2 ’ , 3 ’-Cyclic Nucleotide-3 ’-phosphodiesterase in the Central Nervous System Is Fatty-acylated by Thioester Linkage *

2’,3’-Cyclic nucleotide-3’-phosphodiesterase (CNPI and CNPz with M, of 46,000 and 48,000, respectively) is the major enzyme of central nervous system myelin. It is associated with oligodendroglial plasma membrane and uncompacted myelin (myelin-like fraction), which are in contact with glial cytoplasm. Proteins of the myelin-like fraction were labeled with [3H]palmitic acid in brain slices from 17-day-old rats and immunoprecipitated with anti-CNP antiserum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material revealed intense acylation of CNPl and CNP2, and radioactivity was released by hydroxylamine. Palmitic acid was covalently bound to CNP because radioactivity was not removed by extraction of immunoprecipitated CNP with organic solvent or by boiling in sodium dodecyl sulfate and dithiothreitol. However, treatment of immunoprecipitated CNP with (a) hydroxylamine-released palmitohydroxamate and palmitic acid, (b) sodium borohydride-released hexadecanol, and (c) methanolic-KOH-released methyl palmitate. Synthesis, acylation, or transport of CNP was not affected by monensin or colchicine. However, acylation of CNP was inhibited 24-32s by cycloheximide. These results provide conclusive evidence that CNPl and CNPz are fatty acid acylated with palmitate through a thioester linkage and is posttranslationally modified sometime after synthesis.

and coworkers (4, 5) isolated a myelin-like fraction from developing brain and demonstrated that this fraction (a) floated at a higher density of sucrose than myelin, (b) contained predominantly single vesicles, instead of compact myelin, and (c) was almost devoid of cerebroside and was highly enriched in CNP. These membrane and the compact myelin sheath in the CNS" (5). Subsequently, these results were confirmed (reviewed in Ref. 6). In some preparations, the specific activity of CNP in the myelin-like membranes was greater than in myelin (7, 8). Recent immunohistochemical studies at electron microscopic level provided conclusive evidence that CNP was localized to single membranes, loosely wrapped glial membranes, and structures enriched in glial cell cytoplasm (9). Purified enzyme from rat brain migrates with an A4, of 46,000 (CNP,) and 48,000 (CNPJ on SDS-PAGE and the ratio of CNPl to CNPB is about 1O:l (10, 11). The complete amino acid sequences of CNP in four species have been deduced from their cDNA sequences (12-16). Results of cDNA sequencing suggest that CNP is synthesized from a single gene and the two forms of CNP may result from alternative splicing of mRNA (12) and/ or posttranslational modifications (2, 3, 12). We recently provided the first direct immunochemical evidence that both CNPl and CNP, in the peripheral nervous system undergo phosphorylation.' In this communication, we report that CNP in the myelin-like fraction is fatty acylated and the fatty acids appear to be linked to this enzyme by thioester linkage. ml each of KRB buffer containing 1% fatty acid free bovine serum albumin and pelleted at 1,500 x g, 4 "C. All operations were carried out at O-4 "C. Brain slices were homogenized in 0.88 M sucrose (5%, w/v). Aliquots were removed to determine protein concentration, incorporation of "C-amino acids into total proteins (20), and for immunoprecipitation. The remaining homogenate was overlayered with 0.32 M sucrose and centrifuged at 93,000 X g for 1 h at 4 "C (21). Myelin floating at the interface was collected, homogenized, diluted with ice-cold water, and pelleted at 44,000 X g for 30 min. Pellets were homogenized in ice-cold water, incubated in an ice chest for 30 min, and then centrifuged at 12,000 X g for 10 min (5). The turbid supernatant containing the myelin-like fraction was collected and centrifuged at 93,000 x g for 1 h. The pellets were homogenized again in water and the myelin-like fraction was sedimented by centrifugation at 93,000 x g for 1 h. Myelin was further purified by recycling on sucrose density gradient and washed with water as described (21). Both myelin and myelin-like fractions were each suspended in water, protein concentrations were determined (22), and aliquots were stored at -80 "C to measure CNP activity (23) and for immunoprecipitation. The specific activity of CNP is expressed as micromoles of 2'-AMP produced per mg protein/h. Results are the mean of three separate experiments + S.D. also treated with hydroxylamine as described above and then examined by SDS-PAGE and fluorography.

Reagent
Immunoprecipitation and SDS-PAGE-For immunoprecipitation, SDS was added to brain homogenates (700 fig of proteins) or myelinlike fraction (100 pg of proteins) to a final concentration of 1% and the samples were boiled for 5 min. Four to six volumes of immunoprecipitation buffer (50 mM Tris/HCl, pH 8, 150 mM NaCl, 10 mM EDTA, 0.5 mM dithiothreitol, and 1% Triton X-100), 5 ~1 of Trasylol (21,000 units), and polyclonal or monoclonal antibodies to CNP were added and samples were incubated for 18 h at 4°C with gentle rotation. When monoclonal antibodies to CNP were used, the Staph A was first coated with rabbit anti-mouse IgG (ICN Radiochemicals). The immunoprecipitation was performed as described (24) using 100 ~1 of Staph A. Precipitated proteins were eluted from the washed Staph A pellet by boiling for 5 min in 40 ~1 of sample buffer (125 mM Tris/HCI, pH 6.8, 2.5% SDS, 3% dithiothreitol, 10% glycerol, and 0.01% bromphenol blue) and analyzed on 12% polyacrylamide-SDS gels (25). Proteins were fixed by immersion of gels for 2 h in 10% trichloroacetic acid. Gels were then stained, destained, and prepared for fluorography (26). Stained gels were treated with 1 M hydroxylamine, pH 10, for 18 h (27) and then fluorographed. Absolute amount of radioactivity was determined by liquid scintillation spectrometry. Methanolic-KOH-Myelin-like fractions were immunoprecipitated and each immunoprecipitate was eluted by boiling for 5 min in 80 ~1 of sample buffer. Eluates were divided into 3 aliquots (640 ~1 each) after adding fatty acid-free bovine serum albumin (2 rg/pl). Each aliquot was successively extracted 3 times with 30 volumes of chloroform/methanol (2:1, v/v) and one time each with chloroform/ methanol (1:2), chloroform/methanol/H20 (1:1:0.3, v/v/v) and acetone (28), and then dried under N2. Furthermore, extraction of the protein pellet with organic solvents did not release any radioactivity, indicating that noncovalently bound lipids were removed by this procedure. The lipid specie linked to CNP was identified by treating with (i) 0.2 N KOH in methanol for 18 h at 25 "C, (ii) with 1 M hydroxylamine, pH 9.8 (29), or (iii) with sodium borohydride in 30% tetrahydrofuran for 40 min at 37 "C (30). Palmitic acid, methyl palmitate, and hexadecanol were applied to silica gel plates along with authentic [3H]palmitic acid, [3H]methyl palmitate, and ['"Cl hexadecanol. The TLC plate was developed in benzene/ether/acetic acid (90:10:2, v/v/v), and the chromatogram was sprayed with EN3HANCE (Du Pont-New England Nuclear) and then fluorographed. Palmitic acid and palmitohydroxamate were separated by using toluene/acetic acid/water (80:20:1, by volume) as described (31). The plates were sprayed with 2',7'-dichlorofluorescein to reveal the spot under long wavelength UV light (27). The plates were scraped into l-cm sections and radioactivity in each section was determined by scintillation spectrometry in 10 ml of 3a70. Palmitohydroxamate was synthesized as described (32). After immunoprecipitation, Staph A pellet were treated with 1 M hydroxylamine, pH 10, for 4 h at room temperature. After washing the Staph A with phosphate-buffered saline (0.15 M NaCl, 0.01 M phosphate buffer, pH 7.4) CNP was eluted, analyzed by SDS-PAGE, transferred to nitrocellulose sheets and immunostained with a monoclonal antibody to CNP (33). Likewise, immunopreripitated CNP from radiolabeled brain proteins was RESULTS

AND DISCUSSION
The specific activity of CNP in the myelin-like fraction (2960 f 34) was 3.8-fold higher than myelin (764 + 92) isolated from pons-medulla of 17-day-old rats. These results were further confirmed by immunostaining CNP from myelin and the myelin-like fraction by the immunoblot technique (33). The amount of CNP present in the myelin-like fraction is at least 3-4-fold higher than in purified myelin (data not shown). These findings are consistent with the immunohistochemical localization of CNP to the oligodendroglial plasma membrane and noncompacted regions of myelin membrane (9).
To characterize the fatty acid acylation of CNP, brain slices were incubated with ["Hlpalmitic acid, and the myelin-like fraction was prepared. CNP was isolated by immunoprecipitation from the myelin-like fraction labeled with ["Hlpalmitic acid with polyclonal antisera to CNP, and on fluorography, CNP, and CNPz were acylated (Fig. 1, lane 2). Analysis of 2mm gel sections containing immunoprecipitated CNP with a monoclonal antibody also revealed a solitary peak of radioactivity (Fig. 2). Fatty acids were removed by treatment of immunoprecipitated CNP in the gel with 1 M hydroxylamine, pH 10 (Fig. 1, lane 3). These results provided strong evidence that fatty acids were tightly bound to CNP.
Treatment of immunoprecipitated CNP with 1 M hydroxylamine did not result in hydrolysis of the protein as determined by immunostaining of CNP (Fig. 3A, lane 2) and by the retention of radioactivity associated with CNP (Fig. 3B, lane 2). However, it is apparent from Fig. 3B, lane 2, that there was a slight reduction of radioactivity from CNP labeled with Y-amino acid mixture by hydroxylamine when compared to control (Fig. 3B, lane I). The decrease is in all probability due to metabolic conversion of some W-amino acids into fatty acids which were removed by hydroxylamine. In addition, 4,17,40,76, 78, and 80% of the radioactivity was released after treatment of CNP in the gel section by hydroxylamine between pH 5-10, respectively. Immunoprecipitated CNP was exhaustively delipidated. The delipidated enzyme   to CNP. Fatty acylated CNP was immunoprecipitated from the myelin-like fraction, analyzed by SDS-PAGE, and radioactivity associated with 2-mm gel section was determined as described in the text. A, myelinlike fraction (20 pg of protein) was prepared from the pons-medulla of 17-day-old rats. CNP was immunoprecipitated with a polyclonal anti-CNP antiserum. The Staph A pellets were incubated with 500 ~1 of freshly prepared 1 M hydroxylamine, pH 10, for 4 h with gentle rotation at 23 "C. The Staph A was pelleted in a Microfuge. The pellets were washed 3 times with phosphate-buffered saline, eluted with Laemmli sample buffer, separated by SDS-PAGE, transferred t.o NCS, and immunostained with a monoclonal antibodv to CNP. Lanes 1 .and 2 are control CNP and CNP treated with 1 ti hydroxylamine, pH 10, respectively. B, brain slices were incubated in KBB buffer containing 200 &i of 'C-amino acid mixture for 2.5 h at 37 "C. Brain homogenate (700 ~g of protein) was immunoprecipitated, treated with hydruxylamine, and then examined by SDS-PAGE and fluorography. Lanes 1 and 2 are control CNP and CNP treated with 1 M hydroxylamine, pH 10, respectively. was then treated with 1 M hydroxylamine, pH 10, for varying periods of time at 23 "C to determine the kinetics of release of fatty acid. Lipids were extracted with chloroform/methanol and the radioactivity was determined. Forty percent of radioactivity was released from CNP in 50 min after treatment with hydroxylamine (Fig. 4) Myelin-like fraction was labeled with ["Hlpalmitic acid in brain slices. CNP was immunoprecipitated, eluted with the sample buffer, and then exhaustively extracted with organic solvents (see the text). CNP adhering to glass surface was treated with 3 ml of 1 M hydroxylamine, pH 10, for varying periods of time at 23 "C. Palmitohydroxamate and palmitic acid were extracted with chloroform/ methanol (2:1, v/v). The organic phase was dried under Nf and the radioactivity was determined. Forty percent of radioactivity was released from CNP after 50 min. The results are the mean of two experiments at each time point.
The release of fatty acid from CNP by hydroxylamine suggests that ["Hlpalmitic acid may be conjugated to CNP by thioester and not by O-ester linkages (34-36).
To demonstrate directly the fatty acid bound to immunoprecipitated CNP and the nature of the protein-fatty acid bond, immunoprecipitated CNP was exhaustively extracted with organic solvents to remove noncovalently attached lipids as described above and then digested with 1 M hydroxylamine, pH 9.8 (29), sodium borohydride (30), or methanolic-KOH (37). The extracted radioactive materials were analyzed by TLC. The released radioactivity (85-95%) was associated with palmitohydroxamate and palmitic acid after treatment of CNP with hydroxylamine (Fig. 5A). The presence of free palmitic acid may result from hydrolysis of covalently bound palmitate under the alkaline conditions used (31).
CNP was also cleaved with sodium borohydride, which simultaneously hydrolyzes acylthioesters and reduces the acyl group to an alcohol (30, 34). The released radioactivity (83%) was extracted with toluene and all the radioactivity co-chromatographed with authentic hexadecanol (Fig. 5B, lane 2). These results further suggests thioester linkage of palmitic acid with cysteine residues of CNP. Sodium borohydride cleaves fatty acids linked by thioester bond to cysteine in proteins (34,38,39). Finally, nearly all the radioactivity released from CNP after alkaline hydrolysis (97%) was identified as methyl palmitate (Fig. 5B, lane 5).
CNP is probably synthesized on free ribosomes and newly synthesized enzyme is rapidly incorporated into the glial cell plasma membrane (40, 41). Brain slices were incubated with YZ-amino acids in the presence or absence of colchicine and monensin to determine if the synthesis or transport of CNP was affected. Incorporation of label into CNP, or CNP, was not impaired by colchicine (Fig. 6, lane 2) or by monensin (Fig. 6, lane 3) suggesting that neither microtubule nor Golgi complex-mediated (40) transport of the newly synthesized enzyme occurred. Likewise, neither the incorporation of ["HI palmitic acid into CNP immunoprecipitated from the brain homogenate (Fig. 7)  not shown) was affected by colchicine ( Fig. 7, lane 2) or monensin (Fig. 7, lane 3). These results indicated that microtubules or the Golgi complex are not involved in the acylation or the transport of acylated enzyme to myelin membrane. To determine if CNP acylation occurs cotranslationally or posttranslationally, brain slices were incubated with cycloheximide (0.4 mM) for 2.5 h and acylation of CNP determined after immunoprecipitation.
Cycloheximide blocked incorporation of 14C-amino acids into CNP by 98% after 2.5 h, as might be expected (data not shown). However, palmitoylation of CNP in the brain homogenate (Fig. 7, lane 4)  Thus, this is the first reported evidence of the covalent linkage of fatty acid to the major enzyme (CNP) of central nervous system myelin.
The functional significance of CNP in the central nervous system is unknown because the physiological substrate for this enzyme has not been found in the oligodendrocytes or myelin-like membranes. However, the appearance of CNP is an early event during oligodendrocyte differentiation (reviewed in Refs. 2 and 3). This early appearance of CNP is followed by expression of myelin basic proteins and proteolipid proteins, the major structural proteins of central nervous system myelin. In addition, CNP is also associated with the periaxonal region and is excluded from compact myelin, like myelin-associated-glycoprotein (9). These two findings lead us to postulate that in the central nervous system, CNP may be involved in glial-neuron interaction and in the initial stages of myelination.
The exact role of palmitoylation of CNP is unknown. However, linkage of fatty acids to CNP may be necessary for it to interact with specific lipids or proteins within the hilayer or glial cytoplasm. Furthermore, the posttranslational addition of fatty acids to CNP may be a means by which this enzyme acquires an affinity for oligodendroglial plasma membrane. It is also feasible that CNP is synthesized initially as a soluble protein and later becomes associated with membranes after fatty acylation, like pGosrc and pzl viral proteins (48). Alternatively, hydrophobicity conferred by covalent linkage of palmitic acid to CNP might permit it to play specific roles in cell-cell interaction during development of the central nervous system as it has been postulated for fatty acylated proteins of sea urchins during embryogenesis (35). Finally, it is tempting to speculate that acylation and/or phosphorylation of GNP may regulate its activity or facilitate differentiation of glial precursor (progenitor) cells into mature oligodendrocytes.