribution of myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), and myelin/oligodendroglial glycoprotein (MOG) within CNS myelin sheaths and oligodendrocytes of adult Sprague-Dawley rats. Ultrastructural immunocytochemistry allowed quantitative analysis of antigen density in different myelin and oligodendrocyte zones: MBP was detectable in high density over the whole myelin sheath, but not in regions of loops, somata, or the oligodendrocyte plasma membrane. CNP reactivity was highest at the myelin/axon interface, and found in lower concentration over the outer lamellae of myelin sheaths, at the cytoplasmic face of oligodendrocyte membranes, and throughout the compact myelin. MOG was preferentially detected at the extracellular surface of myelin sheaths and oligodendrocytes and in only low amounts in the lamellae of compacted myelin and the myelin/axon border zone. Our studies, thus, indicate further the presence of different molecular domains in compact myelin, which may be functionally relevant for the integrity and maintenance of the myelin sheath.     

The brain-specific protein, p42IP4 (ADAP 1) is localized in mitochondria and involved in regulation of mitochondrial Ca2+

In brain, p42^IP4 (centaurin‐α1; recently named ADAP 1, which signifies ADP ribosylation factor GTPase activating protein with dual PH domains 1, within the large family of Arf‐GTPase activating proteins) is mainly expressed in neurons. p42^IP4 operates as a dual receptor recognising two second messengers, the soluble inositol(1,3,4,5)tetrakisphosphate and the lipid phosphatidylinositol(3,4,5)trisphosphate. We show here for the first time that p42^IP4 is localized in mitochondria, isolated from rat brain and from cells transfected with p42^IP4. In rat brain mitochondria we additionally found interaction of p42^IP4 with 2′, 3′‐cyclic nucleotide 3′‐phosphodiesterase and α‐tubulin by pull‐down binding assay and by immunoprecipitation. In mitochondria from Chinese hamster ovary cells, p42^IP4 is predominantly associated with the intermembrane space and the inner membrane. This localization of p42^IP4 indicates that p42^IP4 might have a still unknown mitochondrial function. We studied whether p42^IP4 is involved in Ca²⁺‐induced permeability transition pore opening, which is important in mitochondrial events leading to programmed cell death. We used mouse neuroblastoma cells as a model for the functional studies of p42^IP4 in mitochondria. In mitochondria isolated from p42^IP4‐transfected mouse neuroblastoma cells, over‐expression of p42^IP4 significantly decreased Ca²⁺ capacity and lag time for Ca²⁺ retention. Thus, we suggest that p42^IP4 is involved in the regulation of Ca²⁺ transport in mitochondria. We propose that p42^IP4 promotes Ca²⁺‐induced permeability transition pore opening and thus destabilizes mitochondria.     

Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse

In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous [³²P]ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using [³H]methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the [³H] methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane.     

Chromosome assignments of the genes for glucocorticoid receptor,myelin basic protein,leukocyte common antigen,and TRPM2 in the rat

We have utilized rat-mouse somatic cell hybrids to make chromosomal assignments for the glucocorticoid receptor (GR), myelin basic protein (MBP), leukocyte common antigen (LCA), and testosterone-repressed prostate message-2 (TRPM2) genes in the rat. The genes for GR and MBP both map on chromosome 18 of the rat, which corresponds to the mapping of both genes on chromosome 18 of the mouse. The gene for LCA maps on chromosome 13, which is where C4b-binding protein -chain (C4BPB), coagulation factor V (F5), and renin have previously been assigned. This linkage group appears to be homologous to a substantial portion of mouse chromosome 1 and human chromosome 1q. Finally, the TRPM2 gene has been assigned to rat chromosome 15.This project was supported by Grants RG 1877-A-1 from the National Multiple Sclerosis Society and P50 DE09164 from the NIH, by grants from the Swedish Cancer Society, the Erik Philip-Sörensen Foundation, the Trygger Foundation, the IngaBritt and Arne Lundberg Research Foundation, CANCIRCO, and BioVast (Gothenbrug), by the Belgian program on Interuniversity Attraction Poles initiated by the Belgian State-Prime Minister's Office-Science Policy Programming, and by a grant from the CGER-ASLK (Brussels). C.S. is a Senior Research Associate of the National Fund for Scientific Research (FNRS, Belgium).     

Antibodies to Myelin/Oligodendrocyte-Specific Protein and Myelin/Oligodendrocyte Glycoprotein Signal Distinct Changes in the Organization of Cultured Oligodendroglial Membrane Sheets

Abstract: Cultured murine oligodendrocytes elaborate extensive membrane sheets that, unlike multilamellar myelin in vivo, allow the study of interactions between myelin proteins and cytoskeletal elements. This article describes the events that occur due to the interaction of specific antibodies with their respective antigens, myelin/oligodendrocyte-specific protein (MOSP) and myelin/oligodendrocyte glycoprotein (MOG), which are expressed uniquely by oligodendrocytes. After antibody binding, surface anti-MOSP:MOSP complexes redistribute over those cytoplasmic microtubular veins that have 2',3'-cyclic nucleotide 3'-phosphohydrolase colocalized along them. In contrast, surface anti-MOG-MOG complexes redistribute over internal myelin basic protein domains. Long-term anti-MOSP IgM exposure results in an apparent increase in number as well as thickness of microtubular structures in oligodendrocyte membrane sheets, whereas long-term anti-MOG exposure causes depolymerization of microtubular veins in membrane sheets. These data suggest that antibody binding to these two surface proteins elicits signals that have opposite effects on the cytoskeleton in oligodendroglial membrane sheets. Thus, it is possible that signals transduced via antibody binding may contribute to the pathogenesis of diseases affecting CNS myelin.     

Differential regulation of cerebroside sulfotransferase and 2',3'-cyclic nucleotide 3'-phosphodiesterase by basic fibroblast growth factor in relation to proliferation in rat oligodendrocyte cultures.

Previous results (Fressinaud, C., Sarliève, L.L., and Labourdette, G. J. J. Cell. Physiol., 141:667-674, 1989b) have shown that cerebroside sulfotransferase (CST; EC 2.8.2.11) is enriched in pure rat oligodendrocyte (OL) cultures and that its activity is increased by factors mitogenic for OL precursors and galactocerebroside (GC) expressing OL, such as basic fibroblast growth factor (bFGF), platelet-derived growth factor, and high insulin concentrations. In contrast, transforming growth factor beta or low insulin concentrations were found to be ineffective in this culture system. As bFGF mainly enhanced the proliferation of OL precursors (GC negative cells) rather than that of differentiated (GC+) cells, a relationship between OL precursor proliferation and CST increase was suggested. This hypothesis was first tested in 20-day-old OL cultures grown in chemically defined medium. The dose-response curve of [125I] Iododeoxyuridine ([125I]dUrd) incorporation toward bFGF was parallel to that of CST specific activity, and maximal stimulation was reached at 5 ng/ml bFGF for both. In contrast, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP; EC 3.1.4.37) specific activity decreased after bFGF treatment. To determine if CST increase was linked to the proliferation of OL precursors induced by bFGF, cell proliferation was blocked by cytosine arabinoside (ARA-C). From 10(-8) to 10(-5) M ARA-C there was a dose-dependent inhibition of cell proliferation and a decrease in CST specific activity, whereas CNP specific activity was enhanced. When the cells were treated with bFGF and 10(-6) M ARA-C together, the proliferation was completely blocked and CST activity decreased by 72% below control values, whereas CNP activity was not significantly decreased. Immunocytochemical studies showed that the number of sulfatide-expressing cells and the number of cycling cells were increased after bFGF treatment, but that there was no overlapping between these two populations. Taken together these results suggest that CST activity and sulfatide expression appear shortly after the arrest of OL precursor division.     

Susceptibility of myelin proteins to a neutral endoproteinase: The degradation of myelin basic protein (MBP) and P2 protein by purified bovine brain multicatalytic proteinase complex (MPC)

Multicatalytic proteinase complex (MPC) was isolated from bovine brain and the susceptibility of myelin basic protein (MBP) and P₂ protein of bovine central and peripheral nervous system was examined. SDS-polyacrylamide electrophoretic analysis of purified MPC revealed protein bands of molecular weight ranging from 22–35 kDa. The enzyme is activated by SDS at a concentration less than 0.01%. Upon incubation with MPC, purified MBP and P₂ proteins were degraded into smaller fragments. There was a 57% and 100% loss of MBP at 2 and 6 hours of incubation. The P₂ protein which is not susceptible to any endogenous non-lysosomal enzyme thus far studied was digested into small peptide fragments only in the presence of SDS (0.01%) and not in its absence. These results indicate that MPC which is active at physiological conditions may have a role in the turnover of myelin proteins and in demyelinating diseases.     

Metamorphic Changes in Glycolipids and Myelin Proteins and 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase in Bullfrog and Axolotl Brains

Abstract: The metamorphic changes in levels of glycolipids and myelin proteins and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) in the brains of bullfrog tadpoles, adult frogs, and axolotls were investigated, with particular emphasis on myelin maturation. The concentrations of cerebroside. sulfatide, and galactosyldiacylglycerol gradually increased from the onset of prometamorphosis throughout the active metamorphic period and then greatly increased after metamorphosis was completed. The ratio of glucocerebroside to galactocerebroside increased greatly in the prometamorphic period and then rapidly decreased to the frog level during the climax period. The fatty acid compositions of cerebroside and sulfatide showed a developmental change, with 24:1 being more predominant in the later metamorphic stage. The proportion of hydroxy fatty acids increased up to the onset of the prometamorphic stage and thereafter remained constant at ∼ 50% of the total. The CNP activity remained unchanged throughout metamorphosis at 60% that in frog myelin and increased in the adult frog. The composition of tadpole myelin proteins remained constant during metamorphosis, with large basic protein being the most abundant, and in the frog, proteolipid protein and large basic protein were present in comparable amounts. The two adult forms of axolotl, i.e., the neotenous and metamorphosed forms, exhibited almost identical myelin constituents, and CNP activity in the neotenous form amounted to one-fifth that in the bullfrog. These results indicate that active biosynthesis of myelin marker components occurs as metamorphosis proceeds, but more pronounced changes of myelin components occur after metamorphosis is completed.     

Thr94 in bovine myelin basic protein is a second phosphorylation site for 42-kDa mitogen-activated protein kinase (ERK2)

Treatment of bovine brain myelin basic protein with 42-kDa mitogen-activated protein kinase [p42 MAPK or extracellular signal-regulated kinase 2 (ERK2)] in the presence of ATP and Mg²⁺ results in phosphorylation of Thr94 and Thr97. Thr94 is not previously known to be an ERK2 phosphorylation site. Both residues are phosphorylated to about the same extent and are in the highly conserved segment Asn⁹¹-Ile-Val-Thr⁹⁴-Pro-Arg-Thr⁹⁷-Pro-Pro-Pro-Ser¹⁰¹. MALDI mass spectrometry before and after ERK2 treatment revealed the addition of two phosphate groups to the protein. Tryptic cleavage resulted in a single fragment (positions 91–104) carrying the observed mass increase. Tandem mass spectrometry applied to the tryptic peptide showed that both Thr94 and Thr97 are acceptors of phosphate. A singly phosphorylated species could not be detected. Identification of the ERK2 phosphorylation site Thr94 in bovine myelin basic protein reveals a nontraditional phosphate acceptor position, preceded by three noncharged residues (Asn-Ile-Val). Proline at position –2 or –3 from the phosphorylation site, typical for the recognition sequence of proline-directed kinases, is missing. The results provide information for delineation of a further substrate consensus motif for ERK2 phosphorylation.     

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-Dicarboxycyclopropyl)glycine Binding in Rat Brain

Abstract: [(2S,2′R,3′R)-2-(2′,3′-[³H]Dicarboxycyclopropyl)glycine ([³H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K_D value of 180 ± 33 nM and a B_max of 780 ± 70 fmol/mg of protein. The nonspecific binding, measured using 100 µM LY354740, was <30%. NMDA, AMPA, kainate, l (?)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [³H]DCG IV binding up to 1 mM. However, several compounds inhibited [³H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1′S,2′S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1′S,2′S)-2-methyl-2-(2-carboxycyclopropyl)glycine > l -glutamate = ibotenate > quisqualate > (RS)-α-methyl-4-phosphonophenylglycine = l (+)-2-amino-3-phosphonopropionic acid > (S)-α-methyl-4-carboxyphenylglycine > (2S)-α-ethylglutamic acid > l (+)-2-amino-4-phosphonobutyric acid. N-Acetyl-l -aspartyl-l -glutamic acid inhibited the binding in a biphasic manner with an IC₅₀ of 0.2 µM for the high-affinity component. The binding was also affected by GTPγS, reducing agents, and CdCl₂. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPγS show that [³H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.     

Molecular mimicry between Mycobacterium leprae proteins (50S ribosomal protein L2 and Lysyl-tRNA synthetase) and myelin basic protein: a possible mechanism of nerve damage in leprosy

Autoantibodies against various components of host are known to occur in leprosy. Nerve damage is the primary cause of disability associated with leprosy. The aim of this study was to detect the level of autoantibodies and lympho-proliferative response against myelin basic protein (MBP) in leprosy patients (LPs) and their correlation with clinical phenotypes of LPs. Further, probable role of molecular mimicry in nerve damage of LPs was investigated. We observed significantly high level of anti-MBP antibodies in LPs across the spectrum and a positive significant correlation between the level of anti-MBP antibodies and the number of nerves involved in LPs. We report here that 4 B cell epitopes of myelin A1 and Mycobacterium leprae proteins, 50S ribosomal L2 and lysyl tRNA synthetase are cross-reactive. Further, M. leprae sonicated antigen hyperimmunization was responsible for induction of autoantibody response in mice which could be adoptively transferred to naive mice. For the first time our findings suggest the role of molecular mimicry in nerve damage in leprosy.