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Specific tritium labeling of glucosyl- and galactosylceramides at the 6-position of the carbohydrate moiety using CrO3-graphite
ANALYTICAL BIOCHEMISTRY152, 172- 177 ( 1986)
Specific Tritium Labeling of Glucosyl- and Galactosylceramides at the 6-Position of the Carbohydrate Moiety Using Cr03-Graphite SEIGOU USUKI AND YOSHITAKA Department
of Biochemistry, Faculty Department of Neurobiology,
NAGAI’
of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Brain Research Institute, Niigata University, Niigata,
Tokyo 113. and Japan
Received July 8, 1985 A new procedure for introducing tritium into the carbohydrate portions of glucosyl- and galactosylceramides was developed using a new catalyst, CrQ-graphite, which specifically oxidizes the primary alcohol group to the aldehyde. About 10% of the glycolipid was converted to the aldehyde and the aldehyde produced was then reduced back to the original form with KB’H4. After methanolysis, more than 96.7% of the radioactivities of [3H]gIucosyl- and [‘H]galactosylceramides were found to be located in the carbohydrate portions, and the specific activities of the [3H]galactosyland [3H]glucosy]ceramides were 2.08 to 4.30 X IO4 cpm/nmol, which could be increased greatly by purifying the aldehydes and reducing them with KE%‘H4. In addition, B-galactosidase activity was successfully determined with [‘H]galactosylceramide as the enzyme substrate; the K,,, was 18.73 mM and the V,, was 11.63 nmol/mg/h, indicating that no significant structural modification occurs during the oxidation. 0 1986 Academic Press, Inc. KEY WORDS: glycosphingolipids; CMH; carbohydrate; radiolabeling; Cr03-graphite.
Glycosphingolipids are known to be localized on the outer leaflet of the plasma membrane (1) and to function as cell surface markers (2,3) or receptors for bioactive molecules such as toxins or hormones (4,5). Therefore, detection of these molecules and investigation of their metabolism using as little material as possible have become increasingly important. The use of radiolabeled materials is essential for such studies. Various labeling methods for glycosphingolipids have been developed by several investigators (6-22). Although various methods for labeling the hydrophobic portion are available (9-l 8), methods for the carbohydrate portion are limited (6-8, 19-22). The most convenient and widely applied procedure for labeling carbohydrate is galactose oxidaseNaB3H4 reduction to label the C-6 position of galactose or N-acetylgalactosamine of glycosphingolipids (6-8) but glycosphingolipids without galactose are not labeled by this procedure. In addition, there is no method avail’ To whom correspondence should be addressed. 0003-2697186 $3.00 CopyrigJo 0 1986 by Academic Press, Inc. AIL rights of reproduction in any form reserved.
able for labeling all carbohydrates of glycosphingolipids. Therefore, we tried to develop a new method for labeling the sugar portion of glycosphingolipids without any structural change of the lipid portion and found that the use of CrOs-graphite oxidation (23) followed by ISB3H4 reduction was suitable for this purpose. MATERIALS
AND
METHODS
Materials. Galactosylceramide (kerasin) and glucosylceramide were purified from human brain and bovine spleen, respectively, according to the method described by Radin et al. (24), and glucosylceramide was completely freed from contaminating galactosylceramide by column chromatography on Iatrobeads (Iatron Co., Tokyo, Japan) containing sodium borate. The following materials were purchased from commercial sources: Cr03graphite from Alfa Division (Danvers, Ill.), Sep-Pak silica gel cartridges from Waters Associates (Milford, Conn.), KB3H4 (25) (specific radioactivity, 1.13 Ci/mmol) from New En172
SPECIFIC RADIOLABELING
gland Nuclear (Boston, Mass.), galactose oxidase (0.46 units/mg) from Sigma (St. Louis, MO.), P-galactosidase (jack bean, 0.55 units/ mg) from Seikagaku Kogyo (Tokyo, Japan), precoated thin-layer plates (silica gel 60,0.25mm thick) from E. Merck (Darmstadt, Germany), and Iatrobeads (6RS 8060) for column chromatography from Iatron. Tetrahydrofuran was from Wako Chemical Company (Tokyo, Japan) and was redistilled once over LiA1H4 immediately before use. Preparation of [‘H]CMH2 by the Cr03graphite method. One milligram of GalCer or GlcCer was dissolved in 2 ml of toluene-isopropylalcohol (1: 1, v/v) in a screw-capped tube. Cr03-graphite (150 mg) was added to the tube and, after reaction at 110°C for 4 h, the catalyst was pelleted by centrifugation at 3000 rpm for 10 min and the residue was then washed successively with 2 ml of chloroformmethanol at 2: 1, 1: 1, and 1:2 (v/v). The combined supematant was passed through a SepPak silica gel cartridge. Elution of oxidized glycolipid from the cartridge was carried out with 20 ml of chloroform-methanol (2: 1, v/ v), and the eluate was evaporated to dryness and dissolved in 0.5 ml tetrahydrofuran. Reduction of the oxidized CMH was performed with 1 mCi of KB3H4 in 10 ~1 of 0.1 M KOH with shaking at room temperature overnight ( 10 to 12 h). An additional 10 mg of solid NaBH4 was then added and the shaking was continued for 2 h. The excess NaBH4 was eliminated by addition of a few drops of acetic acid. The products were developed on TLC with chloroform-methanol-water (60:35:8, v/ v/v) as the developing solvent and examined with a radiochromatoscanner (Aloka JTC201) at a C/M of 10 K, and with a time constant of 3 s. The radiolabeled CMH was further 2 Abbreviations used: GIc, C-gIucose; Gal, D-galactose; GalCer, galactosylceramide; GlcCer, glucosylceramide; GalCer (C16:0), galactosylceramide containing palmitic acid; NFA-GalCer, galactosylceramide containing nonhydroxy fatty acid; CMH, monohexaosylceramide; GalCer-aldehyde, aldehyde derivative at C-6 of the galactose residue of GalCer; GlcCer-aldehyde, aldehyde derivative at C-6 of the glucose residue of GlcCer.
OF GLYCOSPHINGOLIPIDS
173
purified by preparative TLC with the same developing solvent and extraction of the product from silica gel was performed with chloroform-methanol-water (1: 1:O. 1, v/v/v). Also, to increase the specific radioactivity, the aldehyde of CMH produced on CrO3graphite oxidation was purified by Iatrobeads column chromatography (1 X 35 cm) packed with 10 g of Iatrobeads (6RS-8060) with a linear gradient of 60 ml each of chloroform-isopropylalcohol-water (85: 15:0.2 and 40:60:2, v/v/v) at a flow rate of 0.44 ml/min and the aldehyde obtained was reduced with KB3H4 as described above. Labeling by the galactose oxidase-KB3H4 method. [3H]NFA-GalCer was prepared according to the procedure described by Suzuki and Suzuki (6). Distribution of the radioactivity in the labeled CMH. The distribution of the radioactivity in the labeled CMH was determined by methanolysis with 3% methanolic HCl at 110°C for 3 h. The fatty acid methyl esters liberated were extracted with n-hexane. The hexane layer was subjected to TLC with chloroform-methanol-water (3:3: 1, v/v/v) as the developing solvent and iodine was used for detection. An aliquot of the methanol layer was subjected to TLC with chloroform-methanol-water (3:3: 1, v/v/v) as the developing solvent and 0.2% orcinol in 2 N sulfuric acid was used for detection. The remaining methanolic layer was then converted to a basic solution with aqueous NaOH solution and the free sphingosine base was extracted with diethyl ether and examined by TLC with chloroform-methanol-ammonia (4:4: 1, v/v/v) as the developing solvent and with ninhydrin for detection. Autoradiograms of the plates of fatty acid methyl esters, methyl glycoside, and long-chain base were obtained on Kodak Xray film (XRP- 1) after spraying with an enhancer (Aquasol-2, New England Nuclear) and the radioactivity of each spot was determined with a liquid scintillation counter (Aloka LSC900). Also, NaBZH4, instead of KB3H4, was used for reduction and the distribution of *H in CMH was determined with a mass spectro-
174
USUKI
AND NAGAl
meter (Shimadzu, QP- 1000) after converting CMH to the trimethylsilyl derivative. @-Galactosidase treatment. The susceptibility of [3H]GalCer obtained above to &galactosidase was examined as follows (26). t3H]GalCer (1 to 20 rg) was dissolved in 0.1 ml of sodium citrate buffer (pH 4.5) containing 1 mg of sodium deoxytaurocholate, and then 0.15 unit of P-galactosidase was added to the solution. After 1 h at 37°C the reaction was terminated by adding 0.3 ml of galactose solution (1 mg/ml in water) and 2.5 ml of chloroform-methanol (2: 1, v/v). The upper layer was washed three times with 1.0 ml of chloroform-methanol-water (86: 14: 1, v/v/v) and transferred to a scintillation vial, and the radioactivity was determined with a liquid scintillation counter. Quantitation of CMH. To determine the recovery through the procedure, along with determination of the radioactivity, the amount of [3H]CMH was determined densitometritally with a TLC densitometer (CS-910, Shimadzu Co.) at an analytical wavelength of 540 nm and a control wavelength of 710 nm. Standard CMHs, 0.2 to 3.0 cLg,were spotted on the same plate and the spots were located by spraying with 0.2% orcinol-2 N sulfuric acid reagent after development with chloroformmethanol-water (60:35:8, v/v/v). In addition, the amount of CMH was estimated by determining the fatty acid methyl esters derived from CMH by the hydroxyl amine-ferric perchlorate method (27).
conditions, at least four spots were detected above the major GalCer spot, and the rather broad radioscan peak corresponds to these minor by-products (Fig. 1). However, [3H]GalCer purified by preparative TLC gave a single orcinol-positive and radioactive spot. To increase the specific radioactivity, the GalCer-aldehyde was separated from the untreated GalCer as shown in Fig. 2. After purification of the GalCer-aldehyde followed by reduction, the specific radioactivity was increased 20-fold compared to the case of direct labeling. Similarly, for GlcCer, the specific radioactivity was increased, by 1O-fold. The specific radioactivity of the labeled CMH and the distribution of the radioactivity in the fatty acid, sphingosine, and hexose moieties are presented in Table 1. The specific radioactivities were from 2.0 to 4.0 X IO4 cpm/nmol,
RESULTS
Radiolabeling of GalCer by oxidation with Cr03-graphite and reduction with KB3Hd. GalCer was oxidized with CrQ3-graphite in various solvents: tetrahydrofuran, isopropylalcohol, chloroform, dichloromethane, toluene, and so on. Among them, toluene-isopropylalcohol ( 1: 1, v/v) was found to be the best solvent system. The maximum reaction conditions were 4 h at 110°C. Reaction at more than 15O“C resulted in an increase in the byproducts. After reaction under the optimum
I 3 8 FIG. 1. TLC of [‘H]NFA-GalCer (lane 1) obtained by the galactose oxidase method; NFA-GalCer (lane 2). and GalCer (C16:O) (lane 3) obtained by the CrO&raphite method; and a radioscan of lane 2. TLC was performed with chloroform-methanol-water (60~398, v/v/v) and the spots were located with orcinol reagent.
SPECIFIC RADIOLABELING
-\
Standard
GalCer
123
I
,--
175
OF GLYCOSPHINGOLIPIDS
45
6
I 100
Fraction
number
2. (A) Separation of GalCer-aldehyde from GalCer by Iatrobeads column chromatography. The conditions for the column chromatography are described in the text. Each of the fractions eluted from the column were applied to TLC. TLC was developed with chloroform-methanol-water (60:35:8, v/v/v) and the spots were located with orcinol reagent. (B) GalCer and GlcCer before (lane 3 and 4) and after oxidation (lane 1 and 6) and the GalCer-aldehyde and GlcCer-aldehyde (lane 2 and 5) after purification by the Iatrobeads column chromatography. FIG.
and the specific localization of radioactivity in the hexose moiety was evident. The structure was also confirmed by analyzing the deuterated CMH with a mass spectrophotometer, and the rate of conversion to the derivative was estimated to be 10% from the ion intensity of the fragment ion containing the carbohydrate moiety.
P-Galactosidase treatment of [‘H]GalCer prepared by the Cr&graphite method. The susceptibility of [3H]GalCer prepared by the Cr03-graphite method to 8-galactosidase was compared with the frequently used substrate prepared by the galactose oxidase method. The Km and Max values of P-galactosidase with both substrates were almost identical, that is,
TABLE 1 SPECIFIC RADIOACTIVITIES AND DISTRIBUTION OF THE RADIOACTIVITY OF [‘H]CMH THE CIO~-GRAPHITE AND GALACTOSE OXIDASE METHODS
PREPARED
BY
Distribution of the radioactivity after methanolysis (%) Glycolipid
Specific activity (cpm/nmol)
Fatty acid
Sphingosine
Hexose
Labeling by Crop-graphite oxidation, KB3H, reduction, and preparative TLC GalCer (C 16:O) NFA-GalCer GlcCer
2.08 x IO4 4.29 X IO4 4.30 x IO”
0.08 0.32 I .09
1.37 0.87 0.68
Labeling after separating CMH-aldehyde prepared by the CrQ-graphite GalCer (C 160) GlcCer
6.43 X IO> 4.19 x IOJ
NFA-GalCer
1.63 X lo5
0.73 1.19
98.6 98.8 98.2
method
0.69 1.15
98.6 97.7
2.85
96.7
Labeling by the galactose oxidase method 0.46
176
USUKI AND NAGAl
K,,, of 18.73 and 22.43 mM, and I’,,,,, of 11.63 and 9.36 nmol/mg/h for the substrates prepared by the CrO,-graphite and galactose oxidase methods, respectively, indicating that no significant structural modification occurs in the oxidation reaction with CrOJ-graphite. DISCUSSION
To introduce radioactivity to the hydrophobic ceramide portion of glycosphingolipid, the following methods are usually used: reduction of double bonds in sphingosine residues by 3Hz irradiation (9) or with palladium chloride plus NaB3H4 ( IO,1 I), oxidation of allyl hydroxy groups in sphingosine residues with 2,3-dichloro-5,6-dicyanobenzoquinone followed by reduction with NaB3H4 (12- 17), and acylation of deacylated glycosphingolipids such as psychosine with “C-labeled acyl chloride (18). On the other hand, the procedures used to radiolabel the carbohydrate moiety are as follows: oxidation of the hydroxy group at the C-6 position of galactose or N-acetyl galactosamine with galactose oxidase followed by reduction to the original form with KB3H4 (6-8), N-acetylation of the hydrazinolyzed glycosphingolipids with [14C]acetic anhydride to introduce radioactivity to the IV-acetyl hexosamine residue ( 19), and oxidation of the hydroxyl groups of sialic acid residues with periodate followed by reduction with NaB3Hp (20,2 1). A similar but complicated method for labeling the sugar residue was reported by Mcmaster and Radin (22). They first prepared the 6-trityl ether of GlcCer, acetylated the remaining hydroxyl groups, removed the trityl group, oxidized the free 6-hydroxyl group to the aldehyde, reduced the aldehyde with NaB3H4, and finally removed the acetyl groups to obtain the original glycolipid. The method was complicated due to the lack of a procedure for labeling the sugar residue of GlcCer. We tried to develop a more convenient procedure using the unique catalyst chromic trioxide in graphite, which oxidizes the primary alcohols selectively. The unique structure of graphite, that is, carbon atoms tightly bound in planes
and loosely stacked one upon the other, provides a space with a high electrical conductivity and a high crystal anisotropy, and shows selective catalytic and chemical reactions, and other interesting phenomena. Chromic acid can also be intercalated in graphite. Lalancette et al. (23) showed the Cr03-graphite was a selective oxidizing agent for primary alcohols. The good selectivity of the reagent for primary alcohols must be related to its particular structure, as access to Cr03 is restricted by the planes of the graphite. After oxidation, the resulting aldehyde must be recovered in the reaction solvent under the experimental conditions in the absence of water. By application of this oxidizing agent, we succeeded in selective oxidation at the C-6 position of the carbohydrate moiety of CMH. Thus even GlcCer was labeled by this procedure. A more detailed discussion of the possible application and limitations of this method to the labeling of more complex glycosphingolipids, while not essential, would be of interest. ACKNOWLEDGMENTS This work was supported by grants from the Ministries of Education, Science and Culture, and Health and Welfare of Japan, a research grant from the Yamanouchi Foundation for Research on Metabolic Disorders, a grant-inaid from the Naito Foundation, and the Special Coordination Funds for promoting science and technology from the Science and Technology Agency of Japan.
REFERENCES 1. Hakomori, S., and Kanfer, J. N. (1983) in Handbook of Lipid Research, Sphingolipid Biochemistry (Hanahan, D. J., ed.), pp. 327-379, Plenum Press, New York/London. 2. Feizi, T. (1985) Nature (London) 314, 53-57. 3. Hakomori,S.,and Kannagi, R. (1983)JNC171,231251. 4. Fishman, P. H., and Brady, R. 0. (1976) Science (Washington
D. C.) 194.906-9
15.
5. Hakomori, S. (1981) Annu. Rev. Biochem. 50, 733764. 6. Suzuki, Y., and Suzuki, K. (1972) J. Lipid Res. 13, 687-690. 7. Leskawa, K. C., Dasgupta, S., Chien, J.-L., and Hogan, E. L. (1984) Anal. Biochem. 140, 172-177.
SPECIFIC RADIOLABELING 8. Hajra, A. K., Bowen, D. M., Kishimoto, Y., and Radin, N. S. (1966) J. Lipid Res. 7, 379-386. 9. Seyama, Y., Yamakawa, T., and Komai, T. (1968) J. Biochem.
14,133-137.
Biophys.
19. Higashi, H., and Basu, S. (1982) Anal. Biochem.
25,620-628.
15. Iwamori, M., Moser, H. W., and Kishimoto, Y. (1975) Res. 16, 332-336.
Veh, R. W., Corlield, A. P., Sander, M., and Schauer, R. (1977) Biochim. Biophys. Acla 486, 145-160. 2 1. Valenten, L., and Aschwell, G. (197 1) J. Biol. Chem. 20.
246, 1889-1894. 22.
17. Gazzoti, G.. Sonnino, S., Ghidoni, R., Orlando, P.,
Mcmaster, M. C., and Radin, N. S. (1977) J. Lube/led Compds.
Radiopharm.
8,353-351.
23. Lalancette, J.-M., Rollin, G., and Dumas, P. (1972) Canad.
J. Chem.
50,3058-3062.
24. Radin, N. S., Brown, J. R., and Lavin, F. B. (1956) J. Biol. Chem.
219, 977-983.
25. Murtiashaw, M. H., Thorpe, S. R., and Baynes, J. W. ( 1984) Anal. Biochem. 140, 112- 177. 26. Li, Y.-T., and Li, S.-C. (1968) J. Biol. Chem. 243,
16. Iwamori, M., and Nagai, Y. (1977) Chem. Phys. Lipids 20, 193-203.
120,
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161,426-434.
13. Ghidoni, R., Sonnino, S., Masserini, M., Orlando, P., and Tettamanti, G. (198 1) J. Lipid Res. 22, 12861295. 14. Sonnino, S., Ghidoni, R., Gazzotti, G., Kirschner, G., Galli, G., and Tettamanti, G. (1984) J. Lipid Res.
J. Lipid
and Tettamanti, G. (1984) Glycoconjugates 1, I 1I121. 18. Erikson, J. S., and Radin, N. S. (1973) J. Lipid Res.
64,487-493.
10. Schwartzmann, G. (1978) Biochim. Biophys. Ada 529, 106-I 14. I 1. Momoi, T., and Wiegandt, H. (1980) Hoppe-Seyler’s Z. Physiol. Chem. 361, 1201-1210. 12. Kishimoto, Y., and Mitry, M. T. (1974) Arch. Biochem.
177
OF GLYCOSPHINGOLIPIDS
3994-3998. 27.
Renkonen, 0. (1961) B&him. 362.
Biophys.
Acta 54,36
l-
Specific Tritium Labeling of Glucosyl- and Galactosylceramides at the 6-Position of the Carbohydrate Moiety Using Cr03-Graphite SEIGOU USUKI AND YOSHITAKA Department
of Biochemistry, Faculty Department of Neurobiology,
NAGAI’
of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Brain Research Institute, Niigata University, Niigata,
Tokyo 113. and Japan
Received July 8, 1985 A new procedure for introducing tritium into the carbohydrate portions of glucosyl- and galactosylceramides was developed using a new catalyst, CrQ-graphite, which specifically oxidizes the primary alcohol group to the aldehyde. About 10% of the glycolipid was converted to the aldehyde and the aldehyde produced was then reduced back to the original form with KB’H4. After methanolysis, more than 96.7% of the radioactivities of [3H]gIucosyl- and [‘H]galactosylceramides were found to be located in the carbohydrate portions, and the specific activities of the [3H]galactosyland [3H]glucosy]ceramides were 2.08 to 4.30 X IO4 cpm/nmol, which could be increased greatly by purifying the aldehydes and reducing them with KE%‘H4. In addition, B-galactosidase activity was successfully determined with [‘H]galactosylceramide as the enzyme substrate; the K,,, was 18.73 mM and the V,, was 11.63 nmol/mg/h, indicating that no significant structural modification occurs during the oxidation. 0 1986 Academic Press, Inc. KEY WORDS: glycosphingolipids; CMH; carbohydrate; radiolabeling; Cr03-graphite.
Glycosphingolipids are known to be localized on the outer leaflet of the plasma membrane (1) and to function as cell surface markers (2,3) or receptors for bioactive molecules such as toxins or hormones (4,5). Therefore, detection of these molecules and investigation of their metabolism using as little material as possible have become increasingly important. The use of radiolabeled materials is essential for such studies. Various labeling methods for glycosphingolipids have been developed by several investigators (6-22). Although various methods for labeling the hydrophobic portion are available (9-l 8), methods for the carbohydrate portion are limited (6-8, 19-22). The most convenient and widely applied procedure for labeling carbohydrate is galactose oxidaseNaB3H4 reduction to label the C-6 position of galactose or N-acetylgalactosamine of glycosphingolipids (6-8) but glycosphingolipids without galactose are not labeled by this procedure. In addition, there is no method avail’ To whom correspondence should be addressed. 0003-2697186 $3.00 CopyrigJo 0 1986 by Academic Press, Inc. AIL rights of reproduction in any form reserved.
able for labeling all carbohydrates of glycosphingolipids. Therefore, we tried to develop a new method for labeling the sugar portion of glycosphingolipids without any structural change of the lipid portion and found that the use of CrOs-graphite oxidation (23) followed by ISB3H4 reduction was suitable for this purpose. MATERIALS
AND
METHODS
Materials. Galactosylceramide (kerasin) and glucosylceramide were purified from human brain and bovine spleen, respectively, according to the method described by Radin et al. (24), and glucosylceramide was completely freed from contaminating galactosylceramide by column chromatography on Iatrobeads (Iatron Co., Tokyo, Japan) containing sodium borate. The following materials were purchased from commercial sources: Cr03graphite from Alfa Division (Danvers, Ill.), Sep-Pak silica gel cartridges from Waters Associates (Milford, Conn.), KB3H4 (25) (specific radioactivity, 1.13 Ci/mmol) from New En172
SPECIFIC RADIOLABELING
gland Nuclear (Boston, Mass.), galactose oxidase (0.46 units/mg) from Sigma (St. Louis, MO.), P-galactosidase (jack bean, 0.55 units/ mg) from Seikagaku Kogyo (Tokyo, Japan), precoated thin-layer plates (silica gel 60,0.25mm thick) from E. Merck (Darmstadt, Germany), and Iatrobeads (6RS 8060) for column chromatography from Iatron. Tetrahydrofuran was from Wako Chemical Company (Tokyo, Japan) and was redistilled once over LiA1H4 immediately before use. Preparation of [‘H]CMH2 by the Cr03graphite method. One milligram of GalCer or GlcCer was dissolved in 2 ml of toluene-isopropylalcohol (1: 1, v/v) in a screw-capped tube. Cr03-graphite (150 mg) was added to the tube and, after reaction at 110°C for 4 h, the catalyst was pelleted by centrifugation at 3000 rpm for 10 min and the residue was then washed successively with 2 ml of chloroformmethanol at 2: 1, 1: 1, and 1:2 (v/v). The combined supematant was passed through a SepPak silica gel cartridge. Elution of oxidized glycolipid from the cartridge was carried out with 20 ml of chloroform-methanol (2: 1, v/ v), and the eluate was evaporated to dryness and dissolved in 0.5 ml tetrahydrofuran. Reduction of the oxidized CMH was performed with 1 mCi of KB3H4 in 10 ~1 of 0.1 M KOH with shaking at room temperature overnight ( 10 to 12 h). An additional 10 mg of solid NaBH4 was then added and the shaking was continued for 2 h. The excess NaBH4 was eliminated by addition of a few drops of acetic acid. The products were developed on TLC with chloroform-methanol-water (60:35:8, v/ v/v) as the developing solvent and examined with a radiochromatoscanner (Aloka JTC201) at a C/M of 10 K, and with a time constant of 3 s. The radiolabeled CMH was further 2 Abbreviations used: GIc, C-gIucose; Gal, D-galactose; GalCer, galactosylceramide; GlcCer, glucosylceramide; GalCer (C16:0), galactosylceramide containing palmitic acid; NFA-GalCer, galactosylceramide containing nonhydroxy fatty acid; CMH, monohexaosylceramide; GalCer-aldehyde, aldehyde derivative at C-6 of the galactose residue of GalCer; GlcCer-aldehyde, aldehyde derivative at C-6 of the glucose residue of GlcCer.
OF GLYCOSPHINGOLIPIDS
173
purified by preparative TLC with the same developing solvent and extraction of the product from silica gel was performed with chloroform-methanol-water (1: 1:O. 1, v/v/v). Also, to increase the specific radioactivity, the aldehyde of CMH produced on CrO3graphite oxidation was purified by Iatrobeads column chromatography (1 X 35 cm) packed with 10 g of Iatrobeads (6RS-8060) with a linear gradient of 60 ml each of chloroform-isopropylalcohol-water (85: 15:0.2 and 40:60:2, v/v/v) at a flow rate of 0.44 ml/min and the aldehyde obtained was reduced with KB3H4 as described above. Labeling by the galactose oxidase-KB3H4 method. [3H]NFA-GalCer was prepared according to the procedure described by Suzuki and Suzuki (6). Distribution of the radioactivity in the labeled CMH. The distribution of the radioactivity in the labeled CMH was determined by methanolysis with 3% methanolic HCl at 110°C for 3 h. The fatty acid methyl esters liberated were extracted with n-hexane. The hexane layer was subjected to TLC with chloroform-methanol-water (3:3: 1, v/v/v) as the developing solvent and iodine was used for detection. An aliquot of the methanol layer was subjected to TLC with chloroform-methanol-water (3:3: 1, v/v/v) as the developing solvent and 0.2% orcinol in 2 N sulfuric acid was used for detection. The remaining methanolic layer was then converted to a basic solution with aqueous NaOH solution and the free sphingosine base was extracted with diethyl ether and examined by TLC with chloroform-methanol-ammonia (4:4: 1, v/v/v) as the developing solvent and with ninhydrin for detection. Autoradiograms of the plates of fatty acid methyl esters, methyl glycoside, and long-chain base were obtained on Kodak Xray film (XRP- 1) after spraying with an enhancer (Aquasol-2, New England Nuclear) and the radioactivity of each spot was determined with a liquid scintillation counter (Aloka LSC900). Also, NaBZH4, instead of KB3H4, was used for reduction and the distribution of *H in CMH was determined with a mass spectro-
174
USUKI
AND NAGAl
meter (Shimadzu, QP- 1000) after converting CMH to the trimethylsilyl derivative. @-Galactosidase treatment. The susceptibility of [3H]GalCer obtained above to &galactosidase was examined as follows (26). t3H]GalCer (1 to 20 rg) was dissolved in 0.1 ml of sodium citrate buffer (pH 4.5) containing 1 mg of sodium deoxytaurocholate, and then 0.15 unit of P-galactosidase was added to the solution. After 1 h at 37°C the reaction was terminated by adding 0.3 ml of galactose solution (1 mg/ml in water) and 2.5 ml of chloroform-methanol (2: 1, v/v). The upper layer was washed three times with 1.0 ml of chloroform-methanol-water (86: 14: 1, v/v/v) and transferred to a scintillation vial, and the radioactivity was determined with a liquid scintillation counter. Quantitation of CMH. To determine the recovery through the procedure, along with determination of the radioactivity, the amount of [3H]CMH was determined densitometritally with a TLC densitometer (CS-910, Shimadzu Co.) at an analytical wavelength of 540 nm and a control wavelength of 710 nm. Standard CMHs, 0.2 to 3.0 cLg,were spotted on the same plate and the spots were located by spraying with 0.2% orcinol-2 N sulfuric acid reagent after development with chloroformmethanol-water (60:35:8, v/v/v). In addition, the amount of CMH was estimated by determining the fatty acid methyl esters derived from CMH by the hydroxyl amine-ferric perchlorate method (27).
conditions, at least four spots were detected above the major GalCer spot, and the rather broad radioscan peak corresponds to these minor by-products (Fig. 1). However, [3H]GalCer purified by preparative TLC gave a single orcinol-positive and radioactive spot. To increase the specific radioactivity, the GalCer-aldehyde was separated from the untreated GalCer as shown in Fig. 2. After purification of the GalCer-aldehyde followed by reduction, the specific radioactivity was increased 20-fold compared to the case of direct labeling. Similarly, for GlcCer, the specific radioactivity was increased, by 1O-fold. The specific radioactivity of the labeled CMH and the distribution of the radioactivity in the fatty acid, sphingosine, and hexose moieties are presented in Table 1. The specific radioactivities were from 2.0 to 4.0 X IO4 cpm/nmol,
RESULTS
Radiolabeling of GalCer by oxidation with Cr03-graphite and reduction with KB3Hd. GalCer was oxidized with CrQ3-graphite in various solvents: tetrahydrofuran, isopropylalcohol, chloroform, dichloromethane, toluene, and so on. Among them, toluene-isopropylalcohol ( 1: 1, v/v) was found to be the best solvent system. The maximum reaction conditions were 4 h at 110°C. Reaction at more than 15O“C resulted in an increase in the byproducts. After reaction under the optimum
I 3 8 FIG. 1. TLC of [‘H]NFA-GalCer (lane 1) obtained by the galactose oxidase method; NFA-GalCer (lane 2). and GalCer (C16:O) (lane 3) obtained by the CrO&raphite method; and a radioscan of lane 2. TLC was performed with chloroform-methanol-water (60~398, v/v/v) and the spots were located with orcinol reagent.
SPECIFIC RADIOLABELING
-\
Standard
GalCer
123
I
,--
175
OF GLYCOSPHINGOLIPIDS
45
6
I 100
Fraction
number
2. (A) Separation of GalCer-aldehyde from GalCer by Iatrobeads column chromatography. The conditions for the column chromatography are described in the text. Each of the fractions eluted from the column were applied to TLC. TLC was developed with chloroform-methanol-water (60:35:8, v/v/v) and the spots were located with orcinol reagent. (B) GalCer and GlcCer before (lane 3 and 4) and after oxidation (lane 1 and 6) and the GalCer-aldehyde and GlcCer-aldehyde (lane 2 and 5) after purification by the Iatrobeads column chromatography. FIG.
and the specific localization of radioactivity in the hexose moiety was evident. The structure was also confirmed by analyzing the deuterated CMH with a mass spectrophotometer, and the rate of conversion to the derivative was estimated to be 10% from the ion intensity of the fragment ion containing the carbohydrate moiety.
P-Galactosidase treatment of [‘H]GalCer prepared by the Cr&graphite method. The susceptibility of [3H]GalCer prepared by the Cr03-graphite method to 8-galactosidase was compared with the frequently used substrate prepared by the galactose oxidase method. The Km and Max values of P-galactosidase with both substrates were almost identical, that is,
TABLE 1 SPECIFIC RADIOACTIVITIES AND DISTRIBUTION OF THE RADIOACTIVITY OF [‘H]CMH THE CIO~-GRAPHITE AND GALACTOSE OXIDASE METHODS
PREPARED
BY
Distribution of the radioactivity after methanolysis (%) Glycolipid
Specific activity (cpm/nmol)
Fatty acid
Sphingosine
Hexose
Labeling by Crop-graphite oxidation, KB3H, reduction, and preparative TLC GalCer (C 16:O) NFA-GalCer GlcCer
2.08 x IO4 4.29 X IO4 4.30 x IO”
0.08 0.32 I .09
1.37 0.87 0.68
Labeling after separating CMH-aldehyde prepared by the CrQ-graphite GalCer (C 160) GlcCer
6.43 X IO> 4.19 x IOJ
NFA-GalCer
1.63 X lo5
0.73 1.19
98.6 98.8 98.2
method
0.69 1.15
98.6 97.7
2.85
96.7
Labeling by the galactose oxidase method 0.46
176
USUKI AND NAGAl
K,,, of 18.73 and 22.43 mM, and I’,,,,, of 11.63 and 9.36 nmol/mg/h for the substrates prepared by the CrO,-graphite and galactose oxidase methods, respectively, indicating that no significant structural modification occurs in the oxidation reaction with CrOJ-graphite. DISCUSSION
To introduce radioactivity to the hydrophobic ceramide portion of glycosphingolipid, the following methods are usually used: reduction of double bonds in sphingosine residues by 3Hz irradiation (9) or with palladium chloride plus NaB3H4 ( IO,1 I), oxidation of allyl hydroxy groups in sphingosine residues with 2,3-dichloro-5,6-dicyanobenzoquinone followed by reduction with NaB3H4 (12- 17), and acylation of deacylated glycosphingolipids such as psychosine with “C-labeled acyl chloride (18). On the other hand, the procedures used to radiolabel the carbohydrate moiety are as follows: oxidation of the hydroxy group at the C-6 position of galactose or N-acetyl galactosamine with galactose oxidase followed by reduction to the original form with KB3H4 (6-8), N-acetylation of the hydrazinolyzed glycosphingolipids with [14C]acetic anhydride to introduce radioactivity to the IV-acetyl hexosamine residue ( 19), and oxidation of the hydroxyl groups of sialic acid residues with periodate followed by reduction with NaB3Hp (20,2 1). A similar but complicated method for labeling the sugar residue was reported by Mcmaster and Radin (22). They first prepared the 6-trityl ether of GlcCer, acetylated the remaining hydroxyl groups, removed the trityl group, oxidized the free 6-hydroxyl group to the aldehyde, reduced the aldehyde with NaB3H4, and finally removed the acetyl groups to obtain the original glycolipid. The method was complicated due to the lack of a procedure for labeling the sugar residue of GlcCer. We tried to develop a more convenient procedure using the unique catalyst chromic trioxide in graphite, which oxidizes the primary alcohols selectively. The unique structure of graphite, that is, carbon atoms tightly bound in planes
and loosely stacked one upon the other, provides a space with a high electrical conductivity and a high crystal anisotropy, and shows selective catalytic and chemical reactions, and other interesting phenomena. Chromic acid can also be intercalated in graphite. Lalancette et al. (23) showed the Cr03-graphite was a selective oxidizing agent for primary alcohols. The good selectivity of the reagent for primary alcohols must be related to its particular structure, as access to Cr03 is restricted by the planes of the graphite. After oxidation, the resulting aldehyde must be recovered in the reaction solvent under the experimental conditions in the absence of water. By application of this oxidizing agent, we succeeded in selective oxidation at the C-6 position of the carbohydrate moiety of CMH. Thus even GlcCer was labeled by this procedure. A more detailed discussion of the possible application and limitations of this method to the labeling of more complex glycosphingolipids, while not essential, would be of interest. ACKNOWLEDGMENTS This work was supported by grants from the Ministries of Education, Science and Culture, and Health and Welfare of Japan, a research grant from the Yamanouchi Foundation for Research on Metabolic Disorders, a grant-inaid from the Naito Foundation, and the Special Coordination Funds for promoting science and technology from the Science and Technology Agency of Japan.
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3994-3998. 27.
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l-