Topology of wolfgram proteins and 2′, 3′-cyclic nucleotide 3′-phosphodiesterase in CNS myelin: Studies with proteases

The topological disposition of Wolfgram proteins (WP) and their relationship with 2, 3-cyclic nucleotide 3-phosphodiesterase (CNPase) in human, rat, sheep, bovine, guinea pig and chicken CNS myelin was investigated. Controlled digestion of myelin with trypsin gave a 35KDa protein band (WP-t) when electrophoresed on dodecyl sulfate-polyacrylamide gel in all species. Western blot analysis showed that the WP-t was derived from WP. WP-t was also formed when rat myelin was treated with other proteases such as kallikrein, thermolysin and leucine aminopeptidase. Staining for CNPase activity on nitrocellulose blots showed that WP-t is enzymatically active. Much of the CNPase activity remained with the membrane fraction even after treatment with high concentrations of trypsin when WP were completely hydrolysed and no protein bands with M.W>14KDa were detected on the gels. Therefore protein fragments of WP with M.W<14KDa may contain CNPase activity. From these results, it is suggested that the topological disposition of all the various WP is such that a 35KDa fragment is embedded in the lipid bilayer and the remaining fragment exposed at the intraperiod line in the myelin structure which may play a role in the initiation of myelinogenesis.     

Distribution of lipid synthesizing enzymes, 2′,3′-cyclic nucleotide 3′-phosphodiesterase,and myelin proteins in rat forebrain subfractions during development

The distribution of UDP-galactose: ceramide galactosyltransferase (CGalT) was studied in subcellular fractions of rat forebrain during development using zonal centrifugation on linear gradients. Specialized subfractions: SN 1, a microsomal fraction, SN 4, a myelin-related fraction, and purified myelin were also used for this study. For comparison, two microsomal lipid synthesizing enzymes, a myelin-specific enzyme, 2,3-cyclic nucleotide 3-phosphodiesterase and myelin proteins were measured in the same subfractions. UDP-glucose: ceramide glucosyltransferase and cerebroside sulfotransferase were confined to microsomes. CGalT was ferase and cerebroside sulfotransferase were confined to microsomes. CGalT was localized in microsomes, but also in myelin and myelin-related fractions. The developmental change in distribution of CGalT in adult animals toward myelin containing fractions could indicate that the replacement of galactosylceramide in compact myelin could be carried out in close proximity to compact myelin (mesaxon, paranodal loops) rather than in the distant oligodendrocyte perikaryon.     

Differential expression of 2′∶3′-cyclic nucleotide 3′-phosphodiesterase in cultured central,peripheral, and extraneural cells

The relative levels of the central nervous system myelin marker enzyme 2:3-cyclic nucleotide 3-phosphodiesterase (EC 3.1.4.37, CNPase) were determined in neuroblastoma, astrocyte, oligodendrocyte and Schwann cell cultures and in freshly isolated human lymphocytes and platelets. The highest specific activities were associated with the cells that elaborate myelin membrane in the central and peripheral nervous system, oligodendrocytes and Schwann cells, respectively. Antiserum to bovine CNPase recognized both CNP1 and CNP2 in CNS myelin and human oligodendroglioma. In addition, a 53,000 dalton protein was evident on autoradiographs of immunoblotted PNS myelin and human oligodendroglioma proteins. Cultured rat oligodendrocyte, C₆ and mouse NA neuroblastoma CNPase appear to share common determinants with the corresponding normal rat CNS enzyme.     

Interaction of myelin basic protein and 2′,3′-cyclic nucleotide phosphodiesterase with mitochondria

The content and distribution of myelin basic protein (MBP) isoforms (17 and 21.5 kDa) as well as 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) were determined in mitochondrial fractions (myelin fraction, synaptic and non-synaptic mitochondria) obtained after separation of brain mitochondria by Percoll density gradient. All the fractions could accumulate calcium, maintain membrane potential, and initiate the opening of the permeability transition pore (mPTP) in response to calcium overloading. Native mitochondria and structural contacts between membranes of myelin and mitochondria were found in the myelin fraction associated with brain mitochondria. Using Western blot, it was shown that addition of myelin fraction associated with brain mitochondria to the suspension of liver mitochondria can lead to binding of CNPase and MBP, present in the fraction with liver mitochondria under the conditions of both closed and opened mPTP. However, induction of mPTP opening in liver mitochondria was prevented in the presence of myelin fraction associated with brain mitochondria (Ca²⁺ release rate was decreased 1.5-fold, calcium retention time was doubled, and swelling amplitude was 2.8-fold reduced). These results indicate possible protective properties of MBP and CNPase.     

Identification of cytidine 2′,3′-cyclic monophosphate and uridine 2′,3′-cyclic monophosphate in Pseudomonas fluorescens pfo-1 culture

Cytidine 2′,3′-cyclic monophosphate (2′,3′-cCMP) and uridine 2′,3′-cyclic monophosphate (2′,3′-cUMP) were isolated from Pseudomonas fluorescens pfo-1 cell extracts by semi-preparative reverse phase HPLC. The structures of the two compounds were confirmed by NMR and mass spectroscopy against commercially available authentic samples. Concentrations of both intracellular and extracellular 2′,3′-cCMP and 2′,3′-cUMP were determined. Addition of 2′,3′-cCMP and 2′,3′-cUMP to P. fluorescens pfo-1 culture did not significantly affect the level of biofilm formation in static liquid cultures.     

Density distribution of 2′,3′-cyclic nucleotide 3′-phosphodiesterase and myelin proteins in particulate material from myelin deficient (mld) mutant and control brains

Total particulate material from control and myelin deficient (mld) brains was subjected to density centrifugation on a continuous sucrose gradient. Particles from control brains distributed in a bell-shaped mode with a peak density near 0.64 M-sucrose. In mld material only a slight elevation of optical density was observed near 0.8 M-sucrose. The highest specific activities of 2′,3′-cyclic nucleotide 3′-phosphodiesterase were observed at densities of 0.63 and 0.71 M-sucrose for mld and control brains, respectively. The peak of myelin basic protein in control fractions was near 0.60 M-sucrose. In mld fractions no peak was observed. Proteolipid and Wolfgram proteins had a maximum near 0.65 and 0.73 M-sucrose in control and mld fractions, respectively. The absence of myelin basic proteins in all the fractions makes it unlikely that, in mld mice, myelin basic proteins are synthesized but not incorporated into myelin.     

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.     

Generation of a monoclonal antibody against the myelin protein CNP (2′,3′-cyclic nucleotide 3′-phosphodiesterase) suitable for biochemical and for immunohistochemical investigations of CNP

The functional role of CNP (2,3-cyclic nucleotide 3-phosphodiesterase), a minor component of central and peripheral myelin is still unclear. Here we describe preparation of a monoclonal antibody directed against CNP. The antibody, of the immunoglobulin IgG₁ type, raised with a basic 46 kDa membrane-associated protein solubilized from pig cerebellar membranes, can be used to detect immunoreactivity in solubilized brain homogenates from pig, mouse, rat, sheep, cow and man, in cerebrum and cerebellum, but not in other tissues such as liver, skeletal and heart muscle. The antibody recognizes the CNP doublet band and shows no cross-reactivity with any of the other brain proteins solubilized. In tissue sections from paraformaldehyde-fixed rat brain the antigen was localized in oligodendrocytes. In cultured glial cells from newborn mice the antibody stained cells which were identified as oligodendrocytes by co-localization of myelin basic protein. Even cells from a C6 rat glioma cell line, which contain very little of CNP, were labeled by the monoclonal antibody. Thus the monoclonal antibody recognizing CNP from several species is suitable for immunocytochemical investigations and also for biochemical studies of CNP, since the antibody has been employed for immunoprecipitation and immunopurification of CNP in crude brain homogenates.     

Localization of 3′, 5′-cyclic nucleotide phosphodiesterase activity in isolated thyroid cells and intact thyroid tissue

Summary Cytochemical localization of 3,5-cyclic nucleotide phosphodiesterase (cPDEase) has been investigated by light and electron microscopy in dissociated bovine thyroid cells and in intact bovine thyroid tissue. By light microscopy in isolated thyroid cells reaction product deposition associated with cPDEase activity was localized at the level of the plasma membrane. In intact cryostat cut thyroid tissue, the activity was primarily observed in the cytoplasm and to a lesser extent at the level of the plasma membrane. By electron microscopy, cPDEase activity in isolated cells was found on the plasma membrane and was also encountered on the inner surface of membrane bound vacuoles, presumably pinocytic in origin. In intact tissue, cPDEase activity appeared mostly localized on the apical and lateral plasma membranes and was also present on the outer surface of the endoplasmic reticulum (ER).Even though cPDEase and 5-AMPase did share the same plasma membrane localization, the inhibitory response to theophylline and stimulation with Imidazole permitted the dissociation of their respective activities. 5-AMPase failed to respond to either theophylline or Imidazole suggesting absence of cross reactivity between 5-AMP and cyclic AMP. Thyrotropin (TSH) had no effect on cPDEase activity.We conclude that: (1) regardless of the nature of the material used, the cytomembranes of thyroid cells possess cPDEase activity; and that (2) the variability in distribution as well as in staining intensity recorded by light and electron microscopy between isolated thyroid cells and cryostat cut thyroid tissue is probably inherent to the methodology used.This paper was presented, in part, at the 60th Annual Meeting of the International Academy of Pathology, Montreal, Canada, March 1971 and was initiated in the Department of Pathology, Rhode Island Hospital, Providence, R.I., supported from a Grant-in-Aid of the American Cancer Society, Rhode Island Division, Inc. and the Brown-Hazen Fund.     

Simultaneous inhibition of guinea pig brain 2′, 3′-cyclic nucleotide 3′-phosphohydrolase and myelin protein synthesis by 2′-adenosine monophosphate

Although the function of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) in myelin is unknown, the enzyme has been implicated in the metabolism of myelin proteins. Using 2′-AMP to inhibit CNPase, we examined the effect of reduced enzyme activity on the $\text{in vitro}$ incorporation of ¹⁴C-leucine into brain proteins. The results of this study revealed that (1) guinea pig brain homogenates incorporate leucine into protein from a sucrose medium in a linear fashion, (2) all brain fractions (cytosol, myelin, and microsomes) are labelled within 1 hr, (3) 2′-AMP inhibition of CNPase by 50% results in a similar inhibition of brain protein synthesis, and (4) the reduced protein synthesis is accompanied by a shift in label from myelin proteins to those found in the microsomes. These results are consistent with a role for CNPase in myelin protein synthesis.     

Immunohistochemical localization of thymosin β9 in bovine tissues

Summary Using an indirect fluorescent antibody technique with frozen sections, the localization of thymosin ₉ was investigated for the first time in bovine thymus, spleen, lung, muscle and liver. The antibodies used have been raised against the N-terminal fragment 1–14 of thymosin ₉ in order to minimize the cross-reactivity with thymosin ₄ which was found to be also present in bovine tissues. The specific antibodies against thymosin ₉ raised in our laboratory allowed us to localize this peptide in presence of the highly homologous and always accompanying thymosin ₄ in different tissues. Although thymosin ₉ was first isolated from calf thymus, it could be also detected in other bovine organs. The highest density of positive immunoreaction was found to be in spleen sections. In the muscle tissue a pronounced fluorescence intensity was present in the region of the sarcolemn.     

2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) activity in cultured nerve cell lines from central nervous system: Comparison of proliferating and resting growth states and cell cycle-dependent activity changes

1. The present communication is concerned with the expression and cell cycle-dependent regulation of the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in cultured nerve cell lines derived from the rat central nervous system (CNS). 2. The enzyme activity was measured in relation to two reversible serum-controlled growth states (exponentially growing/quiescent) including a comparison of the enzyme activities in cell lines of neuronal and glial origin as well as in fibroblasts. CNPase is present in all cell types tested, but the enzyme activity is very sensitive to changes in the cellular growth state. Nerve cell lines in exponentially growing cultures express a 3 to 15 times higher specific CNPase activity than the nonneural cell types. In serum-starved quiescent cultures, the differences in specific enzyme activity between the nerve cell lines and the fibroblasts are enlarged even more up to a ratio of about 50 to 150, indicating a specific function of this enzyme within the central nervous system. 3. Neuron-like B104 cells could be stimulated to synchronized growth by serum readdition to quiescent cultures. A series of ordered activity changes of CNPase has been observed after the reinitiation of cell growth. The enzyme is stimulated at two particular stages during the cell cycle, leading to a biphasic activity profile. Maximum stimulation of CNPase correlates with the G1 phase. 4. Hydroxyurea-induced blockage of synchronized B104 cells to traverse the S phase also prevents the subsequent stimulation of CNPase activity. Therefore, we conclude that a correlation exists between the periodic activity changes of CNPase and particular phases of the B104 cell cycle.     

Syn- and anti-conformations of 5′-deoxy- and 5′-O-methyl-uridine 2′,3′-cyclic monophosphate

Two uridine 2′,3′-cyclic monophosphate (cUMP) derivatives, 5′-deoxy (DcUMP) and 5′-O-methyl (McUMP), were studied by means of quantum chemical methods. Aqueous solvent effects were estimated based on the isodensity-surface polarized-continuum model (IPCM). Gas phase calculations revealed only slight energy differences between the syn- and anti-conformers of both compounds: the relative energies of the syn-structure are −0.9 and 0.2 kcal mol^-1 for DcUMP and McUMP, respectively. According to the results from the IPCM calculations, however, both syn-conformers become about 14 kcal mol^-1 more stable in aqueous solution than their corresponding anti-structures. Additionally, the effects of a countercation and protonation on DcUMP were studied, revealing that the syn-structure is also favored over the anti-one for these systems.     

Partial purification and properties of a multifunctional 3′,5′-cyclic nucleotide phosphodiesterase from Lactuca cotyledons

The extraction, partial purification and properties of a 3′,5′-cyclic nucleotide phosphodiesterase from lettuce cotyledons is described. Purification involved fractional precipation with (NH₄)₂SO₄, chromatography on Sephadex G-200, affinity chromatography on Affi-Gel Blue and non-denaturing polyacrylamide gel electrophoresis. The behaviour of the final enzyme preparation on SDS-polyacrylamide gel electrophoresis was examined and inidcated an M, of ca 62 000. The enzyme from 3′,5′-cyclic nucleotide phosphodiesterases previously isolated from plant tissues in that it exhibits activity towards pyrimidine as well as purine cyclic nucleotides. Furthermore, it hydrolyses cyclic CMP at a comparable rate to that with which it hydrolyses cyclic AMP and cyclic GMP. Both 3′- and 5′-AMP were released, with the 5′-nucleotide being the major product. Whereas the K_m with all three substrates remained constant during the purification procedure, V_max with cyclic AMP was lower than that for cyclic CMP but increased as purification proceeded. The effects were examined of a range of di- and trivalent metal ions on the enzyme activity. Fe³⁺ significantly stimulated the activity, more so when cyclic GMP was the substrate. Cu²⁺ inhibited the activity.     

Geometry of the dry-state oligomerization of 2′,3′-cyclic phosphates

Summary Evaporation of a solution of thymidine plus either theexo or theendo diastereomer of uridine cyclic 2,3-O, O-phosphorothioate (U > p(S) in 1,2-diaminoethane hydrochloride buffer gave the 2,5 and 3,5 isomers of (P-thio) uridylylthymidine (Up(S)dT) in a ratio of 1:2 with a combined yield of about 20%. These isomers were re-converted to U > p(S) and dT by a reaction that is known to proceed by an in-line mechanism. Both the 2,5 and 3,5 isomers gave as product the same diastereomer of U > p(S) that had been used originally in their formation. These dry-state prebiotic reactions (Verlander, Lohrmann, and Orgel 1973) are thus shown to be stereospecific, and both the 2,5 and 3,5 internucleotide bonds are formed by an in-line mechanism.Abbreviations DAE 1,2-diaminoethane - HPLC high pressure liquid chromatography - RNase bovine pancreatic ribonuclease A, EC 3.1.4.22 - TEAB triethylammonium bicarbonate - tris tris(hydroxymethyl)aminomethane - UMP(S) uridine monophosphorothioate - U > p uridine cyclic 2,3-phosphate - U > p(S) uridine cyclic 2,3-O, O-phosphorothioate - Up(S)dT (P-thio)uridylylthymidine - U2p(R_p-S)5dT (P-thio)uridylylthymidine with theR configuration at phosphorous, and a 2,5 internucleotide linkage     

Structure and mechanism of E. coli RNA 2′,3′-cyclic phosphodiesterase

2H (two-histidine) phosphoesterase enzymes are distributed widely in all domains of life and are implicated in diverse RNA and nucleotide transactions, including the transesterification and hydrolysis of cyclic phosphates. Here we report a biochemical and structural characterization of the Escherichia coli 2H protein YapD, which was identified originally as a reversible transesterifying “nuclease/ligase” at RNA 2′,5′-phosphodiesters. We find that YapD is an “end healing” cyclic phosphodiesterase (CPDase) enzyme that hydrolyzes an _HORNA>p substrate with a 2′,3′-cyclic phosphodiester to a _HORNAp product with a 2′-phosphomonoester terminus, without concomitant end joining. Thus we rename this enzyme ThpR (two-histidine 2′,3′-cyclic phosphodiesterase acting on RNA). The 2.0 Å crystal structure of ThpR in a product complex with 2′-AMP highlights the roles of extended histidine-containing motifs ⁴³HxTxxF⁴⁸ and ¹²⁵HxTxxR¹³⁰ in the CPDase reaction. His43-Nε makes a hydrogen bond with the ribose O3′ leaving group, thereby implicating His43 as a general acid catalyst. His125-Nε coordinates the O1P oxygen of the AMP 2′-phosphate (inferred from geometry to derive from the attacking water nucleophile), pointing to His125 as a general base catalyst. Arg130 makes bidentate contact with the AMP 2′-phosphate, suggesting a role in transition-state stabilization. Consistent with these inferences, changing His43, His125, or Arg130 to alanine effaced the CPDase activity of ThpR. Phe48 makes a π–π stack on the adenine nucleobase. Mutating Phe28 to alanine slowed the CPDase by an order of magnitude. The tertiary structure and extended active site motifs of ThpR are conserved in a subfamily of bacterial and archaeal 2H enzymes.     

2′,3′-Cyclic nucleotide 3′-phosphohydrolase in rat liver mitochondrial membranes

2′,3′-Cyclic nucleotide 3′-phosphohydrolase (nucleoside-2′:3′-cyclic-phosphate 2′-nucleotidohydrolase, EC 3.1.4.37) activity has been demonstrated in rat liver mitochondria. The enzyme was localized in both the outer and inner mitochondrial membranes but was absent from the intermembrane space and matrix. The mitochondrial (cyclic nucleotide) phosphohydrolase was activated by freezing and thawing and by treatment with digitonin or detergents. It is suggested that (cyclic nucleotide) phosphohydrolase is an integral membrane protein which is buried to a significant degree within the membrane. Atractyloside was found to be a noncompetitive inhibitor of the enzyme both in intact mitochondria and in preparations of the mitochondrial membranes. The enzyme substrate, 2′,3′-cyclic adenosine monophosphate, had no effect on the oxidation of exogenous β-hydroxybutyrate or succinate by intact mitochondria. These findings suggest that 2′,3′-cyclic nucleotide 3′phosphohydrolase is more widely distributed than was previously thought and that the enzyme may play a fundamental role in membranes, independent of their specialized structure or functions.