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Immunochemical studies of organ and tumor lipids
ARCHIVES
OF
BIOCHEMISTRY
AND
Immunochemical XIII. isolation
BIOPHYSICS
106,
Studies
of Cytolipin
431-438
of Organ
From
the Division
of Virology Department
and
K, a Glycosphingolipid Kidney’1
RI. >I. RAPPORT,3
(1964)
LISELOTTE
Tumor
Hapten
Lipids
Present
’
GRAF
AND
H. SCHNEIDER
and Immunology, Sloan-Kettering Institute for Cancer of Biochemistry, Albert Einstein College of Medicine, Yeshiva Universit?y, New York, New York Received
November
in Human
Research,
and
28, 1963
A neutral glycosphingolipid containing fatty acid-sphingosine (long chain base)glucose-galactose-galactosamine in the molar proportions 1: 1: 1: 2: 1 has been isolated from normal human kidney by successive chromatographic separations on silicic acid, magnesium silicate, and silicic acid. The isolated substance, designated cytolipin K, was shown to have haptenic properties by screening it for reactivity with a large number of rabbit antisera prepared against diverse human tissues. Good reactions were observed in a small number of instances. The two most reactive antisera were obtained by combination immunization with lipids from a multiple myeloma-affected kidney and a pyelonephritic kidney, and with these antisera and optimal addition of auxiliary lipid, 0.003-0.005 pg. of cytolipin K was readily measured. It was shown that the immunological reactivity of cytolipin K was distinct with respect to five other lipid haptens present in human tissues, namely, cytolipin H, cytolipin G, galactocerebroside, cardiolipin, and Forssman hapten.
Knowledge of the chemical structure of lipids that display immunological activity has been scant until recently. The first definitive work resulted from the isolation of cardiolipin (l), whose structure is now considered to be that of a complex phosphatidic acid, diphosphatidyl glycerol (2). The reactivity of cardiolipin with specific antibody has made it a substance of considerable value in diagnosis of disease, but cardiolipin has not provided a general model of lipid structures with immunological activity. In contrast, many of the earlier studies of reactive
lipids such as Forssman hapten and the brain specific hapten suggested that lipids containing carbohydrate residues were involved in immunological reactions (3). These studies were brought into sharp focus with isolation of cytolipin H from human cancer tissue (4) and its subsequent structural identification as ceramide lactoside (5). Cytolipin H is the molecule that accounts most frequently for the substantial differences observed between the reactions of lipid extracts of normal tissues and those of cancer tissues when studied with antisera to the latter (6). Immunological specificity of this molecule is conferred principally, if not exclusively, by the two carbohydrate residues (glucose and galactose). The two lipid residues (fatty acid and sphingosine) play their role most probably in the localization of the molecule within cell membranes. Very recently it was demonstrated that galactocerebrosides such as phrenosine and
1 This work was presented in part at the 47th annual meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April, 1963. * Supported by grants from the National Cancer Institute (C-2316), the American Cancer Society, and the National Science Foundation (G-20025). 3 American Cancer Society Professor of Biochemistry. 431
432
RAPPORT,
GRAF.
cerasine are also haptens, and that these molecules account for the organ specificity observed with some anti-brain sera (7). This solution to a long-standing problem provides a firm generalization of the relation of immunological reactivity to the chemical structure of lipids, since cerebrosides are naturally occurring glycosphingolipids with the simplest molecular design: they contain a single monosaccharide residue bound in glycosidic linkage to ceramide. This generalization leads to the prediction that all glycosphingolipids have haptenic function, and differences in specificity are attributable to differences in carbohydrate structure. In the course of an investigation of the lipids of human kidney, a glycosphingolipid of more complex structure than cerebroside or cytoside was detected chemically. The substance was purified and screened for its reactivity with about 70 different rabbit antisera prepared against 20 different specimens of human tissue. It was found to react with eight of these antisera. The subsequent isolation of this substance, for which we propose the name cytolipin K, and its chemical and immunological reactions are the subject of this report. MATERIALS
AND
METHODS
The methods used in this laboratory have been adequately described in previous papers of this series (4, 5, 6, 9, 15, 18, 23). Lipid Extracts. Total lipid extracts were prepared from human tissues, obtained at autopsy or surgery, by homogenization with 20 volumes of chloroform-methanol (2: 1) followed by washing with water (8). For isolation studies involving large quantities of kidney tissue, the tissue was first dehydrated with acetone so that smaller volumes of chloroform-methanol would be required (9). Chromatography. Chromatographic separations on columns of either silicic acid or magnesium silicate (Florisil) were carried out as described earlier (4,9). Thin-layer chromatography on silica gel G (Merck) was done in the usual way (9, 10) with chloroform-methanol-water (75:25:4) as developing solvent. Lipids were detected with iodine vapor or by the charring technique using sulfuric acid and heat. Analytical Methods. Phosphorus was determined by a modification of the method of Beveridge and Johnson (II), anthrone by the method of Radin et al. (12), carbohydrate reses reiduleased on hy-
AND
SCHNEIDER
drolysis by densitometric evaluation against standard substances after separation by paper chromatography (13), and long-chain base by the method of Lauter and Trams (14). Fatty acids were determined by gas-liquid chromatography of the methyl esters, with a 6-foot column of 15% ethylene glycol succinate on Chromosorb W (80-100 mesh), and a Barber-Colman model 10 apparatus containing a strontium-90 detector. The column temperature was 185”C., and argon pressure was 30 lbs. per square inch. Immunological Methods. The preparation of particulate fractions of tissues used as immunizing antigens, immunization schedules, preparation and preservation of antisera, and formulation of lipid test antigens were described previously (6). F or combination immunization with total tissue lipid, 15-20 mg. of water-washed lipid, combined with 1 ml. of swine serum diluted 1:5 with saline, was used for each of the six intravenous injections administered over a a-week period. Serological analysis by complement fixation was performed in the usual way (6, 15). The method of transforming complement-fixation data into isofixation curves has been described in detail (15). RESULTS
CHEMICAL
DETECTION OF A NOVEL IN KIDNEY
LIPID
Silicic acid column chromatography was carried out on total lipids extracted from human kidney. The experiment was designed to purify the component responsible for reactions observed between the total lipid and an antiserum against lung carcinoma (16). Column fractions were monitored by thin-layer chromatography and showed the presence of a slow-moving component (Rf - 0.17) that was eluted by TiO% ethanol in hexane but not by 40 % ethanol in hexane (Fig. 1). This component appeared to be a glycolipid from the character of its reaction with sulfuric acid. The lipid fraction did not, however, react with this particular antiserum reagent, even in the presence of auxiliary lipid. SCREENIXG
WITH AXTISERA
The lipid fraction containing this component was purified further by chromatography on Florisil. A test antigen was formulated by addition of lecithin-cholesterol (1: 1 w/w) in a proportion of 100 parts by weight. Antisera prepared against human tissue particulates or human tissue lipids (com-
IMMUNOCHEMICAL
STUDIES
OF
ORGAN
AND
TUMOR
LIPIDS.
XIII
433
FIG. 1. Thin-layer chromatogram on silica gel G of human kidney lipids separated on a column of silicic acid. Lanes 1 through 9 respectively are fractions eluted successively with hexane-ethanol mixtures lOO:O, 90:10,80:20,70:30,60:40,50:50,40:60,30:70, andO:lOO. Lane 10 is a fraction eluted with methanol; the three spots just above the origin correspond to lecithin, sphingomyelin, and lysolecithin. Lane PN contains a sample of phrenosine; lane S contains a specimen of crude sulfatide. Cytolipin K is seen in lane 6. The method of detection was sulfuric acid spray followed by heating at 140”. The developing solvent was chloroform-methanol-water 75:25:4.
bin&ion immunization) were then tested for their reactivity by complement fixation at a sensitivity level of 6 units of complement. The test antigen was not anticomplemen-
tary. Tests were conducted with 11 antisera to lung carcinoma, 9 to myeloid tumor, 8 to hypernephroma, 6 to breast carcinoma, 3 to reticulum cell sarcoma, 2 to multiple
434
RAPPORT,
GRAF,
myeloma-kidney, 2 to multiple myelomaspleen, 2 to malignant melanoma, 1 to larynx carcinoma, 1 to choriocarcinoma, 1 to neuroblastoma, 2 to pyelonephritic kidney, 9 to “normal” kidney, 4 to “normal” small intestine, 3 to “normal” spleen, 3 to “normal” brain, 3 to “normal” stomach, and 2 to “normal” colon. A very strong reaction was obtained with one of the antisera to multiple myeloma-kidney (Anti-MM Kid), good reactions were obtained with an antiserum to pyelonephritic kidney (Anti-P?J Kid) and a second antiserum to multiple myelomakidney. Weaker reactions were seen with a second antiserum to pyelonephritic kidney and four antisera to “normal” human kidantisera were ney, and the remaining negative. ISOLATION
OF CYTOLIPIN
K
Step 1. h’xtraction of Lipid. Human kidney tissue from 27 autopsy caseswas trimmed, and the resulting 2915 g. were cubed, washed three times with 4-liter volumes of distilled water, and then homogenized (Servall Omnimixer) with 4 liters of acetone. The residue, collected by filtration, was retreated with 4 liters of acetone, filtered, and dried in air for 5 hours. The dry tissue (493 g.) was extracted with 4 liters of chloroform-methanol (2:l v/v) containing 200 ml. of water, and then again with the same solvent. After filtering, the combined filtrates were washed with ten volumes of water at 4” (8). The interfacial fluff was separated by freezing and filtration, and the chloroform was removed to give 34.1 g. of lipid. Step b. First Chromatographic Separation on Silicic Acid. A portion of kidney lipid (5.4 g.) was processedat Ti“ on a column prepared from 450 g. of silicic acid (Baker’s) and 45 g. of Celite 535. Several fractions eluted with hexane-ethanol mixtures varying from 100: 0 through 60: 40 were discarded, and the fraction eluted with 2500 ml. of hexane-ethanol (50: 50) was collected (425 mg.; P, 2.69%). Step 3. Chromatographic Separation on Magnesium Silicate (Florisil). This lipid fraction (420 mg.) was processed at 5” on a column of 100 g. of Florisil. Fractions eluted with chloroform-methanol mixtures varying
AND
SCHNEIDER
from 100:0 through 45: 55 were discarded. Fractions eluted with 2000 ml. of chloroformmethanol mixtures varying from 40: 60 to 0: 100 were collected and combined (228 mg.; P, 0.4%). Step 4. SecondChromatographic Separation on Silicic Acid. The fraction obtained from Florisil (227 mg.) was processed at 5” on a column of 50 g. of silicic acid (“Unisil”) measuring 35 X 2% cm. The lipid was placed on the column in chloroform solution; several fractions eluted with chloroformmethanol mixtures varying from 100:0 to 78: 22 were discarded. The cytolipin K was collected in 21 fractions of 20 ml. each with chloroform-methanol 74 :26 and 72 :28. The material, chromatographically homogeneous on thin layer plates, weighed 73 mg., and corresponded to a yield of 160 pg. of cytolipin K per gram of fresh kidney tissue. Step 5. Recrystallization and Reprecipitation. The above fraction was recrystallized from 5 ml of hot ethanol between 80” and 5”, and the 39 mg. of crystals were reprecipitated four times from pyridine-acetone (1 ml. + 6 ml., 1 ml. + 3 ml., 1 ml. + 3.5 ml., 1 ml. + 3.5 ml.). The final supernatant fraction (2.8 mg.) then appeared as homogeneousas the precipitate (32.2 mg.) indicating the purity of the final fraction (17). A summary of the isolation steps is shown in Table I. PHYSICAL
PROPERTIES
Cytolipin K is soluble in chloroform, pyridine, hot methanol, and hot ethanol. It is insoluble in ether, acetone, and poorly soluble in cold methanol and cold ethanol. It forms transparent gelsin water and turbid solutions when these are warmed. The specific rotation at 22” and 589 rnp (Keston polarimeter) was f28” (c, 0.76% in pyridine). Migration behavior compared with other lipids is shown in Fig. 2; the migration rate relative to that of phrenosine (Rphren)was 0.24 with CHClz-CHSOH-Hz0 (75:25:4). CHEMICAL
PROPERTIES
Chemical analysis showed phosphorus, < 0.03 %; carbohydrate, strongly positive (anthrone absorbancy, l%, 1 cm., at 625
IMMUSOCHEMICAL
STIJI)IES
OF
ORGAX
TABLE METHOD
OF ISOL.YTION
AND
TUMOR
LIPII>S.
435
XIII
I OF CYTOLIPIN
K Lipid recovery (mg./kg. fresh kidney tissue)
Procedure
1. Human kidney is dehydrated with acetone. Lipids are extracted from the dry residue with chloroform-methanol-water and then washed with water. 2. Chromatography on silicic acid at 5°C. Elution with ethanolhexane (1:l). 3. Chromatography on magnesium silicate (Florisil) at 5°C. Elution with chloroform-methanol (40:60 to 0: 100). 4. Chromatography on silicic acid at 5°C. Elution with chloroformmethanol (74:26 to 72:28). Chromatographically homogeneous fraction obtained. 5. Recrystallization and reprecipitation (a) ethanol (b) pyridineacetone (4X).
11,700
Phosphorus content
(70)
2.14
020
2.69
499
0.41
160
<0.03
mp : 128) ; ninhydrin reaction (18), negative ; sialic acid (19),
acids (CWO, GUI,
CXA, CIH,
C&:2, CWI,
C?z+), 10.3. No hydroxy fatty acids mere detected. These properties indicate that cytolipin K is a neutral glycosphingolipid having the structure of a ceramide tetrasaccharide. I~~N~cHE~~L
PROPERTIES
Isofixation curves with two antisera to human tissues (anti-RIM Kid and anti-W
Kid) are shown in Fig. 3. For these tests the cytolipin K test antigen was formulated by combining it with 100 parts by weight of a 1: 2 (w/w) mixture of lecithin and cholesterol as auxiliary lipid. These curves show reactions with cytolipin K, in the region of antibody excess, at a level of 0.003-0.005 pg., a range very similar to that obtained in the cytolipin H-anti-cytolipin H and galactocerebroside - anti - galactocerebroside systems. n’either antiserum reacted with cytolipin H, galactocerebroside, or cardiolipin, and neither contained Forssman antibody. Both sera reacted with a purified preparation of cytolipin G (23), but an antiserum containing anti-cytolipin G did not react with cytolipin K. The following auxiliary lipid mixtures were tested with both antisera: lecithincholesterol 2: 1, 1: 1, and 1: 2 (w/w). These were combined in weight proportions of 40, 80, 100, and 120 times that of cytolipin K. Although the two antisera gave a different pattern of response to these changes in auxiliary lipid, optimal reactivity (based both on intensity as well as sensitivity) was found with lecithin-cholesterol 1: 2 at a level of 100 times the quantity of hapten. Cytolipin K was subsequently found to react well with 6 of 8 anti-kidney sera prepared against total kidney lipid plus swine serum. The antibodies produced this way appeared to be relatively unstable. No reactions were observed between cytolipin K
436
RAPPORT,
GRAF,
FIG. 2. Thin-layer chromatogram of cytolipin K and several reference substances. Sp is sphingomyelin (GO pg.); K, H, and GC are cytolipin K, cytolipin H, and spinal cord galactocerebrosides, respectively (40 pg.). G is a highly purified intestinal lipid fraction with cytolipin G activity (the upper spot gave no reactions with antisera containing anti-cytolipin K; cytolipin G activity was restricted to the lower spot). Same conditions as Fig. 1.
and five antisera prepared against a particulate fraction of the same kidney. In Ouchterlony gel-diffusion plates, single lines of precipitate were observed (6-7 days) between undiluted anti-MM Kid serum and cytolipin K to a limiting dilution approximating 20 pg. per milliliter without addition of auxiliary lipid.
AND
SCHNEIDER
. I 0.01 QUANTITY
*
I I I ? 0.02 0.03 0.04 0.05 OF ANTIGEN, MICROGRAMS
FIG. 3. Isofixation curves of cytolipin K with anti-human tissue sera: (0) antiserum against lipids from the kidney of a patient with multiple myeloma (anti-MM Kid); (0) antiserum against lipids from the kidney of a patient with pyelonephritis (anti-PN Kid). Tests conducted with 6 units of complement (0.0075 ml. of guinea pig serum).
with human tissue fractions. The method by means of which this haptenic function was established is novel. The usual order of events is to detect immunological activity DISCUSSION through the reactions of a particular antiThe observations described in this paper serum with a mixture of tissue components, show that in normal human kidney tissue and then to isolate the reactive antigen using there is present a ceramide tetrasaccharide the antiserum as a reagent. In the present containing a fatty acid, a long-chain base instance, the presence of cytolipin K was (sphingosine), glucose, galactose, and galac- first detected by chemical methods, the tosamine (probably as the N-acetyl deriva- substance was isolated, and then its immunotive) in the molar proportions 1: 1: 1:2: 1 logical activity was established by studying respectively, and that this substance has its reactions with a large number of antisera. The principle on which this inversion in haptenic function since it reacts with antibodies that may arise by injecting rabbits procedure was based was that all glyco-
IMMUNOCHEMICAL
STUDIES
OF
sphingolipids may be expected to function as haptens, a principle that was a direct consequence of the finding that galactocerebroside, the simplest model, had such activity (7). This study of cytolipin K serves to confirm the principle, and establishes a general method for the detection of lipid haptens in which the initial efforts can be restricted to chemical methods. The isolation of cytolipin K brings the number of immunologically distinctive lipid haptens known to be present in human t.issues to at least six: cytolipin K, cytolipin H, cytolipin G, galactocerebroside, cardiolipin, and Forssman hapten. Of these compounds, three, cytolipin H, galactocerebroside, and cytolipin K are known to be neutral glycosphingolipids. E’orssman hapten is very likely to be in this group. Cytolipin G is still undefined chemically. Brain ganglioof compounds whose side, a mixture immunological properties have recently been described (24), is not included since the serological activity is weaker by two orders of magnitude, and the distinctiveness (or specificity) of its reactions cannot be assessed. Molecules with a residue composition similar to cytolipin Ii (ceramide + glucose + galactosamine + 2 galactose) have been found previously to originate from two different sources. One of these is the substance isolated frown human red cells and called “globoside” (25), and the other has been obtained from brain ganglioside by mild acid hydrolysis to remove all sialic acid residues (“asialoganglioside”) (26). Structural relationships among these molecules must still be established, and immunological methods nlay provide a useful tool for this purpose. A recent characterization of the glycosphingolipid content of human kidney in a lipidosis called E’abry’s disease showed the lipid to contain ceramide-glucose-galactosegalactose (27). The fatty acid content of this compound was similar to that found for cytolipin Ii (81 C;: as CZ2:” + C,,:, + C,,:,). If this subst,ance is directly related to cytolipin K metabolically, then the galactosamine residue of the latter should occupy a terminal position, and the structure of
ORGAN
AND
TUMOR
LIPIDS.
437
XIII
cytolipin K would thus be much more closely related to globoside than to “asialoganglioside.” Since terminal carbohydrate residues have such an important influence on serological specificity, it may also be predicted that a substantial degree of cross-reactivity between cytolipin K and globoside will be found, in the event that they do not prove to be identical. ACKNOWLEDGMENTS We wish to thank Dr. Lewis Gidez for the analysis of fatty acids. We also gratefully acknowledge the technical assistance of Mrs. Anna Pollard and Miss Loretta Giuffra. REFERENCES 1. P.\NGBORN, (1942).
M.
C.,
d.
Riol.
Chem.
143,
247
M. G., AVuture 182, 946 (1958). M., J. Lipid Kes. 2, 25 (1961). 4. ROI’ORT, M. M., GRAF, L., SKII’SKI, T’. P., aND ALO~YZO, N. F., Cancer 12, 438 (1959). 5. RAITORT, M. M., GR~F, L., AND YARIV, J., Arch. Biochem. Biophys. 92,438 (1961). G. GRAF, L., BND RMTORT, M. M., Cuncer IZes. 20, 546 (1960). 7. JOFFE, S., RAIVORT, &I. M., ,~ND GR.ZY, L., :Vatzrre 197, 60 (1963). 8. FOLCH, J., AGCOLX, I., LEES, M., ME~TH, J. A., .ZND LEBARON, F. N., J. Biol. Chem. 191,833 (1951). 9. RAPPORT, M. M., SCHNEIDER, H., AND GR~F, L., J. Hiol. Chem. 237, 1056 (1962). 10. MANGOLD, H. K., J. Am. Oil Chem. Sot. 38, 708 (1961). 11. BEVERIDGE, J. M. R., END JOHNSON, 9. E., Can. J. Res. 27 (Sect. E), 159 (1949). 12. RADIN, N. S., LZVIN, F. B., .ZND BROWN, ,J. R., J. Bid. Chem. 217, 789 (1955). 13. NOLAN, C., AND SMITH, E. L., J. Viol. Chem. 237, 44G (1962). 14. hLTER, c. J., ,\ND TRS~, E. G., J. Lipid Res. 3, 136 (1962). 15. R.WPORT, M. M., ,UYD GR~F, L., Ann. X. Y. Acad. sci. 69,608 (1957). 16. R\PIWRT, M. M., .UXD GRAF, L., Cancer lies. 21, 1225 (1961). 17. Itl~ppo~~r, M. M., .IND LERNER, B., J. Biol. Chew. 232, G3 (1958). 18. R.\~JIY~I~, M. M., GRM, L., ‘YND ALONZO, X. F., J. Lipid Res. 1,301 (1960). 19. W.YRREN, I,., J. 13iol. Chem. 234, 1971 (1959). 2. MACF~RL.ZNE,
3. RAPI’ORT,
M.
438
RAPPORT,
GRAF,
20. JEANES, A., WISE, C. S., AND DIMLER, R. J., Anal. Chem. 23,415 (1951). 21. STOFFYN, P. J., AND JEANLOZ, R. W., Arch. Biochem. Biophys. 62, 373 (1954). 22. SWEELEY, C. C., AND MOSCATELLI, E. A., J. Lipid Res. 1, 40 (1959). 23. GRAF, L., RAPPORT, M.M., AND BR.4NDT, R., Cancer Res. 21,1532 (1961).
AND
SCHNEIDER
24. YOKOY.~M~, M., TR.IMS, E. G., AND BRADY, R. O., J. Immunol. 90,372 (1963). 25. Y.4~~1c.4w.4, T., YOKOYAMA, S., AND HANDA, N., J. Biochem. (Tokyo) 63, 28 (1963). 26. SVENNERHOLM, L., Biochem. Biophys. Res. Commun. 9, 436 (1962). 27. SWEELEY, C. C., .INU KLIONSKY, B., J. Biol. Chem. 238, PC3148 (1963).
OF
BIOCHEMISTRY
AND
Immunochemical XIII. isolation
BIOPHYSICS
106,
Studies
of Cytolipin
431-438
of Organ
From
the Division
of Virology Department
and
K, a Glycosphingolipid Kidney’1
RI. >I. RAPPORT,3
(1964)
LISELOTTE
Tumor
Hapten
Lipids
Present
’
GRAF
AND
H. SCHNEIDER
and Immunology, Sloan-Kettering Institute for Cancer of Biochemistry, Albert Einstein College of Medicine, Yeshiva Universit?y, New York, New York Received
November
in Human
Research,
and
28, 1963
A neutral glycosphingolipid containing fatty acid-sphingosine (long chain base)glucose-galactose-galactosamine in the molar proportions 1: 1: 1: 2: 1 has been isolated from normal human kidney by successive chromatographic separations on silicic acid, magnesium silicate, and silicic acid. The isolated substance, designated cytolipin K, was shown to have haptenic properties by screening it for reactivity with a large number of rabbit antisera prepared against diverse human tissues. Good reactions were observed in a small number of instances. The two most reactive antisera were obtained by combination immunization with lipids from a multiple myeloma-affected kidney and a pyelonephritic kidney, and with these antisera and optimal addition of auxiliary lipid, 0.003-0.005 pg. of cytolipin K was readily measured. It was shown that the immunological reactivity of cytolipin K was distinct with respect to five other lipid haptens present in human tissues, namely, cytolipin H, cytolipin G, galactocerebroside, cardiolipin, and Forssman hapten.
Knowledge of the chemical structure of lipids that display immunological activity has been scant until recently. The first definitive work resulted from the isolation of cardiolipin (l), whose structure is now considered to be that of a complex phosphatidic acid, diphosphatidyl glycerol (2). The reactivity of cardiolipin with specific antibody has made it a substance of considerable value in diagnosis of disease, but cardiolipin has not provided a general model of lipid structures with immunological activity. In contrast, many of the earlier studies of reactive
lipids such as Forssman hapten and the brain specific hapten suggested that lipids containing carbohydrate residues were involved in immunological reactions (3). These studies were brought into sharp focus with isolation of cytolipin H from human cancer tissue (4) and its subsequent structural identification as ceramide lactoside (5). Cytolipin H is the molecule that accounts most frequently for the substantial differences observed between the reactions of lipid extracts of normal tissues and those of cancer tissues when studied with antisera to the latter (6). Immunological specificity of this molecule is conferred principally, if not exclusively, by the two carbohydrate residues (glucose and galactose). The two lipid residues (fatty acid and sphingosine) play their role most probably in the localization of the molecule within cell membranes. Very recently it was demonstrated that galactocerebrosides such as phrenosine and
1 This work was presented in part at the 47th annual meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April, 1963. * Supported by grants from the National Cancer Institute (C-2316), the American Cancer Society, and the National Science Foundation (G-20025). 3 American Cancer Society Professor of Biochemistry. 431
432
RAPPORT,
GRAF.
cerasine are also haptens, and that these molecules account for the organ specificity observed with some anti-brain sera (7). This solution to a long-standing problem provides a firm generalization of the relation of immunological reactivity to the chemical structure of lipids, since cerebrosides are naturally occurring glycosphingolipids with the simplest molecular design: they contain a single monosaccharide residue bound in glycosidic linkage to ceramide. This generalization leads to the prediction that all glycosphingolipids have haptenic function, and differences in specificity are attributable to differences in carbohydrate structure. In the course of an investigation of the lipids of human kidney, a glycosphingolipid of more complex structure than cerebroside or cytoside was detected chemically. The substance was purified and screened for its reactivity with about 70 different rabbit antisera prepared against 20 different specimens of human tissue. It was found to react with eight of these antisera. The subsequent isolation of this substance, for which we propose the name cytolipin K, and its chemical and immunological reactions are the subject of this report. MATERIALS
AND
METHODS
The methods used in this laboratory have been adequately described in previous papers of this series (4, 5, 6, 9, 15, 18, 23). Lipid Extracts. Total lipid extracts were prepared from human tissues, obtained at autopsy or surgery, by homogenization with 20 volumes of chloroform-methanol (2: 1) followed by washing with water (8). For isolation studies involving large quantities of kidney tissue, the tissue was first dehydrated with acetone so that smaller volumes of chloroform-methanol would be required (9). Chromatography. Chromatographic separations on columns of either silicic acid or magnesium silicate (Florisil) were carried out as described earlier (4,9). Thin-layer chromatography on silica gel G (Merck) was done in the usual way (9, 10) with chloroform-methanol-water (75:25:4) as developing solvent. Lipids were detected with iodine vapor or by the charring technique using sulfuric acid and heat. Analytical Methods. Phosphorus was determined by a modification of the method of Beveridge and Johnson (II), anthrone by the method of Radin et al. (12), carbohydrate reses reiduleased on hy-
AND
SCHNEIDER
drolysis by densitometric evaluation against standard substances after separation by paper chromatography (13), and long-chain base by the method of Lauter and Trams (14). Fatty acids were determined by gas-liquid chromatography of the methyl esters, with a 6-foot column of 15% ethylene glycol succinate on Chromosorb W (80-100 mesh), and a Barber-Colman model 10 apparatus containing a strontium-90 detector. The column temperature was 185”C., and argon pressure was 30 lbs. per square inch. Immunological Methods. The preparation of particulate fractions of tissues used as immunizing antigens, immunization schedules, preparation and preservation of antisera, and formulation of lipid test antigens were described previously (6). F or combination immunization with total tissue lipid, 15-20 mg. of water-washed lipid, combined with 1 ml. of swine serum diluted 1:5 with saline, was used for each of the six intravenous injections administered over a a-week period. Serological analysis by complement fixation was performed in the usual way (6, 15). The method of transforming complement-fixation data into isofixation curves has been described in detail (15). RESULTS
CHEMICAL
DETECTION OF A NOVEL IN KIDNEY
LIPID
Silicic acid column chromatography was carried out on total lipids extracted from human kidney. The experiment was designed to purify the component responsible for reactions observed between the total lipid and an antiserum against lung carcinoma (16). Column fractions were monitored by thin-layer chromatography and showed the presence of a slow-moving component (Rf - 0.17) that was eluted by TiO% ethanol in hexane but not by 40 % ethanol in hexane (Fig. 1). This component appeared to be a glycolipid from the character of its reaction with sulfuric acid. The lipid fraction did not, however, react with this particular antiserum reagent, even in the presence of auxiliary lipid. SCREENIXG
WITH AXTISERA
The lipid fraction containing this component was purified further by chromatography on Florisil. A test antigen was formulated by addition of lecithin-cholesterol (1: 1 w/w) in a proportion of 100 parts by weight. Antisera prepared against human tissue particulates or human tissue lipids (com-
IMMUNOCHEMICAL
STUDIES
OF
ORGAN
AND
TUMOR
LIPIDS.
XIII
433
FIG. 1. Thin-layer chromatogram on silica gel G of human kidney lipids separated on a column of silicic acid. Lanes 1 through 9 respectively are fractions eluted successively with hexane-ethanol mixtures lOO:O, 90:10,80:20,70:30,60:40,50:50,40:60,30:70, andO:lOO. Lane 10 is a fraction eluted with methanol; the three spots just above the origin correspond to lecithin, sphingomyelin, and lysolecithin. Lane PN contains a sample of phrenosine; lane S contains a specimen of crude sulfatide. Cytolipin K is seen in lane 6. The method of detection was sulfuric acid spray followed by heating at 140”. The developing solvent was chloroform-methanol-water 75:25:4.
bin&ion immunization) were then tested for their reactivity by complement fixation at a sensitivity level of 6 units of complement. The test antigen was not anticomplemen-
tary. Tests were conducted with 11 antisera to lung carcinoma, 9 to myeloid tumor, 8 to hypernephroma, 6 to breast carcinoma, 3 to reticulum cell sarcoma, 2 to multiple
434
RAPPORT,
GRAF,
myeloma-kidney, 2 to multiple myelomaspleen, 2 to malignant melanoma, 1 to larynx carcinoma, 1 to choriocarcinoma, 1 to neuroblastoma, 2 to pyelonephritic kidney, 9 to “normal” kidney, 4 to “normal” small intestine, 3 to “normal” spleen, 3 to “normal” brain, 3 to “normal” stomach, and 2 to “normal” colon. A very strong reaction was obtained with one of the antisera to multiple myeloma-kidney (Anti-MM Kid), good reactions were obtained with an antiserum to pyelonephritic kidney (Anti-P?J Kid) and a second antiserum to multiple myelomakidney. Weaker reactions were seen with a second antiserum to pyelonephritic kidney and four antisera to “normal” human kidantisera were ney, and the remaining negative. ISOLATION
OF CYTOLIPIN
K
Step 1. h’xtraction of Lipid. Human kidney tissue from 27 autopsy caseswas trimmed, and the resulting 2915 g. were cubed, washed three times with 4-liter volumes of distilled water, and then homogenized (Servall Omnimixer) with 4 liters of acetone. The residue, collected by filtration, was retreated with 4 liters of acetone, filtered, and dried in air for 5 hours. The dry tissue (493 g.) was extracted with 4 liters of chloroform-methanol (2:l v/v) containing 200 ml. of water, and then again with the same solvent. After filtering, the combined filtrates were washed with ten volumes of water at 4” (8). The interfacial fluff was separated by freezing and filtration, and the chloroform was removed to give 34.1 g. of lipid. Step b. First Chromatographic Separation on Silicic Acid. A portion of kidney lipid (5.4 g.) was processedat Ti“ on a column prepared from 450 g. of silicic acid (Baker’s) and 45 g. of Celite 535. Several fractions eluted with hexane-ethanol mixtures varying from 100: 0 through 60: 40 were discarded, and the fraction eluted with 2500 ml. of hexane-ethanol (50: 50) was collected (425 mg.; P, 2.69%). Step 3. Chromatographic Separation on Magnesium Silicate (Florisil). This lipid fraction (420 mg.) was processed at 5” on a column of 100 g. of Florisil. Fractions eluted with chloroform-methanol mixtures varying
AND
SCHNEIDER
from 100:0 through 45: 55 were discarded. Fractions eluted with 2000 ml. of chloroformmethanol mixtures varying from 40: 60 to 0: 100 were collected and combined (228 mg.; P, 0.4%). Step 4. SecondChromatographic Separation on Silicic Acid. The fraction obtained from Florisil (227 mg.) was processed at 5” on a column of 50 g. of silicic acid (“Unisil”) measuring 35 X 2% cm. The lipid was placed on the column in chloroform solution; several fractions eluted with chloroformmethanol mixtures varying from 100:0 to 78: 22 were discarded. The cytolipin K was collected in 21 fractions of 20 ml. each with chloroform-methanol 74 :26 and 72 :28. The material, chromatographically homogeneous on thin layer plates, weighed 73 mg., and corresponded to a yield of 160 pg. of cytolipin K per gram of fresh kidney tissue. Step 5. Recrystallization and Reprecipitation. The above fraction was recrystallized from 5 ml of hot ethanol between 80” and 5”, and the 39 mg. of crystals were reprecipitated four times from pyridine-acetone (1 ml. + 6 ml., 1 ml. + 3 ml., 1 ml. + 3.5 ml., 1 ml. + 3.5 ml.). The final supernatant fraction (2.8 mg.) then appeared as homogeneousas the precipitate (32.2 mg.) indicating the purity of the final fraction (17). A summary of the isolation steps is shown in Table I. PHYSICAL
PROPERTIES
Cytolipin K is soluble in chloroform, pyridine, hot methanol, and hot ethanol. It is insoluble in ether, acetone, and poorly soluble in cold methanol and cold ethanol. It forms transparent gelsin water and turbid solutions when these are warmed. The specific rotation at 22” and 589 rnp (Keston polarimeter) was f28” (c, 0.76% in pyridine). Migration behavior compared with other lipids is shown in Fig. 2; the migration rate relative to that of phrenosine (Rphren)was 0.24 with CHClz-CHSOH-Hz0 (75:25:4). CHEMICAL
PROPERTIES
Chemical analysis showed phosphorus, < 0.03 %; carbohydrate, strongly positive (anthrone absorbancy, l%, 1 cm., at 625
IMMUSOCHEMICAL
STIJI)IES
OF
ORGAX
TABLE METHOD
OF ISOL.YTION
AND
TUMOR
LIPII>S.
435
XIII
I OF CYTOLIPIN
K Lipid recovery (mg./kg. fresh kidney tissue)
Procedure
1. Human kidney is dehydrated with acetone. Lipids are extracted from the dry residue with chloroform-methanol-water and then washed with water. 2. Chromatography on silicic acid at 5°C. Elution with ethanolhexane (1:l). 3. Chromatography on magnesium silicate (Florisil) at 5°C. Elution with chloroform-methanol (40:60 to 0: 100). 4. Chromatography on silicic acid at 5°C. Elution with chloroformmethanol (74:26 to 72:28). Chromatographically homogeneous fraction obtained. 5. Recrystallization and reprecipitation (a) ethanol (b) pyridineacetone (4X).
11,700
Phosphorus content
(70)
2.14
020
2.69
499
0.41
160
<0.03
mp : 128) ; ninhydrin reaction (18), negative ; sialic acid (19),
acids (CWO, GUI,
CXA, CIH,
C&:2, CWI,
C?z+), 10.3. No hydroxy fatty acids mere detected. These properties indicate that cytolipin K is a neutral glycosphingolipid having the structure of a ceramide tetrasaccharide. I~~N~cHE~~L
PROPERTIES
Isofixation curves with two antisera to human tissues (anti-RIM Kid and anti-W
Kid) are shown in Fig. 3. For these tests the cytolipin K test antigen was formulated by combining it with 100 parts by weight of a 1: 2 (w/w) mixture of lecithin and cholesterol as auxiliary lipid. These curves show reactions with cytolipin K, in the region of antibody excess, at a level of 0.003-0.005 pg., a range very similar to that obtained in the cytolipin H-anti-cytolipin H and galactocerebroside - anti - galactocerebroside systems. n’either antiserum reacted with cytolipin H, galactocerebroside, or cardiolipin, and neither contained Forssman antibody. Both sera reacted with a purified preparation of cytolipin G (23), but an antiserum containing anti-cytolipin G did not react with cytolipin K. The following auxiliary lipid mixtures were tested with both antisera: lecithincholesterol 2: 1, 1: 1, and 1: 2 (w/w). These were combined in weight proportions of 40, 80, 100, and 120 times that of cytolipin K. Although the two antisera gave a different pattern of response to these changes in auxiliary lipid, optimal reactivity (based both on intensity as well as sensitivity) was found with lecithin-cholesterol 1: 2 at a level of 100 times the quantity of hapten. Cytolipin K was subsequently found to react well with 6 of 8 anti-kidney sera prepared against total kidney lipid plus swine serum. The antibodies produced this way appeared to be relatively unstable. No reactions were observed between cytolipin K
436
RAPPORT,
GRAF,
FIG. 2. Thin-layer chromatogram of cytolipin K and several reference substances. Sp is sphingomyelin (GO pg.); K, H, and GC are cytolipin K, cytolipin H, and spinal cord galactocerebrosides, respectively (40 pg.). G is a highly purified intestinal lipid fraction with cytolipin G activity (the upper spot gave no reactions with antisera containing anti-cytolipin K; cytolipin G activity was restricted to the lower spot). Same conditions as Fig. 1.
and five antisera prepared against a particulate fraction of the same kidney. In Ouchterlony gel-diffusion plates, single lines of precipitate were observed (6-7 days) between undiluted anti-MM Kid serum and cytolipin K to a limiting dilution approximating 20 pg. per milliliter without addition of auxiliary lipid.
AND
SCHNEIDER
. I 0.01 QUANTITY
*
I I I ? 0.02 0.03 0.04 0.05 OF ANTIGEN, MICROGRAMS
FIG. 3. Isofixation curves of cytolipin K with anti-human tissue sera: (0) antiserum against lipids from the kidney of a patient with multiple myeloma (anti-MM Kid); (0) antiserum against lipids from the kidney of a patient with pyelonephritis (anti-PN Kid). Tests conducted with 6 units of complement (0.0075 ml. of guinea pig serum).
with human tissue fractions. The method by means of which this haptenic function was established is novel. The usual order of events is to detect immunological activity DISCUSSION through the reactions of a particular antiThe observations described in this paper serum with a mixture of tissue components, show that in normal human kidney tissue and then to isolate the reactive antigen using there is present a ceramide tetrasaccharide the antiserum as a reagent. In the present containing a fatty acid, a long-chain base instance, the presence of cytolipin K was (sphingosine), glucose, galactose, and galac- first detected by chemical methods, the tosamine (probably as the N-acetyl deriva- substance was isolated, and then its immunotive) in the molar proportions 1: 1: 1:2: 1 logical activity was established by studying respectively, and that this substance has its reactions with a large number of antisera. The principle on which this inversion in haptenic function since it reacts with antibodies that may arise by injecting rabbits procedure was based was that all glyco-
IMMUNOCHEMICAL
STUDIES
OF
sphingolipids may be expected to function as haptens, a principle that was a direct consequence of the finding that galactocerebroside, the simplest model, had such activity (7). This study of cytolipin K serves to confirm the principle, and establishes a general method for the detection of lipid haptens in which the initial efforts can be restricted to chemical methods. The isolation of cytolipin K brings the number of immunologically distinctive lipid haptens known to be present in human t.issues to at least six: cytolipin K, cytolipin H, cytolipin G, galactocerebroside, cardiolipin, and Forssman hapten. Of these compounds, three, cytolipin H, galactocerebroside, and cytolipin K are known to be neutral glycosphingolipids. E’orssman hapten is very likely to be in this group. Cytolipin G is still undefined chemically. Brain ganglioof compounds whose side, a mixture immunological properties have recently been described (24), is not included since the serological activity is weaker by two orders of magnitude, and the distinctiveness (or specificity) of its reactions cannot be assessed. Molecules with a residue composition similar to cytolipin Ii (ceramide + glucose + galactosamine + 2 galactose) have been found previously to originate from two different sources. One of these is the substance isolated frown human red cells and called “globoside” (25), and the other has been obtained from brain ganglioside by mild acid hydrolysis to remove all sialic acid residues (“asialoganglioside”) (26). Structural relationships among these molecules must still be established, and immunological methods nlay provide a useful tool for this purpose. A recent characterization of the glycosphingolipid content of human kidney in a lipidosis called E’abry’s disease showed the lipid to contain ceramide-glucose-galactosegalactose (27). The fatty acid content of this compound was similar to that found for cytolipin Ii (81 C;: as CZ2:” + C,,:, + C,,:,). If this subst,ance is directly related to cytolipin K metabolically, then the galactosamine residue of the latter should occupy a terminal position, and the structure of
ORGAN
AND
TUMOR
LIPIDS.
437
XIII
cytolipin K would thus be much more closely related to globoside than to “asialoganglioside.” Since terminal carbohydrate residues have such an important influence on serological specificity, it may also be predicted that a substantial degree of cross-reactivity between cytolipin K and globoside will be found, in the event that they do not prove to be identical. ACKNOWLEDGMENTS We wish to thank Dr. Lewis Gidez for the analysis of fatty acids. We also gratefully acknowledge the technical assistance of Mrs. Anna Pollard and Miss Loretta Giuffra. REFERENCES 1. P.\NGBORN, (1942).
M.
C.,
d.
Riol.
Chem.
143,
247
M. G., AVuture 182, 946 (1958). M., J. Lipid Kes. 2, 25 (1961). 4. ROI’ORT, M. M., GRAF, L., SKII’SKI, T’. P., aND ALO~YZO, N. F., Cancer 12, 438 (1959). 5. RAITORT, M. M., GR~F, L., AND YARIV, J., Arch. Biochem. Biophys. 92,438 (1961). G. GRAF, L., BND RMTORT, M. M., Cuncer IZes. 20, 546 (1960). 7. JOFFE, S., RAIVORT, &I. M., ,~ND GR.ZY, L., :Vatzrre 197, 60 (1963). 8. FOLCH, J., AGCOLX, I., LEES, M., ME~TH, J. A., .ZND LEBARON, F. N., J. Biol. Chem. 191,833 (1951). 9. RAPPORT, M. M., SCHNEIDER, H., AND GR~F, L., J. Hiol. Chem. 237, 1056 (1962). 10. MANGOLD, H. K., J. Am. Oil Chem. Sot. 38, 708 (1961). 11. BEVERIDGE, J. M. R., END JOHNSON, 9. E., Can. J. Res. 27 (Sect. E), 159 (1949). 12. RADIN, N. S., LZVIN, F. B., .ZND BROWN, ,J. R., J. Bid. Chem. 217, 789 (1955). 13. NOLAN, C., AND SMITH, E. L., J. Viol. Chem. 237, 44G (1962). 14. hLTER, c. J., ,\ND TRS~, E. G., J. Lipid Res. 3, 136 (1962). 15. R.WPORT, M. M., ,UYD GR~F, L., Ann. X. Y. Acad. sci. 69,608 (1957). 16. R\PIWRT, M. M., .UXD GRAF, L., Cancer lies. 21, 1225 (1961). 17. Itl~ppo~~r, M. M., .IND LERNER, B., J. Biol. Chew. 232, G3 (1958). 18. R.\~JIY~I~, M. M., GRM, L., ‘YND ALONZO, X. F., J. Lipid Res. 1,301 (1960). 19. W.YRREN, I,., J. 13iol. Chem. 234, 1971 (1959). 2. MACF~RL.ZNE,
3. RAPI’ORT,
M.
438
RAPPORT,
GRAF,
20. JEANES, A., WISE, C. S., AND DIMLER, R. J., Anal. Chem. 23,415 (1951). 21. STOFFYN, P. J., AND JEANLOZ, R. W., Arch. Biochem. Biophys. 62, 373 (1954). 22. SWEELEY, C. C., AND MOSCATELLI, E. A., J. Lipid Res. 1, 40 (1959). 23. GRAF, L., RAPPORT, M.M., AND BR.4NDT, R., Cancer Res. 21,1532 (1961).
AND
SCHNEIDER
24. YOKOY.~M~, M., TR.IMS, E. G., AND BRADY, R. O., J. Immunol. 90,372 (1963). 25. Y.4~~1c.4w.4, T., YOKOYAMA, S., AND HANDA, N., J. Biochem. (Tokyo) 63, 28 (1963). 26. SVENNERHOLM, L., Biochem. Biophys. Res. Commun. 9, 436 (1962). 27. SWEELEY, C. C., .INU KLIONSKY, B., J. Biol. Chem. 238, PC3148 (1963).