Demonstration of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase in Cultured Human Schwann Cells

Abstract: Schwann cell cultures were established from adult human sural nerve biopsies. 2'3'-Cyclic nucleotide 3'-phosphohydrolase (CNPase) activity was estimated in the homogenates of those cells by a sensitive isotope assay using [³H]2',3'-cyclic AMP as substrate. A high level of CNPase activity was observed in cultured Schwann cells, whereas cultured human muscle and skin fibroblasts contained negligible levels of CNPase activity. CNPase of human Schwann cells followed typical enzyme-substrate kinetics, with an apparent K _m of 1.6 m M for 2',3'-cyclic AMP, and the enzyme was stimulated by detergents such as Triton X-100 and deoxycholate. It was inhibited by p -chloromercuricbenzoate and 2'-AMP. These properties are typical of CNPase isolated from adult brain and spinal cord. CNPase can serve as a new biochemical marker of normal cultured human Schwann cells and can be useful in analyzing the properties of cultured Schwann cells from patients with dysschwannian neuropathies.     

Molecular cloning of the myelin specific enzyme 2',3'-cyclic-nucleotide 3'-phosphohydrolase

We describe the isolation of cDNA clones for bovine brain 2',3'-cyclic-nucleotide 3'-phosphohydrolase (CNPase, EC 3.1.4.37), the third most abundant protein in central nervous system myelin. The cDNA encodes the complete protein (400 amino acids) and hybridizes to a major size species of mRNA in bovine brain tissue, approx. 2.7 kb in size. CNPase mRNA levels do not appear to be affected in quaking dysmyelinating mutant mice. The sequence reveals probable sites for CNPase phosphorylation by cAMP-dependent protein kinase and a region of homology with haemocyanin.     

Improved Method for Prufication of 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase from Bovine Cerebral White Matter

Abstract: An improved procedure of the solubilization and purification of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) from bovine cerebral white matter is reported. To remove easily extractable protein, the tissue was homogenised in 10 vol. of 0.5 M-ammonium acetate containing 10 mM-Tris. HCI, pH 6.9, at 4°C and centrifuged at 105,000 g for 60 min. The precipitate was extracted with 10 vol. of 0.5% Triton X-100 containing 10 mM-Tris. HCI, pH 6.9, and centrifuged, By this extraction, over 70% soluble protein could be removed in the supernatant and most CNPase activity was kept in the precipitate. The precipitate was extracted with 10 vol. of 1% Triton X-100 and 1 M-ammonium acetate mixture containing 10 mM-Tris.HCI, pH 8.2, and centrifuged at 105,000 g for 60 min. The extract contained 54% of CNPase and the specific activity was fivefold that of the original homogenate. Subsequently, the extractions were carried out with 2% Triton X-100-2 M-ammonium acetate and 4% Triton X-100-4 M-ammonium acetate at pH 8.2. The recovery of CNPase was found to be nearly 90% from the original homogenate, without loss of enzyme activity during extraction, while much CNPase activity was lost when guanidinium chloride was used as the extraction medium. Using the Triton X-100-ammonium acetate extract, several column chromatography techniques were applied to purify the enzyme. In the first step, Phenyl-Sepharose CL-4B column chromatography was performed by eluting with a double-linear gradient of ammonium acetate and Triton X-100. In the second step, the fraction containing CNPase after Phenyl-Sepharose CL-4B column chromatography was applied to a Sepharose 6B column and the enzyme was eluted with 1% Triton X-100- I M-ammonium acetate, pH 8.2. The peak containing CNPase was applied to CM-Sepharose CL-6B column chromatography in the final step. The enzyme was eluted with a linear gradient of KCI. In this step, CNPase eluted as a sharp peak and the specific activity was approximately 2300 pmol 2′-AMP formed/min/mg protein. The recovery of CNPase from the original homogenate was about 50%. By the isoelectrofocusing technique, the pI of CNPase was found to be 8.6. When Reisfeld polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis were carried out on the purified CNPase, only one protein band, corresponding to CNPase activity, was detected. Its molecular weight was estimated to be approximately 51,000 as the active enzyme form. K, value of the purified enzyme for 2′,3′-CAMP calculated from a Lineweaver-Burk plot was 3.13 mM.     

Intracellular Localization of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase in a Neuronal Cell Line as Examined by Immunofluorescence and Cell Fractionation

The enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase, EC 3.1.4.37) occurs not only in myelin fractions and glial cells, but can also be shown to be present in a CNS cell line of neuronal origin (B104). Direct immunofluorescence microscopy of B104 cells with fluorescein isothiocyanate-conjugated rabbit anti-CNPase antibodies shows a discrete and specific intracytoplasmic location of CNPase. Fractionation of the cells was performed by differential centrifugation of a cell homogenate and continuous sucrose density-gradient centrifugation. As monitored by marker enzyme activities, CNPase seems to be associated with endoplasmic reticulum membranes.     

Characterization of 2'':3''-Cyclic Nucleotide 3''-Phosphodiesterase: Limited Proteolytic Digestion, Plant Lectin Affinity Chromatography, and Immunological Identification

Purified bovine brain 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) migrates as a protein double band in SDS-polyacrylamide gel electrophoresis. The positions of the two protein bands correspond to approximate molecular weights (MW) of 56,000 and 53,000. Limited protease treatment of isolated CNPase leads to subsequent degradation of the enzyme into smaller polypeptides having MWs of approximately 40,000, 30,000, and 20,000. During proteolytic digestion CNPase remains enzymatically active. Binding studies with several immobilized plant lectins as well as periodic acid-Schiff reagent (PAS) staining of SDS gels indicate that CNPase is a glycoprotein. An antiserum against purified CNPase, prepared in rabbits, was used to confirm the immunological identity of various CNPase preparations obtained in our laboratory.     

Identification of 2',3'-cyclic nucleotide 3'-phosphodiesterase in bovine adrenal medulla

The adrenal medulla contains an enzyme which catalyzes the hydrolysis of 2',3'-cAMP to 2'-AMP. For the parameters which have been examined, the adrenal medulla 2',3'-cAMP phosphodiesterase appears to be similar to brain 2',3'-cyclic nucleotide 3'-phosphodiesterase (also commonly referred to as CNPase). The apparent Km of the adrenal medulla CNPase for 2',3'-cAMP is 0.88 mM. The enzyme activity is unaltered by either EDTA, MgCl2 or CaCl2 in the presence or absence of calmodulin. The apparent molecular weight is 102,500 daltons. The function of the enzyme in either the brain or the adrenal medulla is, at the present time, unknown.     

Myelin 2',3'-cyclic nucleotide 3'-phosphodiesterase: active-site ligand binding and molecular conformation

The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is a highly abundant membrane-associated enzyme in the myelin sheath of the vertebrate nervous system. CNPase is a member of the 2H phosphoesterase family and catalyzes the formation of 2'-nucleotide products from 2',3'-cyclic substrates; however, its physiological substrate and function remain unknown. It is likely that CNPase participates in RNA metabolism in the myelinating cell. We solved crystal structures of the phosphodiesterase domain of mouse CNPase, showing the binding mode of nucleotide ligands in the active site. The binding mode of the product 2'-AMP provides a detailed view of the reaction mechanism. Comparisons of CNPase crystal structures highlight flexible loops, which could play roles in substrate recognition; large differences in the active-site vicinity are observed when comparing more distant members of the 2H family. We also studied the full-length CNPase, showing its N-terminal domain is involved in RNA binding and dimerization. Our results provide a detailed picture of the CNPase active site during its catalytic cycle, and suggest a specific function for the previously uncharacterized N-terminal domain.     

Characterization of 2'':3''-Cyclic Nucleotide 3''-Phosphodiesterase: Rapid Isolation, Native Enzyme Analysis, Identification of a Serum-Soluble Activity, and Kinetics

The enzyme 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) was isolated from bovine brain white matter by a rapid (72 h) procedure. The minimum molecular weight (MW) of the enzyme was approximately 52,500 as estimated by sucrose density gradient analysis. When this isolated enzyme was stimulated with bovine serum albumin (BSA), the peak of activity was shifted to approximately 90,000 MW. Prior treatment by trypsin blocked the expression of the higher MW form of CNPase, but not the BSA activation of the enzyme. If the trypsin digestion was allowed to progress, the MW was gradually lowered to a broad peak sedimenting between 20,000 and 50,000 MW. An apparently soluble form of CNPase found in serum is described. Kinetic and MW comparisons between the serum soluble enzyme and CNPase isolated from bovine brain, as well as an analysis of substrate specificity, were made and it was concluded that the two enzymes were identical.     

Production of Monoclonal Antibody to 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase from Bovine Cerebral White Matter

Lewis rats were immunized with partially purified 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) from bovine cerebral white matter and the spleen cells were fused with cell of a mouse myeloma cell line (SP-2). The production of monoclonal antibody was detected by enzyme-linked immunoadsorbent assay, immunohistochemical staining of bovine cerebrum, Western blotting analysis, and CNPase binding assay. Monoclonal antibody that specifically binds CNPase molecules was obtained. However, the antibody did not suppress the enzyme activity. Western blotting analysis demonstrated that the monoclonal antibody binds both CNa (Wla) and CNb (Wlb). The monoclonal antibody was identified as being of the IgG2c subclass. Immunohistochemical examination revealed that the myelin sheath in the CNS was heavily stained with the monoclonal antibody in several species (bovine, mouse, rat, and human). In contrast, peripheral nervous system myelin was not stained even in bovine tissue. These results suggest that the monoclonal antibody obtained in the present study specifically recognizes the CNPase molecules in the CNS.     

Immunohistochemical Localization of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase and Myelin Basic Protein in the Central Nervous System of the Jimpy and the Normal Mouse

We describe the immunohistochemical localization of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and myelin basic protein (MBP) in CNS of the jimpy mutant mouse which is characterized by dys- and demyelination. In controls, the CNPase and MBP were localized exclusively in white matter in the CNS. The jimpy mutant mice were severely affected: A very weak reaction was observed in the white matter. Very few CNPase- and MBP-positive myelin sheaths were observed, and some degradation products were also observed after reaction with antisera against both CNPase and MBP. The immunohistochemical reaction in the jimpy mice showed a similar localization in both CNPase and MBP.     

A new spectrophotometric assay for 2',3'-cyclic nucleotide 3'-phosphohydrolase activity in nervous tissue

A new continuous spectrophotometric method for determining 2′,3′-cyclic nucleotide 3′-phosphohydrolase (EC 3.1.4.37) is described. The assay method involves monitoring the decrease in pH which accompanies the hydrolysis of 2′,3′-cyclic AMP. The reaction is performed in the presence of phenol red and the pH change is followed spectrophotometrically by recording the decrease in absorbance of the basic chromophore at 560 nm. The assay method is sufficiently sensitive to make accurate determinations of CNPase activity in 20-μl samples of CNS homogenates containing less than 5 μg protein. The primary advantage of the phenol red CNPase assay is the ease and speed with which it is performed.     

Crystallographic Analysis of the Reaction Cycle of 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase,a Unique Member of the 2H Phosphoesterase Family

2H phosphoesterases catalyze reactions on nucleotide substrates and contain two conserved histidine residues in the active site. Very limited information is currently available on the details of the active site and substrate/product binding during the catalytic cycle of these enzymes. We performed a comprehensive X-ray crystallographic study of mouse 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), a membrane-associated enzyme present at high levels in the tetrapod myelin sheath. We determined crystal structures of the CNPase phosphodiesterase domain complexed with substrate, product, and phosphorothioate analogues. The data provide detailed information on the CNPase reaction mechanism, including substrate binding mode and coordination of the nucleophilic water molecule. Linked to the reaction, an open/close motion of the β5–α7 loop is observed. The role of the N terminus of helix α7—unique for CNPase in the 2H family—during the reaction indicates that 2H phosphoesterases differ in their respective reaction mechanisms despite the conserved catalytic residues. Furthermore, based on small-angle X-ray scattering, we present a model for the full-length enzyme, indicating that the two domains of CNPase form an elongated molecule. Finally, based on our structural data and a comprehensive bioinformatics study, we discuss the conservation of CNPase in various organisms.     

Subcellular distribution of 2′,3′-cyclic nucleotide-3′-phosphodiesterase in cat peripheral nerve

Subcellular distribution of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) in desheathed, saline perfused cat sciatic nerve is reported. CNPase specific activity was enriched in the total particulate (P₂) fraction and was low in the soluble (S₂) fraction. “Light-myelin” floating above the 0.60 M sucrose phase had the highest CNPase activity, 2.5-fold over the crude homogenate (CH). By contrast, enzyme activity in “heavy myelin” floating above the 0.85 M sucrose interface was equal to that of the CH and accounted for only 12% of total activity. CNPase activity in the membranes floating above the 0.25 and 0.60 and 0.85 M sucrose phases comprised nearly 70% of the total CNPase activity. The “light myelin” fraction floating above the 0.60 M sucrose accounted for approx. 51% of the total CNPase activity. SDS-PAGE of membranes individually harvested from above the 0.25 and 0.60 and 0.85 M sucrose phases revealed the presence of myelin-specific proteins (P₀, P₁; and P₂). Electron microscope examination demonstrated the presence of myelin in each membrane fraction. The results of this study show that the majority of CNPase activity is associated with “light myelin” in cat peripheral nerve.     

Simultaneous Measurement of 2':3' Cyclic-Nucleotide 3' Phosphodiesterase and RNase Activities in Sera and Spinal Fluids of Multiple Sclerosis Patients

Abstract: The enzymes 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and RNase were simultaneously measured in the sera and CSF of multiple sclerosis (MS) and non-MS patients. No evidence of increased activity for these enzymes could be found regardless of pathology in either fluid source. Discrepancies between the present results and those from two previous studies that reported significant increases in CNPase activity in the CSF of patients with MS were carefully analyzed. It was concluded that the apparent increased CNPase activity correlated with MS in both previous studies was most probably the result of methodological and computational difficulties.     

The activity of 2'',3''-cyclic nucleotide 3''-phosphohydrolase in the corpus callosum, subcortical white matter, and spinal cord in infants dying from sudden infant death syndrome

Abstract: The activity of 2',3'-cyclic nucleotide 3'-phos-phohydrolase (CNPase) has been determined in corpus callosum, subcortical white matter, and spinal cord of infants whose death was attributed to the sudden infant death syndrome (SIDS), and compared with enzyme activity in other cases in which the cause of death was not associated with respiratory distress. In nearly half the SIDS cases, CNPase activity and oligodendroglial cell numbers were reduced before the onset of myelination, but only in the corpus callosum. In other SIDS cases, enzyme activity and cell numbers were the same as in non-SIDS cases. If the expression of CNPase activity reflects glioblast differentiation to oligodendrocytes with myelinating potential, then this transformation is abnormal in certain SIDS cases, as also evidenced in cases of prolonged neonatal respiratory insufficiency and gives rise to a subsequent deficit of myelin in the corpus callosum.     

2',3'-cyclic nucleotide phosphohydrolase activity determined using an image analyzer detection system

The activity of 2',3'-cyclic nucleotide phosphohydrolase (CNPase) was assayed using high-performance thin-layer chromatography (HPTLC) and an image analyzer detection system. The assay system was used to study a possible inhibitory effect by aminoguanidine on CNPase specific activity. One advantage of using a fixed-time HPTLC system over a real-time spectrophotometric system for an enzyme activity study was that apparent inhibition of the enzyme due to interference of the assay system (chromophore inhibition, etc.) was avoided. In addition, due to the increased accuracy of the image analyzer over conventional methods of TLC plate analysis, a rapid and more accurate measurement of HPTLC plates was possible which required only nanomole amounts of substrate. Also, a digital image of each plate analyzed was stored indefinitely in the computer's memory for future reference. The measurements of CNPase specific activity made using this system compared favorably to those found in recent literature.     

Identification of phosphorylated form of 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) as 46 kDa phosphoprotein in brain non-synaptic mitochondria overloaded by calcium

In our previous studies phosphorylation of several membrane-bound proteins in brain and liver mitochondria were found to be regulated by Ca²⁺ as a second messenger. One of the proteins, the 46 kDa phosphoprotein was found to be highly phosphorylated when Ca²⁺-induced permeability transition pore (mPTP) was opened in rat brain mitochondria (RBM). In the present study the 46 kDa phosphoprotein was identified as 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) after purification by 2D diagonal electrophoresis following mass spectrometric analysis and Western blot probed with anti-CNP antibody. CNPase was discovered in immunoprecipitates of mitochondria, phosphorylated under both conditions (control and with opened mPTP). Status phosphorylation of CNPase was found to be higher in the inmmunoprecipiates of calcium-overloaded RBM. The phospohoserine and phosphotyrosine residues were detected in phosphorylated 46 kDa band (CNPase) as well as in CNPase immunoprecipitates indicating possible participation of tyrosine and serine protein kinases in phosphorylation of CNPase in mitochondria. The levels of phospo-Ser and phospho-Tyr were increased in RBM with mPTP opened. It was found that CNPase substrate, 2′,3′-cAMP (5 μM) and, a non-competitive CNPase inhibitor, atractyloside (5 μM), were able to increase the level of CNPase phosphorylation in calcium-overloaded mitochondria, while CsA (mPTP blocker) was able to strong suppress the phosphorylation of the enzyme. Collectively, our results provide evidence that Ca²⁺-stimulated and mPTP-associated CNPase phosphorylation might be an important stage of mPTP regulation in mitochondria, revealing a new function of CNPase outside of myelin structure.     

Investigations on Myelination In Vitro: Regulation of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase by Thyroid Hormone in Cultures of Dissociated Brain Cells from Embryonic Mice

Abstract: The direct influence of l -3,3',5-triiodothyronine (T₃) on the development of 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37, CNPase) is demonstrated by using an in vitro culture system of dissociated embryonic mouse brain cells. Serum from a thyroidectomized calf, which contained low levels of T₃ (31 ng/100 ml), and thyroxine, T₄ (<1 μg/ml), was used in the culture medium in place of normal calf serum (T₃, 103 ng/100 ml; T₄, 5.7 μg/ml) to render the culture responsive to exogenously added T₃. The lower levels of enzyme activity observed in the presence of such a deficient medium could be restored to normal values by T₃ supplementation. Half-maximal effect was obtained with 2.5 ± 10⁻⁹ m -T₃. Three days of hormone treatment resulted in the maximal stimulation of CNPase. T₄ was less effective in inducing CNPase activity and the inactive analog of the hormone, reverse T₃ (3,3',5'-T₃) was ineffective. The morphological appearance of the cells was characterized by deformed (smaller size and less in number) reaggregates in the cultures, lacking hormone.     

Related domains in yeast tRNA ligase, bacteriophage T4 polynucleotide kinase and RNA ligase, and mammalian myelin 2',3'-cyclic nucleotide phosphohydrolase revealed by amino acid sequence comparison

Related domains containing the purine NTP-binding sequence pattern have been revealed in two enzymes involved in tRNA processing, yeast tRNA ligase and phage T4 polynucleotide kinase, and in one of the major proteins of mammalian nerve myelin sheath, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). It is suggested that, similarly to the tRNA processing enzymes, CNPase possesses polynucleotide kinase activity, in addition to the phosphohydrolase one. It is speculated that CNPase may be an authentic mammalian polynucleotide kinase recruited as a structural component of the myelin sheath, analogously to the eye lens crystallins. Significant sequence similarity was revealed also between the N-terminal regions of yeast tRNA ligase and phage T4 RNA ligase. A tentative scheme of the domainal organizations for the three complex enzymes is proposed. According to this model, tRNA ligase contains at least three functional domains, in the order: N-ligase-kinase-phosphohydrolase-C, whereas polynucleotide kinase and CNPase encompass only the two C-terminal domains in the same order.     

The brain in vivo expresses the 2',3'-cAMP-adenosine pathway

Although multiple biochemical pathways produce adenosine, studies suggest that the 2',3'-cAMP-adenosine pathway (2',3'-cAMP→2'-AMP/3'-AMP→adenosine) contributes to adenosine production in some cells/tissues/organs. To determine whether the 2',3'-cAMP-adenosine pathway exists in vivo in the brain, we delivered to the brain (gray matter and white matter separately) via the inflow perfusate of a microdialysis probe either 2',3'-cAMP, 3',5'-cAMP, 2'-AMP, 3'-AMP, or 5'-AMP and measured the recovered metabolites in the microdialysis outflow perfusate with mass spectrometry. In both gray and white matter, 2',3'-cAMP increased 2'-AMP, 3'-AMP and adenosine, and 3',5'-cAMP increased 5'-AMP and adenosine. In both brain regions, 2'-AMP, 3-AMP and 5'-AMP were converted to adenosine. Microdialysis experiments in 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) wild-type mice demonstrated that traumatic brain injury (controlled cortical impact model) activated the brain 2',3'-cAMP-adenosine pathway; similar experiments in CNPase knockout mice indicated that CNPase was involved in the metabolism of endogenous 2',3'-cAMP to 2'-AMP and to adenosine. In CSF from traumatic brain injury patients, 2',3'-cAMP was significantly increased in the initial 12 h after injury and strongly correlated with CSF levels of 2'-AMP, 3'-AMP, adenosine and inosine. We conclude that in vivo, 2',3'-cAMP is converted to 2'-AMP/3'-AMP, and these AMPs are metabolized to adenosine. This pathway exists endogenously in both mice and humans.