Val. 258, No. 10, Issue of May 25, pp. 6556-6560, 1983 THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S.A.

In Vivo Incorporation of [3H]Palmitic Acid into PO Protein, the Major Intrinsic Protein of Rat Sciatic Nerve Myelin EVIDENCE FOR COVALENT LINKAGE OF FATTY ACID TO PO*

(Received for publication, November 15, 1982)

Harish C. AgrawalS57, Robert E. SchmidtGII, and Daya AgrawalS From the $Departments of Pediatrics and Neurology and the §Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 632 78

Separation of rat sciatic nerve myelin proteins by sodium dodecyl sulfate-slab gel electrophoresis 18 h after injection of [3H]palmitic acid into the nerve dem- onstrated acylation of the PO protein. When sciatic nerve myelin labeled with [3H]palmitic acid was ex- tracted with acidified chloroform/methanol or chloro- form/methanol, the radioactivity associated with PO was retained. These results provided evidence that the radioactivity derived from [3H]palmitic acid in PO pro- tein was not due to labeled phospholipid bound to PO protein by strong physical interaction. Futhermore, the radioactivity associated with purified preparations of PO remained after dialysis and re-electrophoresis, providing additional evidence that [3H]palmitic acid was firmly bound to PO. Treatment of myelin proteins with hydroxylamine at pH 6.6 released most of the radioactivity, indicating that [3H]palmitic acid was covalently bound by ester linkage to PO. Cleavage of purified acylated PO with methanolic NaOH released 85% of the protein-bound radioactivity. Gas-liquid chromatography of the fatty acids released from PO showed labeling of methyl esters of palmitate, stearate, and oleate (56, 16, and 570, respectively). In addition, a small unlabeled peak with retention time identical with methyl linoleate was also observed.

Intraneural injection of a mixture of [3H]palmitic acid and [’4C]fucose or [3H]palmitic acid and [35S]su1- fate in vivo followed by disc gel electrophoresis and determination of incorporated radioactivity clearly showed that PO was glycosylated, sulfated, and ac- ylated. The potential significance of fatty acids linked to PO is discussed.

Myelin isolated from the peripheral nervous system of several animal species has been shown to contain five major proteins, PO, Y, X, P1, and P2 (1). PO is an intrinsic protein of peripheral nervous system myelin and accounts for 50-60% of the total membrane protein (1). Considerable post-trans- lational modification of PO has been described including glycosylation (2-8), sulfation (9, IO), and phosphorylation (1 1). In addition, glycosylation (12-16) and phosphorylation of PO (13,17) have been shown in vitro. In the present study,

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* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll Supported by National Science Foundation Grant BNS 78-24793 and National Institutes of Health Grants NS 13464 and NS 19414.

1) Supported by National Institutes of Health Grant AM 19645 and a grant from the Juvenile Diabetes Foundation.

we demonstrate the acylation of PO in vivo following injection of [“Hlpalmitic acid directly into the rat sciatic nerve. Fur- thermore, evidence is presented that palmitate, stearate, oleate, and linoleate are covalently linked by ester bonds in purified preparations of PO. A preliminary report of this work has recently appeared (18).

MATERIALS AND METHODS

Chemicals-All reagents used for SDS’-polyacrylamide gel electro- phoresis were obtained from Eastman Kodak. 2-Methoxy-2,4-di- phenyl-3-(2H)-furanone was obtained from Hoffmann-LaRoche. In- stafluor, Soluene 350, and 2,5-diphenyloxazole were purchased from Packard. Fatty acid-free bovine serum albumin was from Sigma, and methyl esters of fatty acids were from Supelco, Inc., Bellefonte, PA. Omnifluor, ~-[5,6-~H]fucose (56 Ci/mmol), ~-[ l - ’~C]fucose (56.2 mCi/ mmol), [9,10-3H]palmitic acid (11.8 Ci/mmol), and Na;%O, (766 mCi/mmol) were obtained from New England Nuclear. [3H]Palmitic acid (25 mCi) was evaporated to dryness under nitrogen and sus- pended in 3% (w/v) fatty acid-free bovine serum albumin (0.5 ml).

In Vivo Acylation of PO and Isolation of Myelin-All studies of glycosylation, sulfation, and acylation of PO were carried out on 40-50-day-old Sprague-Dawley albino rats. Rats were anesthetized (Nembutol, 33 mg/kg, intraperitoneally), the sciatic nerves were exposed bilaterally in mid-thigh, and 20 p1 of [3H]palmitic acid (1 mCi) or 10 pl of [3H]fucose (100 pCi) were injected into the epineu- rium. The incision was closed and the animals were killed 18 h later. For double labeling experiments, rats were injected with a mixture of [l-’4C]fucose (6 pCi) and [3H]palmitic acid (320 pCi) or a mixture of [’H]palmitic acid (500 pCi) and Na,35S04 (100 pCi) as described above and killed 18 h later. All subsequent operations were carried out at 0-4 “C. The sciatic nerves were removed, and the epineurium was teased away under a dissecting microscope. The nerves were homog- enized in 0.29 M sucrose (18 mg of tissue/ml) using a Polytron (Brinkmann Instruments) homogenizer. A portion of the homogenate (2 ml) was used to determine the incorporation of radioactivity into total nerve protein as described below. Five ml of the homogenate were layered over 11 ml of 0.85 M sucrose and centrifuged a t 93,000 X g for 1 h, and the floating myelin layer was removed. The myelin was osmotically shocked with 30 volumes of water and centrifuged a t 25,000 X g for 15 min. The pellet was resuspended three times in 10 volumes of water, homogenized, and centrifuged a t 18,000 X g for 15 min. Myelin’ was suspended in water and lyophilized. Lyophilized myelin was stored over Drierite a t -80 “C. The results presented in this paper represent the mean of a t least eight separate experiments, except the double labeling experiment using fucose and sulfate which was performed once.

Incorporation of fH/Palrnitic Acid into Total Protein and Chloro- form/Methanol-insoluble Proteins-All operations were carried out a t 4 “C. A portion of the homogenate (2 ml) was mixed vigorously with an equal volume of 20% (v/v) trichloroacetic acid and kept on ice for 1 h. This suspension was centrifuged a t 10,000 X g for 20 min. The precipitate was washed two more times with 10% trichloroacetic acid as described above. The insoluble residue was extracted three times each with diethyl ether/ethanol (l:l, v/v), chloroform/methanol (21,

’ The abbreviation used is: SDS, sodium dodecyl sulfate.

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Covalent Linkage of Fatty Acids to PO Protein 6557

v/v), and acetone and centrifuged as described. Nerves from six rats were pooled, pulverized in liquid N2 (1). extracted three times with chloroform/methanol (2:l v/v) and acetone and centrifuged as de- scribed above. Proteins insoluble in trichloroacetic acid and chloro- form/methanol were dissolved in a solution containing 10 mM borate buffer (pH 9.0). 1% SDS, 1.5% (w/v) dithiothreitol and heated in a boiling water bath for 4 min.

Conjugation of Myelin Proteins with 2-Methoxy-2,4-diphenyl-3- (2H)-furanone and Purification of PO-Lipids were removed from lyophilized myelin (50 mg) by successive extraction with diethyl ether/ethanol (3:2, v/v) and ether (19). Myelin proteins were imme- diately dissolved in 30 mM borate buffer (pH 9.0) containing 3% (w/ v ) SDS at a concentration of 2.5 mg of protein/ml. The protein concentration in all samples was determined according to the method of Lowry et 01. (20) and the radioactivity by liquid scintillation spectrometry. Myelin proteins were separated by semipreparative SDS-disc gel electrophoresis in a Tris borate gel system (21) after coupling with 2-methoxy-2,4-diphenyl-3-(2H)-furanone (22,23). Only 15% of the total myelin proteins was coupled to the fluorescent reagent. The fluorescent bands corresponding to PO were excised and eluted electrophoretically into dialysis tubing until the gels were completely devoid of fluorescence (23). PO was dialyzed against 0.1% SDS containing 0.01% 2-mercaptoethanol for 24 h at room tempera- ture (23) prior to re-electrophoresis (24).

Purified PO was exhaustively dialyzed against 0.001% SDS and then against distilled water to remove most of the SDS before cleavage

STAIN FLUOROGRAM

n

of fatty acid (23). The retentate was centrifuged at 100.000 X g for 30 min at 20 "C. The clear supernatant containing fluorescent protein was lyophilized, and the pellet was discarded.

SDS-Slab and -Disc Gel Electrophoresis-Myelin proteins (94-100 pg) and purified PO (36 pg) were separated on a Tris glycine SDS- gel system (24). Stained gels (23) were infiltrated with 1 M sodium salicylateand fluorographed (25). Proteins insoluble in trichloroacetic acid and chloroform/methanol (2:1, v/v) were separated on a Tris borate SDS-gel system (26) and fluorographed (24). Untreated gels and gels treated with 1 M hydroxylamine (pH 6.6) (26) were fluoro- graphed using the technique of Bonner and Laskey (27) as described in detail (23). Myelin proteins (160 pg) labeled with ["C]fucose and ['HHjpalmitic acid were separated by SDS-disc gel electrophoresis (28). The gels were stained with 1% (w/v) fast green in methanol/acetic acid/water (45:15:40, v/v/v) and destained in decreasing concentra- tions of methanol (1). The entire gel was sliced into 1-mm sections, and the radioactivity was determined by liquid scintillation spec- trometry (28). Similarly, myelin proteins (150 pg) labeled with a mixture of ['Hlpalmitic acid and [3sS]sulfate were examined by SDS- slab gel electrophoresis (24), stained, and destained and the radioac- tivity in each 1-mm section was determined as described (28).

Cleavage of Protein-bound Fatty Acids from Purified PO-Two samples each of PO (3.0 mg) and eluate from the blank gel were hydrolyzed with 2 ml of methanolic NaOH (0.2 N) at room tempera- ture for 24 h to cleave the fatty acids. The procedures for the transmethylation of fatty acids, the separation of methyl esters of fatty acids by thin layer chromatography, and the determination of radioactivity associated with each fatty acid have been described in detail (23). The methyl esters of fatty acids were separated using a gas-liquid chromatograph equipped with an effluent divider (29). Eighty per cent of the fatty acids were collected for the determination

S T A l N FLUOROGRAM

- - -

LIPID- I 2 3 4

FIG. 1. SDS-slab gel electrophoresis of proteins of rat pe- ripheral nervous system myelin and purified PO on a Tris glycine gel showing acylation of PO protein. The gels were stained with Coomassie blue. Lane 1, myelin proteins (94 pg); lane 2, purified PO (36 pg); lanes 3 and 4, fluorograph of lanes I and 2. The gel was fluorographed for 82 days at -80 "C. The labeling of basic proteins (lane 3) is in all probability due to the recycling of [3H] palmitic acid into amino acids in vivo and the subsequent incorpora- tion of these amino aicds into the myelin basic proteins. Identical results were obtained in six separate experiments. 21K and 17K, myelin basic proteins with molecular weights of 21,000 and 17,000, respectively; LBP, large basic protein; SBP, small basic protein.

PO -

21 K - LBP- 17 K' SBP\ p2 '

E LIPID- -a

1 2 3 4 5 6 7 8 FIG. 2. Protein profile and fluorography of acylated and

fucosylated peripheral nerve and myelin proteins. SDS-gel electrophoresis was carried out in a Tris borate gel system. Gels were stained with Coomassie blue and the same stained gel was fluoro- graphed. Lane I, proteins insoluble in trichloroacetic acid (57 pg); lane 2, proteins insoluble in chloroform/methanol (154 pg); lane 3, myelin proteins labeled in oivo with [3H]palmitic acid (100 pg); lane 4, myelin proteins labeled in vivo with ['H]fucose (94 pg); lanes 5-8, fluorograph of lanes 1-4, respectively. The gel was fluorographed for 32 days at -80 "C. Identical results were obtained in two separate experiments. 21K and 17K, myelin basic proteins with molecular weights of 21,000 and 17,000, respectively; LBP, large basic protein; SBP, small basic protein.

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6558 Covalent Linkage of Fatty Acids to PO Protein

I20.000 t

G E L SLICE N U M B E R

FIG. 3. Electrophoresis of proteins acylated and fucosylated in vivo. Rat peripheral nervous system myelin proteins (160 pg) were separated on a 12% (w/v) cylindrical SDS-polyacrylamide gel. Electrophoresis was carried out at 1.5 mA/gel for 8 h, and the radioactivity in the gel after protein staining was determined as described in the text. The radioactivity due to ["C]fucose and ['HI palmitic acid co-migrated with PO. 21K and 17K, myelin basic pro- teins with molecular weights of 21,000 and 17,000, respectively; LBP, large basic protein; SBP, small basic protein.

of radioactivity associated with each fatty acid, and the remaining 20% was used for the detection of fatty acids.

RESULTS

Purified PO from rat sciatic nerve myelin migrated as a single band when subjected to re-electrophoresis in a Tris glycine (24) SDS-gel system (Fig. 1, lane 2). The molecular weight of purified PO was 29,500 as determined by SDS-gel electrophoresis; this is similar to values reported by other investigators (2,3).

Radioactivity was predominantly associated with PO when acylated proteins insoluble in trichloroacetic acid or chloro- form/methanol were examined by SDS-slab gel electropho- resis (21) followed by fluorography (Fig. 2, lanes 5-6). Simi- larly, when myelin proteins labeled with [3H]palmitic acid or ['H]fucose were subjected to electrophoresis under identical conditions, PO was both acylated and fucosylated (Fig. 2, lanes 7-8). Furthermore, examination by SDS-disc gel elec- trophoresis of myelin proteins labeled with a mixture of ["C] fucose and ['Hlpalmitic acid demonstrated that the peak of the radioactivity due to ['4C]fucose co-migrated with that of ['Hlpalmitic acid in the region in which PO migrates (Fig. 3). Furthermore, when myelin proteins labeled with a mixture of ['sS]sulfate and [3H]palmitic acid were separated by SDS- slab gel electrophoresis, the radioactivity derived from ["SI sulfate and ['Hlpalmitic acid also co-migrated with PO (data not shown). The radioactivity associated with PO acylated in uiuo was retained when electrophoretically purified PO was exhaustively dialyzed and subjected to re-electrophoresis, and the stained gels were fluorographed (Fig. 1, lane 4) . Radioac- tivity associated with PO was also retained following extrac- tion of myelin with acidified chloroform/methanol or chloro-

TABLE I Incorporation of PHJpalmitic acid into PO after extraction of labeled myelin with diethyl etherlethano1 or acidified chloroform/methanol or

chloroform/methanol (A) and incorporation of 'H-labeled myelin lipids into PO protein after addition of labeled lipids to unlabeled

myelin (B) For A, rat sciatic nerve myelin was labeled in vivo after intraneural

injection of ['Hlpalmitic acid as described in the text. Lipids were removed from lyophilized myelin by three different procedures. Mye- lin proteins were dissolved in SDS and separated by SDS-slab gel electrophoresis, and the radioactivity associated with PO was deter- mined (28). Procedure 1, myelin (20 mg) was successively extracted with diethyl ether/ethanol (3:2, v/v) and ether as described (19). The residue was dissolved in 2% SDS. Procedure 2, myelin (10.5 mg) was extracted three times at 4 "C with 10 ml of acidified chloroform methanol, 12 N HCI (2001001, v/v/v) and centrifuged at 12,000 X g for 10 min. The three supernatants containing the labeled lipids and traces of myelin proteins were combined. The residue was successively extracted with acetone and ether as described above. This residue was dried under Nz and solubilized in 2% SDS. Procedure 3, myelin (13.5 mg) was extracted three times with 14 ml of chloroform/ methanol (2:l. v/v) as described above. The three supernatants con- taining the labeled lipids and traces of myelin proteins were combined. The precipitate was extracted twice with 14 ml of HCI (pH 2.2), and the supernatants were discarded (31). The residue was defatted twice with 14 ml of chloroform/methanol (2:l. v/v), and the pellet was dissolved in 2% SDS. The supernatants resulting from the two chloroform/methanol extractions were partitioned with water and washed three times with the synthetic upper phase (37). The lower phase and the material floating at the interface were dried under N2 and dissolved in 2% SDS. For B, myelin labeled with ['Hlpalmitic acid was extracted with 30 ml of acidified chloroform/methanol and the insoluble residue was removed by centrifugation as described above. Unlabeled myelin (10 mg) was added to the labeled lipids (7.7 X 1O'dpm) soluble in acidified chloroform/methanol. This suspension was vortexed vigorously and allowed to stand at room temperature for 1 h. Myelin proteins of each sample were extracted under condi- tions identical with those described above and subjected to SDS-gel electrophoresis to determine radioactivity nonspecifically bound to PO. A similar experiment was conducted in which another sample of myelin labeled with ['Hlpalmitic was extracted with 42 ml of chlo- roform/methanol (21, v/v). The radioactive lipids (3.9 X 10' dpm) were added to 10 mg of unlabeled myelin and processed as described above. -

[3H]Palmitic acid in P0/50 pg myelin protem

B Fractions" A 3H-laeled

in oioo with ['HI adlded to un- Myelin labeled rn elin llplds

palmitic acid labeled mye- linb

dpm Myelin proteins insoluble in diethyl 9,739

Myelin proteins insoluble in acidified 19,440 125

Myelin proteins insoluble in chloro- 38,064' 463

ether/ethanol

chloroform/methanol

form/methanol, HCI, and chloro- form/methanol

form/methanol "Three separate groups of rats were injected with ['Hlpalmitic

acid, and the myelin was isolated. The myelin protein fractions were prepared as described above.

"The small amount of radioactivity found with PO represents a trace amount of PO solubilized when myelin is extracted with acidi- fied chloroform/methanol or chloroform/methanol. ' These fractions are highly enriched in PO protein; therefore, the

radioactivity is substantially higher in these fractions.

Myelin proteins soluble in chloro- 40,377' 487

form/methanol (Table I). Separation of myelin proteins la- beled with ['Hlpalmitic acid in uiuo followed by SDS-slab gel electrophoresis and treatment of the gel with 1 M hydroxyl- amine and fluorography showed that a negligible amount of radioactivity remained associated with PO (Fig. 4, lanes 1 and

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Covalent Linkage of Fatty Acids to PO Protein

I I

2 FIG. 4. Fluorography of untreated and hydroxylamine

treated SDS-slab gels of acylated proteins on rat peripheral nervous system myelin. Lane I , untreated myelin protein (100 pg); lane 2, myelin proteins (100 pg) treated with 1 M hydroxylamine (pH 6.6) for 12 h. Most of the radioactivity associated with PO was removed after treatment with hydroxylamine. The gel was fluoro- graphed for 32 days a t -80 “C. The persistence of a small amount of radioactivity with PO following treatment with hydroxylamine (lane 2) may represent the recycling of [‘Hlpalmitic acid into amino acids in uiuo and the subsequent incorporation of labeled amino acid into PO.

2) . The persistence of a small amount of radioactivity with PO following treatment with hydroxylamine may represent the recycling of [:’H]palmitic acid into amino acids in uiuo and the subsequent incorporation of these amino acids into PO. This assumption is substantiated by the fact that myelin basic proteins are labeled in uiuo after injection of [“Hlpalmitic acid, although they do not contain bound fatty acids (Fig. 1, lane 3) . Under identical conditions, radioactivity due to [‘HI fucose was not removed from PO by hydroxylamine (data not shown). Cleavage of acylated PO with methanolic NaOH (0.2 N) released almost all the radioactivity (85%) into the organic phase. The fatty acids bound to PO were determined by gas- liquid chromatography (Fig. 5). These results provided defi- nite evidence that PO contains palmitate, stearate, oleate, and possibly linoleate. In two separate experiments, the methyl esters of palmitate, stearate, and oleate were found to contain 56, 16, and 5%, respectively, of the radioactivity as determined by gas-liquid chromatography. Similar results were obtained when fatty acids associated with PO were separated by thin layer chromatography on silver nitrate and reverse phase plates (23).

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I I I I 1 I I

0 2 4 6 e 10 12 RETENTION TIME (MINUTES)

FIG. 5. Gas-liquid chromatography results of authentic methyl esters of fatty acids (- - -) and fatty acids associated with PO (-). Purified PO was cleaved with methanolic NaOH (0.2 N); the released fatty acids were methylated by diazomethane, and the methyl esters of fatty acids were separated by gas-liquid chromatography using a stream split divider as described in the text. The methyl esters of palmitate, stearate, and oleate contain 883,264, and 80 dpm, respectively. The results are the mean of two separate experiments. The peak designated ? was completely devoid of radio- activity, and since a similar peak with identical retention time was also observed from the blank gel eluate, it may be due to residual SDS and/or impurities present in acrylamide.

DISCUSSION

Since PO has not been shown previously to contain fatty acids, it was essential to ascertain that the acylated protein is, in fact, PO. Purified PO migrated as a single band in a Tris glycine system (24), as well as in the SDS-gel system described by Weber and Osborne (30), thus confirming the purity of the preparation. The radioactivity associated with acylated PO was retained after dialysis and re-electrophoresis. These observations provided evidence that [‘Hlpalmitic acid was firmly bound to PO. When sciatic nerve myelin labeled with [‘Hlpalmitic acid was extracted with acidified chloro- form/methanol or exhaustively extracted with chloroform/ methanol (2:1, v/v) as described by Ishaque et al. (31), radio- activity associated with PO was retained (Table I). In con- trast, very little radioactivity was incorporated into PO when “H-labeled myelin lipids were added to unlabeled myelin (Ta- ble I) . This small amount of radioactivity found with PO is present because a trace amount of PO is soluble when myelin is extracted with acidified chloroform/methanol or chloro- form/methanol. These results provide further evidence that the [‘’Hlpalmitate-derived radioactivity in PO was not due to labeled phospholipids associated with PO by strong physical interaction. Since PO has been shown to be fucosylated both in vivo (2-8) and in vitro (12-16) and sulfated in uiuo (9, lo), the co-migration of radioactivity due to radiolabeled fucose and palmitic acid as well as sulfate and palmitic acid provided

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H C Agrawal, R E Schmidt and D Agrawalprotein of rat sciatic nerve myelin.

In vivo incorporation of [3H]palmitic acid into PO protein, the major intrinsic

1983, 258:6556-6560.J. Biol. Chem. 

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