Purification of Rat 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP, EC 3.1.4.37) has been isolated from rat brain myelin by chromatography on successive columns of phenyl-Sepharose CL-4B, CM-Sepharose CL-6B, and 8-(6-aminohexyl) amino-2'AMP-Sepharose 4B. From 15 g of rat brain, approximately 400 micrograms of pure CNP was obtained, with a specific activity of 1,200 (2',3'-cyclic AMP) units/mg protein. The Km of the rat enzyme was 3.7 mM, using 2',3'-cAMP as the substrate. Isoelectric focusing of the enzyme indicated a broad isoelectric range of 8.5-9.0. On SDS polyacrylamide gels, rat CNP appears as two protein bands of approximately 48,000 and 50,000 M.W., with an upper band intensity of about 1/10 that of the lower band. The relative intensities of the bands for CNP and the molecular weights correspond to the Wolfgram proteins W1 and W2 described by other investigators. The amino acid analysis of the purified rat enzyme compared favorably with reported determinations for the bovine enzyme and also with reported values for the rat Wolfgram proteins W1 and W2.     

Susceptibility of the Wolfgram Proteins and Stability of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase of Rat Brain Myelin to Limited Proteolytic Digestion

The susceptibility of proteins in the myelin membrane to proteases was studied. Lyophilized rat brain myelin suspended in water was subjected to controlled proteolytic digestion with pure trypsin (N-tosyl-L-phenylalanine chloromethyl ketone treated, 5 units/mg of myelin), and proteins remaining in the pellet were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under these conditions, large basic protein (LBP) was completely hydrolyzed in 5-10 min, proteolipid proteins remained largely intact until 60 min, whereas Wolfgram protein (WP) was progressively degraded from 10 min onward with the simultaneous appearance of a new protein band with a molecular weight of 35K. A similar pattern was obtained on treatment with chymotrypsin or subtilisin. The 35K protein band was shown to be derived from WP by its immunological cross-reactivity with WP antibodies. Western blot analysis showed that 35K protein is the only major breakdown product of WP under these conditions. Treatment with higher concentrations of trypsin (greater than 20 units/mg of myelin) resulted in the degradation of all myelin proteins. Essentially all the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) activity was observed in the myelin pellet after controlled or drastic digestion with trypsin. It is concluded that the major fragment of WP (35K) is located in the hydrophobic milieu of the bilayer, relatively inaccessible to trypsin, whereas a portion (20K) of the WP is exposed to the cytoplasmic side (major dense line), like LBP, and that peptide fragments (less than 14K) that remained in the myelin membrane lipid bilayer after trypsin digestion could exhibit CNP activity.     

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.     

Cyclic AMP Induction of the Myelin Enzyme 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase in Rat Oligodendrocytes

Cyclic AMP (cAMP) is known to induce the activity of the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37) in C6 rat glioma cells. This report shows that CNP is also inducible in oligodendrocytes explanted from 1-day-old rat cerebrum and grown in tissue culture. Induction was observed after a 1-day treatment with 1 mM N6, O2-dibutyryl cyclic AMP (dbcAMP) and was maximal after 5 days, reaching 200-240% of control. Induction was observed both in mixed cerebral cell cultures containing oligodendrocytes and astrocytes, and in purified cultures of oligodendrocytes prepared by a differential shakeoff procedure. Addition of dbcAMP to the cultures 3-9 days after the cells were explanted from rat brain induced CNP activity, but no induction was observed when dbcAMP treatment was begun 13 or more days after explanation. These results demonstrate that one component of myelin, CNP, is inducible in oligodendrocytes by a cAMP-mediated mechanism, and suggest a role for cAMP in the regulation of the myelin-associated functions of oligodendrocytes.     

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.     

Biosynthesis of the Myelin 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterases

We have investigated the site of synthesis of the 2',3'-cyclic nucleotide 3'-phosphodiesterases (CNPs I and II) in rat brain. Rapid kinetics of incorporation of CNPs into oligodendrocyte plasma membrane in the intact brain are consistent with their synthesis on free polysomes. This hypothesis was confirmed by the translation in vitro of RNA isolated from free and bound polysomes, respectively. Unlike myelin basic protein (MBP) mRNAs, CNP mRNAs are not enriched in a myelin-associated pool of RNA. MBPs, but not CNPs, were found to readily associate in vitro with membrane vesicles derived from rough endoplasmic reticulum. The avidity of MBPs in binding to membranes is probably related to the previously observed spatial segregation of MBP mRNAs into actively myelinating cellular processes of the oligodendrocyte. Such a segregation would ensure that newly synthesized MBPs are immediately incorporated into myelin. In contrast, the CNPs probably associate with the cytoplasmic surface of the oligodendrocyte plasma membrane through interaction with a membrane-bound receptor.     

2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase and 5''-Nucleotidase in the Central Nervous System of a Myelin-Deficient Rat Mutant

2',3'-Cyclic nucleotide 3'-phosphodiesterase activity was examined in brains and spinal cords of normal and myelin-deficient Wistar rats. While the activity in normal brains increased from 0.2 mumol/min/mg protein (units) at 6-10 days to 3.5 units at 25 days of postnatal age, the activity in the myelin-deficient rat remained at 0.2-0.3 units over the same period. In spinal cord, the normal activities were 5.7 and 10.9 units at 12 and 20 days, respectively, whereas they declined in the myelin-deficient rat from 1.06 to 0.79 units for the same age points. 5'-Nucleotidase activities in brain and spinal cord were normal in the myelin deficient rat at both ages.     

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.     

Induction of Myelin Components: Cyclic AMP Increases the Synthesis Rate of 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase in C6 Glioma Cells

In an effort to determine the factors that stimulate myelin synthesis, we investigated the mechanism by which dibutyryl cyclic AMP induces the activity of the myelin enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37), in C6 glioma cells. Immunotitration experiments and measurements of the accumulation of [35S]methionine-labeled CNP showed that dibutyryl cyclic AMP increased the amount of CNP in the cells but not the catalytic activity per molecule of the enzyme. Moreover, inhibition of protein synthesis with cycloheximide abolished induction of enzyme activity. Dibutyryl cyclic AMP doubled the rate of CNP synthesis but had no effect on the half-life of the enzyme (approximately 33 h). The induction was partially blocked by the inhibitors of mRNA synthesis, cordycepin or alpha-amanitin. Thus, cyclic AMP induces the synthesis of CNP.     

The Activity of 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase in Rat Tissues

Abstract: The activity of the myelin-associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) was measured in 14 rat tissues and in subcellular fractions of rat liver by a sensitive fluorometric method, using cyclic NADP as substrate. CNP activity in brain (339 μmol/h/mg protein) was fourfold that of the sciatic nerve. The activities in tissues outside the nervous system ranged from a low of 0.42 μmol/h/mg protein in the unwashed red blood cell to a high of 9.96 in the spleen. The activity was highest in tissues containing cells with membranes capable of undergoing transformation and elaboration (spleen and thymus) and low in those in which the cell membranes are morphologically stable (muscle and red cell). The enzyme was found in all major liver subtractions, with the highest activities in the microsomal and nuclear fractions. Despite the large difference in the maximal velocities of CNP in brain and liver, the affinity of the liver enzyme for the substrate ( k _m) was similar to that of brain enzyme. Brain CNP was stable over a 48-h postmortem period.     

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.     

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.     

Immunohistochemical Localization of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase and Myelin Basic Protein in the Chick Retina

A GTP-dependent regulatory component of adenylate cyclase was found in myelin from rat brain. The fraction solubilized from myelin contained a component that reconstituted guanine nucleotide-responsive adenylate cyclase activity when combined with the catalytic unit of adenylate cyclase prepared from rat brain. Purified myelin demonstrated little adenylate cyclase activity, even in the presence of F- or Mn2+. The reconstituted activity was dependent on the amount of the solubilized myelin fraction and required the presence of 5'-guanylylimidodiphosphate, a hydrolysis-resistant analog of GTP. The elution pattern of the component solubilized from myelin in gel filtration was very similar to that of a GTP-dependent regulatory component from synaptic plasma membranes. The content of the regulatory component-like activity in myelin was estimated to be 50-60% of that in synaptic plasma membranes. Cholera toxin ADP-ribosylated proteins having molecular weights of 48,000, 38,000, 23,000, 20,000, and 15,000 and other minor peptides in myelin, some of which were also present in synaptic plasma membranes. We conclude that myelin contained a GTP-dependent regulatory component of adenylate cyclase despite the apparent lack of adenylate cyclase activity in myelin.     

A Monoclonal Antibody Raised to Corpus Callosum Extract Reacts with 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase

Monoclonal antibody against 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with delipidated white matter from rat corpus callosum. The antibody was characterized by solid-phase radioimmunoassay, immunoblot of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoprecipitation from C6 glioma cells, and indirect immunofluorescence staining of monolayer cultures containing oligodendrocytes. The monoclonal antibody bound specifically to an intracellular antigen of oligodendrocytes, but not to Schwann cells, astrocytes, neurons, or fibroblast cytoplasm. The immunoblot of SDS-PAGE of CNS myelin showed that the antibody identified two protein bands at 48,000 and 50,000 molecular weight. These proteins were not identified in peripheral nervous system myelin. The monoclonal antibody immunoprecipitated CNP enzyme activity from extracts of C6 glioma cells. This monoclonal antibody should prove useful in further study of this myelin-specific enzyme in CNS myelin and in cells responsible for myelin production.     

Evidence for the Association of 2',3'-Cyclic-Nucleotide 3'-Phosphodiesterase with Myelin-Related Membranes in Peripheral Nervous System

Abstract: In PNS, the specific activity of 2′,3′-cyclic nucleotide 3′-phospho–diesterase (CNP) in myelin was not enriched over the starting homogenate. Nevertheless, most of the total activity was recovered in myelin. In myelin-deficient mutants, low CNP activities were measured in sciatic nerves. CNP specific activities were similar in myelinated and non-myelinated nerves but in non-nervous tissues, they were significantly lower than in nervous tissue. There was no indication for the presence of an isoenzyme of CNP in peripheral nerves. These results indicate that CNP is present in PNS myelin and preferentially localized in Schwann cell plasma membranes.     

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.     

Correlation Between 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase Activity and Demyelination In Vitro Using a Syngeneic System

Cultures of myelinated SJL/J fetal mouse spinal cord were incubated with serum and lymphoid cells from syngeneic animals with experimental allergic encephalomyelitis (EAE) induced by syngeneic spinal cord homogenate (SSCH) in complete Freund's adjuvant or others injected with complete Freund's adjuvant alone. After 24 or 48 h of exposure, demyelination was determined by light microscopic examination and quantification of 2',3'-cyclic nucleotide 3'-phosphohydrolase activity. Cultures exposed to spleen or lymph node cells from SSCH-sensitized animals showed the greatest alterations in myelin and decreases in 2',3'-cyclic nucleotide 3'-phosphohydrolase activity whereas serum from these animals had less effect. Cells and serum from complete Freund's adjuvant-injected control animals also induced structural changes in myelin that were significantly less than changes induced by cells and serum from animals with EAE. These experiments show that lymphoid cells and serum obtained from SJL/J mice with acute EAE affected myelin biochemistry and morphology in syngeneic CNS cultures.     

Myelin Proteins, Glycoproteins, and Myelin-Related Enzymes in Experimental Demyelination of the Rabbit Optic Nerve: Sequence of Events

Wallerian degeneration of the rabbit optic nerve was investigated by the technique of retinal ablation which precludes edema, hemorrhage, or macrophage infiltration. After 8 days of degeneration, marked degradation of axons and some myelin abnormalities appeared in the optic nerve, optic chiasma, and optic tract. Myelin lesions were maximal 32 days after retinal destruction. The amount of material stained with a myelin dye decreased drastically between 32 and 90 days after the operation. Biochemical parameters gave the following sequence of events. The concentration of the major periodic acid--Schiff staining glycoproteins was decreased after 2 days, and 6 days later the presence of cholesterol esters was detected in the optic tissue. After 16 days of Wallerian degeneration, the specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase not associated with myelin decreased, indicating a possible de-differentiation of oligodendrocytes. Degradation of myelin basic protein became significant at 32 days and the amount of myelin isolated decreased later. The loss of myelin basic protein coincided with a reduction of myelin periodicity as measured in purified fractions by electron microscopy. These results show that secondary myelin destruction in the absence of edema, hemorrhage, or macrophages is a very slow process, and in this situation myelin undergoes a selective and sequential loss of its constituents.