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Isolation and partial characterization of human kidney gangliosides
284
Biochimica
et Biophysics
0 Elsevier
Scientific
Acta,
Publishing
424 (1976) 284-295 Company, Amsterdam
- Printed
in The Netherlands
BBA 56714
ISOLATION AND PARTIAL CHARACTERIZATION GANGLIOSIDES
HEIKKI
OF HUMAN KIDNEY
RAUVALA
Department of Medical Helsinki 17 (Finland).
(Received
August
Chemistry,
University
of Helsinki,
Siltavuorenpenger
10 A, SF-001
70
4th, 1975)
Summary 1. Eight gangliosides were purified from chloroform/methanol extracts of human kidneys by using modified Folch partition, dialysis, ethanol precipitation, silicic acid column chromatography and preparative thin-layer chromatography. 2. By thin-layer chromatographic behaviour and gas-liquid chromatographic determinations the main gangliosides in human kidney are N-acetylneuraminyllactosylceramide (74% of total) and di-N-acetylneuraminyllactosylceramide (19% of total). 3. Five hexosamine-containing fractions were isolated. Four of them were homogeneous on thin-layer chromatography, and one contained two gangliosides. By gas-liquid chromatography-mass spectrometry it was shown that two gangliosides (together 5% of total) contain glucosamine, and one (1% of total) contains galactosamine. The other of the glucosamine gangliosides contains fucose in addition to the usual sugars found in gangliosides. Of the two remaining hexosamine positive fractions (together 1% of total) one was homogeneous on thin-layer chromatography, the other contained two gangliosides. These two fractions contained both glucosamine and galactosamine. 4. The main long-chain base in all fractions was sphingosine.
Introduction
shown
The complexit,y of the ganglioside patterns in extraneural tissues has been [ 11. Usually, the gangliosides containing lactose as their neutral carAbbreviations GlcNAc,
Svennerholm GMaa
and
nomenclature:
N-acetylglucosamine; [41
is used
Gal,
galactose:
NeuNAc, for
gangliosides.
(N-acetylneuraminyllactosylceramide)
The
nomenclature
sion
[ 5.61.
used
for long-chain
Glc,
glucose;
N-acetylneuraminic The and bases
and
hematosides as GMsb
GalNAc.
acid. of
The
bovine
N-acetylgalactosamine:
shorthand kidney
nomenclature were
designated
(N-glycolylneuraminy~actosylceramide).
oligosaccharides
is that
proposed
by
IUB
Commis-
of as
285
bohydrate portion predominate in extraneural tissues [2,3] . Because of the scarcity of the material and the difficulties in the isolation procedures, relatively few reports have been published concerning the more complex extraneural gangliosides. Wiegandt and Bucking have developed a method to release the sialo-oligosaccharides from gangliosides, and have found a wide variety of gangliosides in several extraneural tissues by their method [3]. In this work, an attempt was made to isolate especially the more complex gangliosides from human kidney as intact molecules. A simplified isolation method was developed, and in addition to the gangliosides containing neuraminyl- and di-neuraminyllactose, five hexosamine-containing ganglioside fractions could be isolated. A preliminary characterization of the ganglioside fractions is presented. Experimental Materials
Human kidneys were obtained at autopsy and stored at -2O”C, if not used immediately. The brain gangliosides used as reference on thin-layer chromatography were made available by Professor E. Klenk (Physiologisch-Chemisches Institut der Universitat Koln, Germany), the hematosides of bovine kidof Medical Chemistry, ney (GM3, and GM3i, ) by Dr. K. Puro (Department University of Helsinki, Finland), the mixture of pig brain gangliosides, and the disialoganglioside of pig brain by Dr. P. Maury (Department of Medical Chemistry, University of Helsinki, Finland). L-fucose, D-galactose, D-glucose, D-mannose, 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, N-acetylneuraminic acid, N-glycolylneuraminic acid, inositol and O-P-D-galactopyranosyl-(1 -+ 4)D-glucopyranose (lactose) were purchased from commercial sources. Other sugars used for standardization of gas-liquid chromatographic determinations were gifts: 2-acetamido2-deoxy-O-P-D-galactopyranosyl( 1 -+ 3)-D-galactopyranose from Dr. D. Shapiro (Rehovoth, Israel), 0-a-L-fucopyranosyl-( 1 + 2)-O-P-D-galactopyranosyl-( 1 + 4)-D-glucopyranose (2’-fucosyllactose), O-O-D-galactopyranosyl-( 1 + 3)-%acetamido-2-deoxy-O-/3-D-glucopyranosyl-(1 + 3)-O-fl-D-galactopyranosyl(1 -+ 4) D-glucopyranose (la&o-N-tetraose) and 0-a-N-acetylneuraminyl-(2 --f 6)-O-p-~galactopyranosyl (1 + 4)-D-glucopyranose (6’-iV-acetylneuraminyllactose) from Dr. A. Gauhe (Heidelberg, Germany). 0-cu-N-acetylneuraminyl-( 2 + 3)-0-@-D -galactopyranosyl-( 1 -+ 4)-D-glucopyranose (3’-N-acetylneuraminyllactose) was prepared in this laboratory [7] . Organic solvents were of analytical reagent quality. Trimethylchlorsilane (Fluka A.G.) and hexamethyldisilazane (Fluka A.G.) were distilled before use. Pyridine was distilled from KOH and stored over a molecular sieve. Absorbents for column chromatography were silicic acid (Anasil-S, Analytical Engineering Laboratories, Inc., Hamden, Conn.) and cellulose (Whatman powder, standard grade, W and P, Bolston, Ltd.), and for thin-layer chromatography silica gel G (E. Merck, A.G., Darmstadt). Dowex 1 X 8 (200/400 mesh) and Dowex 50 X 8 (200/400 mesh) were purchased from Fluka A.G. Preparation
of gangliosides
Capsules and pelvices were removed
from human
kidneys by coarse dissec-
286
tion. The kidney tissue was rinsed and made bloodless by washing several times with 0.9% saline. The rinsed tissue was homogenized in cold acetone and dried to acetone powder. The lipids were extracted and partitioned in the Folch system as described before [8,9]. The lower phases were discarded, the upper phases were dialyzed at -+4”C against running tap water and concentrated by rotary evaporation below 40” C. For further purification, crude ganglioside was dried and precipitated from ethanol at -20°C for 24 h in the concentration of 30 mg of gangliosides per 100 ml of ethanol. In some experiments, cellulose column chromatography [ 91 was used instead of ethanol precipitation. Preparation of gangiioside fractions The gangliosides were separated by chromatography on silicic acid columns using continuous chloroform/methanol/water gradients with a mixing chamber of constant volume [ 91. The separation of gangliosides was monitored by neuraminic acid determinations and by thin-layer chromatography. Final purification was achieved by preparative thin-layer chromatography. Gangliosides were detected by spraying with water. In addition, samples of wet silica gel were transferred to another glass plate, which was sprayed with resorcinol HCl. The more apolar gangliosides were usually well detected already with water, but for more polar gangliosides resorcinol HCl detection was essential. Detection of bands in water sprayed plates was aided by ultraviolet light. Analytical methods Neuraminic acid was determined by the method of Svennerholm [lo], as modified by Miettinen and Takki-Luukkainen [ 111, for crude lipid extracts ion exchange purification was used [ 121. Hexosamine was detected by a modified Elson-Morgan reaction [ 131. Thin-layer chromatography was performed using silica gel G plates. The bands containing neuraminic acid were detected by resorcinol HCl spray [lo] . (NH,),SO,/(NH,)HSO, solution was used as a general spray reagent [14]. The solvents were: (A) chloroform/methanol/water (60/35/B), (B) chloroform/ methanol/2.5 M NH,OH (60/35/B), (C) n-propanollwater (7/3), (D) n-propanel/water/concentrated ammonia (6/2/l) [15]. Gas-liquid chromatography was performed with a Perkin-Elmer 900 gas chromatograph (Perkin-Elmer Corporation, Norrwalk, Conn., U.S.A.) equipped with a flame ionization detector. Gas-liquid chromatography-mass spectrometry was performed with a Varian Aerograph 1700 gas chromatography combined to a Varian MAT CH-7 mass spectrometer equipped with Spectra System 100 MS data processing system (Varian MAT, Bremen, Germany). The energy level of the ions was 70 eV, the ionizing current was 300 pamp. The mass range covered was from m/e 40 to m/e 800. The ratios of fucose, galactose, glucose, galactosamine and glucosamine were determined on 1% OV-225 column (2 m X 3 mm, internal war) as alditol acetates after acetol.ysis and hydrolysis [ 16-191. In addition, the monosaccharides, neuraminic acid and long-chain bases were quantitated in a single gasliquid chromatographic run on 2.2% SE-30 column (2 m X 3 mm, internal diameter) as Me JSi ethers after methanolysis [20-221. Identification of the Me,Si ether peaks was achieved by comparison with standard monosaccharides
and with long-chain bases obtained after methanolysis of bovine kidney hematosides [ 231. Identification of the gas-liquid chromatographic peaks was aided by mass spectrometry. Molar responses of monosaccharides were determined by using di-, tri- and tetrasaccharides as reference compounds. The species of neuraminic acid was determined after weak acid hydrolysis (0.05 M Hz S04, 80°C 90 min) by thin-layer chromatography and by gas-liquid chromatography [ 241. Results Purification
of gangliosides
from other lipids
When extracting the lipids from acetone powder by chloroform/methanol 2/l and l/2 mixtures [9], 1.0 mg neuraminic acid/100 g wet tissue weight was obtained, which corresponds to 5.6 mg/lOO g dry tissue weight. After a modified Folch partition and dialysis [9], the gangliosides were still extensively contaminated with neuraminic acid free lipids. To achieve satisfactory separations on silicic acid columns, it was found necessary to purify the gangliosides further from other lipids. This could be achieved by cellulose column chromatography, as described before [9,25]. The precipitation of gangliosides from cold ethanol was found to be a more convenient method in large scale purification. About 30 mg of gangliosides was precipitated in 100 ml ethanol at -20°C for 24 h, centrifuged and the ethanol-soluble part decanted. 86% of the neuraminic acid was found in the precipitate. The loss was mainly due to hematoside. None of the other gangliosides was selectively lost to the ethanol-soluble fraction. When tested with a mixture of pig brain gangliosides, 93% of neuraminic acid was in the precipitate, and selective loss of any ganglioside was not observed on thin-layer chromatography. The better yield for brain gangliosides probably reflects the fact that gangliosides having longer carbohydrate chains prevail in brain as compared with the kidney gangliosides.
Fig. 1. Separation of human kidney gangliosides (19 mg, as NeuNAc) on silicic acid column (60 9). The followed by elution was carried out by a continuous chloroform/methanol/water gradient (-----_) methanol/water 9/l (v/v) (- - - - - -). Fractions l-5 were collected according to neuraminic acid determinations and thin-layer chromatographic analysis. The ratios of chloroform. methanol and water (v/v/v) were: 200 ml, 64/31/4.8; 600 ml, 64/31/4.9; 1000 ml. 64/31/5.0; 1400 ml 61/33/6.1.
288
Fractionation of gangliosides fractions 70-80 mg of gangliosides
and
carbohydrate
composition
of
individual
was transferred to an Anasil-S column (60 g). After the chloroform/methanol/water gradient the column was eluted with 1 1 of methanol/water (9/l). This step was essential for complete recovery of the most polar fractions, which did not usually separate as distinct peaks. A typical elution pattern of gangliosides in silicic acid column chromatography is shown in Fig. 1. The corresponding 5 fractions from 11 silicic acid chromatographic runs were pooled and rechromatographed. Preparative thin-layer chromatography was used in the final purification in either solvent A or B, except for fraction 5, which was purified in solvent D. Recovery of NeuNAc from preparative thin-layer chromatography was 90% or somewhat more (elution was performed three times with chloroform/methanol/water, 5/4/l). A thin-layer chromatogram of the seven ganglioside fractions purified from human kidney is shown in Fig. 2. The percentage distribution of gangliosides was calculated on the basis of neuraminic acid determinations after silicic acid chromatography and after preparative thin-layer chromatography for those gangliosides, which were not separated with column chrcmatography. The amount of neuraminic acid per mole of ganglioside was determined by gas-liquid chromatography as Me 3Si ethers, and the percentage distribution of gangliosides was finally calculated on the molar basis. Fraction 1. This fraction contained ganglioside I, which was homogeneous on thin-layer chromatography in the four solvents used (see above) and moved with hematoside GM3 a. It was Elson-Morgan negative, and no hexosamines were found in gas-liquid chromatography. The only sialic acid by thin-layer
-SOLVENT
*e: ) ‘dP
Fig. C,
2. GM*:
Thin-layer D.
mixture
chromatogram of
human
of brain
human
kidney
gangliosides.
gangliosides Soivent
B. Spray:
I-VIII.
FRONT
References:
resorcinol
HCl.
A.
GM3a;
B. GM3b;
289
chromatography and gas-liquid chromatography after mild acid hydrolysis was N-acetylneuraminic acid. As Me 3Si derivatives the ratio of galactose : glucose : neuraminic acid was 1.0 : 0.9 : 0.8. This ganglioside is therefore a hematoside, and comprises 74% of human kidney gangliosides. Fraction 2. This fraction was composed of gangliosides II and III. After separation of these gangliosides, they were both homogeneous on thin-layer chromatography in solvents A-D. The properties of these gangliosides on thinlayer chromatography were peculiar. In solvents A and D they moved behind ganglioside IV (which corresponds to di-N-acetylneuraminyllactosylceramide of bovine kidney in its thin-layer chromatographic behaviour and sugar composition). In solvent B ganglioside II moved ahead of ganglioside IV and ganglioside III moved slightly behind ganglioside IV, while in solvent C both ganglioside II and ganglioside III moved ahead of ganglioside IV. Both of these gangliosides were Elson-Morgan positive. In addition to galactose, glucose, and neuraminic acid, II and III contained glucosamine and ganglioside III fucose. Identification of glucosamine was achieved by gas-liquid chromatography-mass spectrometry. In gas-liquid chromatography as Me ,Si derivatives and as alditol acetates, the retention times of the amino sugar peaks corresponded to N-acetylglucosamine. By mass-spectrometry the structure of methyl 2-acetamido-2-deoxy-tri-OMe 3Si-hexoside was established according to the data of Karkkainen and Vihko [26]. The mass spectra of the amino sugar peaks were also found to be identical to those obtained from the gas-liquid chromatographic peaks of authentic N-acetylglucosamine. Three gas-liquid chromatographic peaks identical in the retention times and the ratios of the peak areas to those obtained from fucose were found in ganglioside III. The mass soectral characteristics were also identical to those of fucose. Ganglioside.111 was sprayed with (NH,), SO, /(NH,)HSO 4 solution in addition to recorcinol HCl, to detect any organic impurities which could convey fucose to the fraction. Ganglioside III was homogeneous also by this detection method. N-acetylneuraminic acid was found to be the only sialic acid by thin-layer chromatography and gas-liquid chromatography after mild acid hydrolysis. The ratio of galactose : glucosamine : glucose : neuraminic acid was for ganglioside II (in parentheses as alditol acetates) 2.0 : 1.1 : 0.8 : 1.0 (2.0 : 0.9 ; 0.8). The ratio of fucose : galactose : glucose : glucosamine : neuraminic acid in ganglioside III was 0.6 : 2.0 : 0.9 : 0.7 : 0.9 (0.8 : 2.0 : 1.0 : 0.9). Gangliosides II and III accounted together for 5% of human kidney gangliosides (about equal amounts of both). Fraction 3. This fraction contained ganglioside IV, which was the other major ganglioside in human kidney. Ganglioside IV had the characteristic thinlayer chromatographic properties reported by Puro for di-N-acetylneuraminyllactosylceramide [ 91. On the basis of the Elson-Morgan reaction and gas-liquid chromatography this ganglioside does not contain amino sugars. Only N-acetylneuraminic acid was found in thin-layer chromatography and gas-liquid chromatography. As Me3Si derivatives in gas-liquid chromatography the ratio of galactose : glucose : neuraminic acid was 1.0 : 0.8 : 1.9. The carbohydrate structure of this ganglioside is therefore the same as that of di-hr-acetylneuraminyllactosylceramide isolated by Puro from bovine kidney [ 21. Ganglioside IV comprised 19% of human kidney gangliosides. Fraction 4. Gangliosides IV, V and VI were found in this fraction. Gan-
290
glioside V was purified to give a homogeneous band on thin-layer chromatography. The thin-layer chromatographic properties of V resembled to those of ganglioside GD, b. The Elson-Morgan reaction was positive. The presence of galactosamine was verified as described for glucosamine in gangliosides II and III. As before, N-acetylneuraminic acid was the only sialic acid. The ratio of galactose : glucose : galactosamine : neuraminic acid (as Me,Si derivatives) was 2.0 : 1.0 : 1.0 : 1.9. These data suggest that ganglioside V could have the same kind of carbohydrate structure as the brain disialoganglioside. Ganglioside V comprised 1% of human kidney gangliosides. Fraction 5. Gangliosides VI, VII and VIII were found in this fraction (and a small amount of ganglioside V). Two fractions were well separated in solvent D. Ganglioside VI was purified from the slower moving fraction to give a homogeneous band in solvents A-D. The slower band, though quite homogeneous in solvent D, revealed two recorcinol positive spots close to each other in solvents A-C. These were named gangliosides VII and VIII in order of decreasing thin-layer chromatographic mobility. Because of lack of material, gangliosides VII and VIII could not be purified to homogeneous fractions. The thinlayer chromatographic behaviour of ganglioside VI in the solvents used was near to gangliosides GD, b and GT 1, gangliosides VII and VIII moved near the trisi~ogangliosides of brain on thin-layer chromatography. The Elson-Morgan reaction was positive for ganglioside VI and for the mixture of VI1 and VIII. By gas-liquid chromatography-mass spectrometry, ganglioside VI and the mixture of gangliosides VII and VIII contain both glucosamine and galactosamine. The only sialic acid found on thin-layer chromatography after mild acid hydrolysis was N-acetylneuram.inic acid. The ratio of galactose : glucose : galactosamine : glucosamine : neuraminic acid in ganglioside VI was (as Me,Si ethers) 2.0 : 1.3 : 0.5 : 0.5 : 1.9. Therefore, this fraction contains either a ganglioside with galactosamine and glucosamine in the same molecule or it is still a mixture of two gangliosides. The carbohydrate ratios in the mixture of VII and VIII (as Me,Si derivatives) were galactose : glucose : galactosamine : glucosamine : neuraminic acid, 2.0 : 1.0 : 0.9 : 0.2 : 1.3. About 0.5% of human kidney gangliosides was due to ganglioside VI and about 0.5% to the mixture of VII and VIII. Composition of long-chain bases After methanolysis in dry HCl . methanol, acetylation and trimethylsilylation, long-chain basis were converted mainfy to 1,3-di-0-Me,Si-NAc and 1,3,4tri-O-Me~Si-NAc derivatives, which were eluted in gas-liquid chromato~aphy after neuraminic acid (Fig. 3). Because of by-product formation in dry HCl methanol [27] (mainly 0-methylation), dry methanolysis is not usually used for long-chain base determinations. However, by adding the peaks having m/e 174 in their mass spectra (m/e 174 is common to various species of long-chain bases, Fig. 4), the response of long-chain bases was fairly constant in gas-liquid chromatography (the peak area of long-chain bases as related to glucose was 0.5 in ganglioside I of human kidney). For various species of long-chain bases, a diagnostic peak [28] arises from the cleavage of the molecule between carbon atoms 2 and 3 (m/e 311 for 1,3-di-O-Me 3Si-NAc derivative of sphingosine and m/e 253 for l-0-Me,Si-3-0-Me-NAc derivative of sphingosine, Fig. 4). For tri-
291
Long-than
bases
III
30
50 TIME
(min)
10
Fig. 3. Gas-liquid chromatograms of trimethylsilylated monosaccharides and long-chain bases obtained in dry methanolysis (0.75 M HCl * methanol, 20 h, 80°C). A, hematoside GM3a from bovine kidney; B. ganglioside III from human kidney. I,II,l-O-MesSi-3-0-Me-NAc derivatives of long-chain bases (mainly from sphingosine); lII.IV.1.3-di-0-Me3Si-NAc derivatives of long-chain bases (III, mainly from sphingosine; IV, mainly from dihvdrosphingosine and sphingosine): V.1.3,4-tri-O-Me~Si-NAr derivatives of long-chain bases (mainly from phytosphingosine). Conditions: 2.2% SE-30, temperature programmed at 4”Cimin from 140 to 240°C. inositol as internal standard.
hydroxybases [ 291, a similar cleavage was also observed, but the cleavage between carbon atoms 3 and 4 favoured (for 1,3,4-tri-0Me,Si-NAc derivative of phytosphingosine m/e 401 and m/e 174 from the cleavage between carbons 2 and 3 and m/e 299 and m/e 276 from the cleavage between carbons 3 and 4, Fig. 4). In the conditions used, IV-acetylation was complete. The most prominent by-product was the 30methylated 4-sphingenine (Fig. 4). The long-chain bases of hematoslde GMja from bovine kidney were used as reference in gasliquid chromatography and gas-liquid chromatography-mass spectrometry. The main the long-chain bases in hematoside GM3, were by this method phytosphingosine and sphingosine, which is in agreement with the analysis of the oxidation products by lead tetraacetate [23]. In all fractions of human kidney gangliosides the main long-chain base was spingosine. Trihydroxybases were found in much smaller amounts than in bovine kidney hematosides (Fig. 3 and Fig. 4). Because of lack of material, identification of the minor fractions of long-chain bases was not attempted in this study.
0
50
50.
lLdu,,,l.l.l
/’
13
73
,73
100
!
132
132
‘P
$63 116 \
91 103
116
of long-chain bases. Above, derivative of sphingosine);
L
106~
Fig. 4. Mass spectra 1-0-MejSi-3-0-Me-NAc
F
?.
2
z iii
YD
50.
100
114
,,
!,l
200
&IL.&
167
.l,,,, 185
216
1,
2p .L
276
mle
.._...
241 /
I
253(t$1741
300
]31,,-,74~9o,
299 W2761
1
,:;‘“:‘-“““,,
M-575
L
401 : 174
: tOCH3
; 0-CH2
253 :174 CH=;Wi 3
:
NH : COCH3
OSiMea
400
.L
:
441
426 (r~9o~59)
I 276
500
: COW3
470(M~g0~,5) 1
299
CH3-(CH2),2-CH2-$H; CtifCH-CHpOStMe3 Me3St0 v+lO: NH
KH2),Z
401 (M-1741
366 (y-591;
360lM-32-151 1
CH3-
M=427
Me+O/
560 Ct$ 151
1
peak III/A of Fig. 3 (mainly 1,3-di-0-MejSi-NAc derivative of sphingosine); middle, peak I/B of Fig. 3 (mainly below, peak V/A of Fig. 3 (mainly 1,3,4-tri-O-Me3Si-NAc derivative of phytosphingosine).
147 I=’
141
174
211
311 NM.174) 1 292W90-103)
311 174
+ CH3~(CH2),iCH=CH-“iCH-CH20S,Me3
M=465
293
Discussion Isolation of gangliosides from extraneural tissues in reasonable yields for structural study is laborious and time consuming. In the handling of relatively large amounts of starting material, it is desirable to lessen the number of chromatographic runs. Therefore, an ethanol precipitation step was introduced after the Folch partition. This step gave an 86% recovery of neuraminic acid, and removed small molecular substances which leak into the upper phase during the Folch partition. Dialysis may be used to remove small molecular organic substances, but it was partially ineffective in this case, perhaps because gangliosides tend to form micelles with these [30]. When precipitated in ethanol after the modified Folch partition and dialysis, the gangliosides were fractionated fairly well on silicic acid columns, and showed minimal or no contamination on thin-layer chromatography. Recently, phosphotungstic acid precipitation has been recommended to purify the ganglioside preparation from small molecular organic contaminants in the water layer of chloroform/methanol/water partition [31]. The amount of neuraminic acid extracted from human kidneys by chloroform/methanol 2/l and l/2 mixtures was considerably lower than that reported for rat, rabbit, pig and bovine kidneys [1,9], i.e. 5.6 mg/lOO g dry weight compared with 13, 13, 16 and 16 mg/lOO g dry weight, respectively. This is a real difference, because there were no essential differences in experimental procedures. According to Martensson [ 32,331, the ganglioside content of human kidney is 0.24 pmol/g dry weight, which is somewhat more than the value calculated from the data of this work (0.17 pmol ganglioside/g dry weight). The dominating ganglioside in human kidney was N-acetylneuraminyllactosylceramide (hermatoside), which is in agreement with reports concerning several extraneural tissues [ 341. The corresponding hematoside containing N-glycolylneuraminic acid was another major ganglioside in bovine kidney [2], but could not be found in human kidney. The absence of N-glycolylneuraminic acid was confirmed for all ganglioside fractions of human kidney, which is in accordance with previous studies on extraneural human gangliosides [3] . The hematoside containing N-acetylneuraminic acid constituted about the same percentage of gangliosides in human kidney as the sum of hematosides GM3 a and GMs,, in bovine kidney [9]. The other major fraction was di-N-acetylneuraminyllactosylceramide, which also was the major ganglioside, after hematosides, in bovine kidney [2,9]. A glucosamine-containing ganglioside was first described in bovine erythrocytes and spleen [ 351, Glucosamine-containing oligosaccharides liberated from ganglioside preparations have been found in human spleen, kidney and liver and in bovine extraneural tissues [ 3,34,36]. Glucosamine has also been found in the gangliosides of human erythrocytes [37], human platelets [38], bovine platelets [ 391, human, bovine and rabbit plasma [40], human newborn brain, peripheral nerve, skeletal muscle, serum and red blood cells [41]. In this work, two gangliosides containing glucosamine as their only hexosamine were isolated. The other of these gangliosides contains fucose, which was unequivocally identified by gas-liquid chromatography-mass spectrometry. Fucose could
294
not be separated from this ganglioside by preparative thin-layer chromatography, and the homogeneity could also be confirmed by analytical thin-layer chromatography in four quite different solvents. These facts suggest that fucose exists in the same glycolipid molecule as neuraminic acid, which is an unusual structural feature. Fucose, galactose, glucose, glucosamine and neuraminic acid have also been found in A and B active glycolipid preparations [42,43]. However, the highly purified A and B glycolipids do not contain neuraminic acid [ 441. Wiegandt has found an H active oligosaccharide containing fucose, galactose, glucose, galactosamine and neuraminic acid from gangliosides of bovine liver [36]. In addition to these two glucosamine-containing gangliosides, a disialoganglioside containing galactosamine and a homogeneous ganglioside fraction containing both galactosamine and glucosamine were isolated. Gangliosides with two hexosamine moieties are not known to exist, and the possibility remains that the fraction containing both galactosamine and glucosamine is still a mixture. Ganglioside structures are often inferred from their thin-layer chromatographic mobilities using brain gangliosides as standards. This work shows quite clearly that these kinds of interpretations can lead to erroneous conclusions. For example, ganglioside III was near to ganglioside G, , a in its thin-layer chromatographi~ behaviour, although it, contains only one sialic acid, and gangliosides VII and VIII moved with brain trisialog~~liosides, although the mixture of these gangliosides contains only 1.32 neuraminic acids per molecule. The dominant long-chain base in all ganglioside fractions was 4sphingosine, as in neutral glycolipids of human kidney [45]. Compared with bovine kidney gangliosides, clearly less trihydroxybases were found in all fractions of human kidney gangliosides. Acknowledgements The technical assistance of Mrs. Liisa Kuivalainen and Mrs. Hilkka Rijnkkij is greatly appreciated. This work was supported by the Sigrid Juselius Foundation and the National Research Council for Medical Sciences, Finland. References 1
Pure,
K..
2
Pure,
K.
3
Wiegandt.
4
Svennerholm.
5
IUPAC-IUB Eur.
6
P. and
H. and
Biicking,
7
Huttunen.
8
Folch,
9
Pure,
21.
Svennerholm. Miettineo.
12
Svennerholm,
Eur.
of
10,
Biochim.
Biophys.
Acta
187,
‘23~-235
401-413 J. Biochem.
15.
287.-292
613-623
Biochemical
Nomenclature
The
(CBN)
Nomenclature
the
Nomenclature (CBN),
of
Organic
Tentative
Chemistry
Rules
for
(CNOC)
Carbohydrate
and
(1966)
Ann.
M. and
SIoane
Acta
I3
Miettrnen,
T.A.
Zimiriski.
T. and
Exp.
Stanley, Sand.
Biochim.
24.
(1961)
Acta Sand.
Borowski.
BioI.
Fenniae
G.H.
(1957) Acta
I.-T.
(1959)
Chem. J. Clin.
E. (1966)
44.
Supul.
J. Bioi.
12
Chem.
226,
497-509
13-22
Biophys.
Takki-Luukkainen.
L. (1958)
14
Med.
Chem.
I,. (1957) ‘I’. and
of
IUPAC-IUB
Nomenclature
455--477
(1970)
11
(1970)
187,
Lipids
(1967)
31
Nomenclature
Lees,
10
on
2, 127--l
J.K.
J.. K.
W.W.
(1969)
Acta
J. Neurochem.
Commission
Commission
J.K.
Biophys.
I,. (lV63)
Biochemical
Biochem.
Hut&men,
Biochxm.
J. Riochem.
IUPAC on
Maury. (196V)
Stand. Lab.
12,
24.
604-611
Acta
Chem.
Stand.
547-554
Invest.
J. Chromatog.
13,
Suppl.
23.
13
480.-482
13,
856-858
Commission (1971)
Eur.
J.
295
15
Penick.
16
Sawarderker,
R.J..
17
Kim,
18
Bjiirndal. Young.
20
Sweeley. Bhatti. Pun),
25
T.,
Rouser.
G.. ed.),
R.E.
and
A.
l,edeen. Kntchevsky.
Chem.
J.R.
(1970)
New
Gaver.
R.C.
and
Sweeley.
C.C.
(1965)
J. .4m.
011 Chem.
28
Gaver,
R.C.
and
Sweeley.
C.C.
(1966)
J. Am.
Chem.
29
Thorpe,
Svennerholm. Dunn.
32
MPrtensson,
E..
Percy,
33
MBrtensson.
E.
(1969)
in Progress
in the
367-407.
Pergamon
Press.
X,
Part
4, pp.
34
Wiegandt.
35
Kuhn.
36
Wiegandt,
37
Wherrett.
38
Marcus,
39
Etzrodt.
H. (1968)
40
Yu.
and
41
MPnsson.
and
Adv.
Wiegandt.
H. (1973) J.R. A.J.,
R.K.
Pullman,
Lipid
Res.
II. (1964)
222.
339-347
393-400 Chromatographic
Analysis
(Marinetti.
10,
113-120 Sot.
42.
88,
294-298
3643.
3647
6. 887-897
239~-250
I,. (1966) Chetmstry
of
11.1,. and
&Tier.
Acta L.B.
Univrrslty
(1972)
J. Lipid
J.,
Y.-T..
Li,
Acta
Paedlatr.
Fats
and
Stand.
other
Llpids
55.
l-9
(Holman,
R.T..
ed.),
Vol.
Oxford 19b, Chem.
%. Physiol. Biophys.
R.W.
Acta
9, 249-289
Biochim.
Holmgren.
187.
Sot.
Z. Naturforsch.
Dissertation.
Lcdern.
17.
Svennerholm.
IIoppe-Seyler’s
(1973)
J.-E..
Sand.
258-274
1801-1804
2.3, 293m-295
A. and
H. (1971)
R.
Chem.
J. Neurochem.
Res.
Biochemistry
30
(1974)
Acta
(1967)
31
A.
I,. (1963)
C.C.
Biophys.
in I.ipid
Klrkkiilnen.
Sweeley.
21.
Yorh
26
and
Carbohyd.
20,
Sand.
279.-287
506--516
.4. (1967)
Dekker.
Bmchem.
116.
1602-1607
1009~~1018
Acta
11.
37.
1192-1200
Biochim.
Res.
Actd
Chem.
1461-1466
J. 125.
Biophvs.
Yamamoto.
246.
36.
Blochem.
Marcel
1~. (19ti9)
Chem.
Chem.
27
S.R.
Vihko,
Anal.
Acta
J. Lipid
G. and 99-m162.
Biophys.
Anal.
(1967)
Biochim.
(1970)
Blochim.
(1965)
J.G.
J. Biol.
(1971)
(1969)
A.
S. (1967)
Anal.
Clamp.
J.R.
R.W.
1’01. I. pp. J. and
and
Clamp.
P~ercr.
(1971)
B. (1964)
(1966)
Jeanes,
Svensson.
S.-I.
Walker.
R.H.
and
T.11. and
B. and
Kerlnen.
and
McClucr. J.H.
Liao.
Chambers,
K. nnd
and
Hakomori.
and
R.E.
R.K.
G.\‘..
B..
and
C.C.
Chambers, Yu.
Shame.
H.J.
22 23
M.H.
Sloanecker.
11.. Lindberg.
21
24
d.S.,
J.H.,
19
Melsler.
326.
(1972) of
80~.-81 354.
1049~~1056
63-73 J. Clin.
Invest.
51.
2602--2612
Kdln
Res.
13.
\‘amer.
680-686
M.-T.
and
Svennrrholm,
L.
(1974)
Med.
Biol.
52.
240-243 42
Ilakomori,
S.-l.
and
43
Hakomori.
S.-l.
(1970)
44
Hakomori.
S.-l.
and
Press,
York
45
New
Martensson,
I?. (1965)
Strycharz. Chem. Kobata,
G.D.
(1968)
Phys.
Lipids
A.
Biochim.
(1974) Biophys.
Biochemistry
7. 12’79
-1286
5. 96-115
in The Acta
Antigens 116.
(Sela.
296-308
M..
ed.),
Vol.
Il.
pp.
79-140,
Academic
Biochimica
et Biophysics
0 Elsevier
Scientific
Acta,
Publishing
424 (1976) 284-295 Company, Amsterdam
- Printed
in The Netherlands
BBA 56714
ISOLATION AND PARTIAL CHARACTERIZATION GANGLIOSIDES
HEIKKI
OF HUMAN KIDNEY
RAUVALA
Department of Medical Helsinki 17 (Finland).
(Received
August
Chemistry,
University
of Helsinki,
Siltavuorenpenger
10 A, SF-001
70
4th, 1975)
Summary 1. Eight gangliosides were purified from chloroform/methanol extracts of human kidneys by using modified Folch partition, dialysis, ethanol precipitation, silicic acid column chromatography and preparative thin-layer chromatography. 2. By thin-layer chromatographic behaviour and gas-liquid chromatographic determinations the main gangliosides in human kidney are N-acetylneuraminyllactosylceramide (74% of total) and di-N-acetylneuraminyllactosylceramide (19% of total). 3. Five hexosamine-containing fractions were isolated. Four of them were homogeneous on thin-layer chromatography, and one contained two gangliosides. By gas-liquid chromatography-mass spectrometry it was shown that two gangliosides (together 5% of total) contain glucosamine, and one (1% of total) contains galactosamine. The other of the glucosamine gangliosides contains fucose in addition to the usual sugars found in gangliosides. Of the two remaining hexosamine positive fractions (together 1% of total) one was homogeneous on thin-layer chromatography, the other contained two gangliosides. These two fractions contained both glucosamine and galactosamine. 4. The main long-chain base in all fractions was sphingosine.
Introduction
shown
The complexit,y of the ganglioside patterns in extraneural tissues has been [ 11. Usually, the gangliosides containing lactose as their neutral carAbbreviations GlcNAc,
Svennerholm GMaa
and
nomenclature:
N-acetylglucosamine; [41
is used
Gal,
galactose:
NeuNAc, for
gangliosides.
(N-acetylneuraminyllactosylceramide)
The
nomenclature
sion
[ 5.61.
used
for long-chain
Glc,
glucose;
N-acetylneuraminic The and bases
and
hematosides as GMsb
GalNAc.
acid. of
The
bovine
N-acetylgalactosamine:
shorthand kidney
nomenclature were
designated
(N-glycolylneuraminy~actosylceramide).
oligosaccharides
is that
proposed
by
IUB
Commis-
of as
285
bohydrate portion predominate in extraneural tissues [2,3] . Because of the scarcity of the material and the difficulties in the isolation procedures, relatively few reports have been published concerning the more complex extraneural gangliosides. Wiegandt and Bucking have developed a method to release the sialo-oligosaccharides from gangliosides, and have found a wide variety of gangliosides in several extraneural tissues by their method [3]. In this work, an attempt was made to isolate especially the more complex gangliosides from human kidney as intact molecules. A simplified isolation method was developed, and in addition to the gangliosides containing neuraminyl- and di-neuraminyllactose, five hexosamine-containing ganglioside fractions could be isolated. A preliminary characterization of the ganglioside fractions is presented. Experimental Materials
Human kidneys were obtained at autopsy and stored at -2O”C, if not used immediately. The brain gangliosides used as reference on thin-layer chromatography were made available by Professor E. Klenk (Physiologisch-Chemisches Institut der Universitat Koln, Germany), the hematosides of bovine kidof Medical Chemistry, ney (GM3, and GM3i, ) by Dr. K. Puro (Department University of Helsinki, Finland), the mixture of pig brain gangliosides, and the disialoganglioside of pig brain by Dr. P. Maury (Department of Medical Chemistry, University of Helsinki, Finland). L-fucose, D-galactose, D-glucose, D-mannose, 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, N-acetylneuraminic acid, N-glycolylneuraminic acid, inositol and O-P-D-galactopyranosyl-(1 -+ 4)D-glucopyranose (lactose) were purchased from commercial sources. Other sugars used for standardization of gas-liquid chromatographic determinations were gifts: 2-acetamido2-deoxy-O-P-D-galactopyranosyl( 1 -+ 3)-D-galactopyranose from Dr. D. Shapiro (Rehovoth, Israel), 0-a-L-fucopyranosyl-( 1 + 2)-O-P-D-galactopyranosyl-( 1 + 4)-D-glucopyranose (2’-fucosyllactose), O-O-D-galactopyranosyl-( 1 + 3)-%acetamido-2-deoxy-O-/3-D-glucopyranosyl-(1 + 3)-O-fl-D-galactopyranosyl(1 -+ 4) D-glucopyranose (la&o-N-tetraose) and 0-a-N-acetylneuraminyl-(2 --f 6)-O-p-~galactopyranosyl (1 + 4)-D-glucopyranose (6’-iV-acetylneuraminyllactose) from Dr. A. Gauhe (Heidelberg, Germany). 0-cu-N-acetylneuraminyl-( 2 + 3)-0-@-D -galactopyranosyl-( 1 -+ 4)-D-glucopyranose (3’-N-acetylneuraminyllactose) was prepared in this laboratory [7] . Organic solvents were of analytical reagent quality. Trimethylchlorsilane (Fluka A.G.) and hexamethyldisilazane (Fluka A.G.) were distilled before use. Pyridine was distilled from KOH and stored over a molecular sieve. Absorbents for column chromatography were silicic acid (Anasil-S, Analytical Engineering Laboratories, Inc., Hamden, Conn.) and cellulose (Whatman powder, standard grade, W and P, Bolston, Ltd.), and for thin-layer chromatography silica gel G (E. Merck, A.G., Darmstadt). Dowex 1 X 8 (200/400 mesh) and Dowex 50 X 8 (200/400 mesh) were purchased from Fluka A.G. Preparation
of gangliosides
Capsules and pelvices were removed
from human
kidneys by coarse dissec-
286
tion. The kidney tissue was rinsed and made bloodless by washing several times with 0.9% saline. The rinsed tissue was homogenized in cold acetone and dried to acetone powder. The lipids were extracted and partitioned in the Folch system as described before [8,9]. The lower phases were discarded, the upper phases were dialyzed at -+4”C against running tap water and concentrated by rotary evaporation below 40” C. For further purification, crude ganglioside was dried and precipitated from ethanol at -20°C for 24 h in the concentration of 30 mg of gangliosides per 100 ml of ethanol. In some experiments, cellulose column chromatography [ 91 was used instead of ethanol precipitation. Preparation of gangiioside fractions The gangliosides were separated by chromatography on silicic acid columns using continuous chloroform/methanol/water gradients with a mixing chamber of constant volume [ 91. The separation of gangliosides was monitored by neuraminic acid determinations and by thin-layer chromatography. Final purification was achieved by preparative thin-layer chromatography. Gangliosides were detected by spraying with water. In addition, samples of wet silica gel were transferred to another glass plate, which was sprayed with resorcinol HCl. The more apolar gangliosides were usually well detected already with water, but for more polar gangliosides resorcinol HCl detection was essential. Detection of bands in water sprayed plates was aided by ultraviolet light. Analytical methods Neuraminic acid was determined by the method of Svennerholm [lo], as modified by Miettinen and Takki-Luukkainen [ 111, for crude lipid extracts ion exchange purification was used [ 121. Hexosamine was detected by a modified Elson-Morgan reaction [ 131. Thin-layer chromatography was performed using silica gel G plates. The bands containing neuraminic acid were detected by resorcinol HCl spray [lo] . (NH,),SO,/(NH,)HSO, solution was used as a general spray reagent [14]. The solvents were: (A) chloroform/methanol/water (60/35/B), (B) chloroform/ methanol/2.5 M NH,OH (60/35/B), (C) n-propanollwater (7/3), (D) n-propanel/water/concentrated ammonia (6/2/l) [15]. Gas-liquid chromatography was performed with a Perkin-Elmer 900 gas chromatograph (Perkin-Elmer Corporation, Norrwalk, Conn., U.S.A.) equipped with a flame ionization detector. Gas-liquid chromatography-mass spectrometry was performed with a Varian Aerograph 1700 gas chromatography combined to a Varian MAT CH-7 mass spectrometer equipped with Spectra System 100 MS data processing system (Varian MAT, Bremen, Germany). The energy level of the ions was 70 eV, the ionizing current was 300 pamp. The mass range covered was from m/e 40 to m/e 800. The ratios of fucose, galactose, glucose, galactosamine and glucosamine were determined on 1% OV-225 column (2 m X 3 mm, internal war) as alditol acetates after acetol.ysis and hydrolysis [ 16-191. In addition, the monosaccharides, neuraminic acid and long-chain bases were quantitated in a single gasliquid chromatographic run on 2.2% SE-30 column (2 m X 3 mm, internal diameter) as Me JSi ethers after methanolysis [20-221. Identification of the Me,Si ether peaks was achieved by comparison with standard monosaccharides
and with long-chain bases obtained after methanolysis of bovine kidney hematosides [ 231. Identification of the gas-liquid chromatographic peaks was aided by mass spectrometry. Molar responses of monosaccharides were determined by using di-, tri- and tetrasaccharides as reference compounds. The species of neuraminic acid was determined after weak acid hydrolysis (0.05 M Hz S04, 80°C 90 min) by thin-layer chromatography and by gas-liquid chromatography [ 241. Results Purification
of gangliosides
from other lipids
When extracting the lipids from acetone powder by chloroform/methanol 2/l and l/2 mixtures [9], 1.0 mg neuraminic acid/100 g wet tissue weight was obtained, which corresponds to 5.6 mg/lOO g dry tissue weight. After a modified Folch partition and dialysis [9], the gangliosides were still extensively contaminated with neuraminic acid free lipids. To achieve satisfactory separations on silicic acid columns, it was found necessary to purify the gangliosides further from other lipids. This could be achieved by cellulose column chromatography, as described before [9,25]. The precipitation of gangliosides from cold ethanol was found to be a more convenient method in large scale purification. About 30 mg of gangliosides was precipitated in 100 ml ethanol at -20°C for 24 h, centrifuged and the ethanol-soluble part decanted. 86% of the neuraminic acid was found in the precipitate. The loss was mainly due to hematoside. None of the other gangliosides was selectively lost to the ethanol-soluble fraction. When tested with a mixture of pig brain gangliosides, 93% of neuraminic acid was in the precipitate, and selective loss of any ganglioside was not observed on thin-layer chromatography. The better yield for brain gangliosides probably reflects the fact that gangliosides having longer carbohydrate chains prevail in brain as compared with the kidney gangliosides.
Fig. 1. Separation of human kidney gangliosides (19 mg, as NeuNAc) on silicic acid column (60 9). The followed by elution was carried out by a continuous chloroform/methanol/water gradient (-----_) methanol/water 9/l (v/v) (- - - - - -). Fractions l-5 were collected according to neuraminic acid determinations and thin-layer chromatographic analysis. The ratios of chloroform. methanol and water (v/v/v) were: 200 ml, 64/31/4.8; 600 ml, 64/31/4.9; 1000 ml. 64/31/5.0; 1400 ml 61/33/6.1.
288
Fractionation of gangliosides fractions 70-80 mg of gangliosides
and
carbohydrate
composition
of
individual
was transferred to an Anasil-S column (60 g). After the chloroform/methanol/water gradient the column was eluted with 1 1 of methanol/water (9/l). This step was essential for complete recovery of the most polar fractions, which did not usually separate as distinct peaks. A typical elution pattern of gangliosides in silicic acid column chromatography is shown in Fig. 1. The corresponding 5 fractions from 11 silicic acid chromatographic runs were pooled and rechromatographed. Preparative thin-layer chromatography was used in the final purification in either solvent A or B, except for fraction 5, which was purified in solvent D. Recovery of NeuNAc from preparative thin-layer chromatography was 90% or somewhat more (elution was performed three times with chloroform/methanol/water, 5/4/l). A thin-layer chromatogram of the seven ganglioside fractions purified from human kidney is shown in Fig. 2. The percentage distribution of gangliosides was calculated on the basis of neuraminic acid determinations after silicic acid chromatography and after preparative thin-layer chromatography for those gangliosides, which were not separated with column chrcmatography. The amount of neuraminic acid per mole of ganglioside was determined by gas-liquid chromatography as Me 3Si ethers, and the percentage distribution of gangliosides was finally calculated on the molar basis. Fraction 1. This fraction contained ganglioside I, which was homogeneous on thin-layer chromatography in the four solvents used (see above) and moved with hematoside GM3 a. It was Elson-Morgan negative, and no hexosamines were found in gas-liquid chromatography. The only sialic acid by thin-layer
-SOLVENT
*e: ) ‘dP
Fig. C,
2. GM*:
Thin-layer D.
mixture
chromatogram of
human
of brain
human
kidney
gangliosides.
gangliosides Soivent
B. Spray:
I-VIII.
FRONT
References:
resorcinol
HCl.
A.
GM3a;
B. GM3b;
289
chromatography and gas-liquid chromatography after mild acid hydrolysis was N-acetylneuraminic acid. As Me 3Si derivatives the ratio of galactose : glucose : neuraminic acid was 1.0 : 0.9 : 0.8. This ganglioside is therefore a hematoside, and comprises 74% of human kidney gangliosides. Fraction 2. This fraction was composed of gangliosides II and III. After separation of these gangliosides, they were both homogeneous on thin-layer chromatography in solvents A-D. The properties of these gangliosides on thinlayer chromatography were peculiar. In solvents A and D they moved behind ganglioside IV (which corresponds to di-N-acetylneuraminyllactosylceramide of bovine kidney in its thin-layer chromatographic behaviour and sugar composition). In solvent B ganglioside II moved ahead of ganglioside IV and ganglioside III moved slightly behind ganglioside IV, while in solvent C both ganglioside II and ganglioside III moved ahead of ganglioside IV. Both of these gangliosides were Elson-Morgan positive. In addition to galactose, glucose, and neuraminic acid, II and III contained glucosamine and ganglioside III fucose. Identification of glucosamine was achieved by gas-liquid chromatography-mass spectrometry. In gas-liquid chromatography as Me ,Si derivatives and as alditol acetates, the retention times of the amino sugar peaks corresponded to N-acetylglucosamine. By mass-spectrometry the structure of methyl 2-acetamido-2-deoxy-tri-OMe 3Si-hexoside was established according to the data of Karkkainen and Vihko [26]. The mass spectra of the amino sugar peaks were also found to be identical to those obtained from the gas-liquid chromatographic peaks of authentic N-acetylglucosamine. Three gas-liquid chromatographic peaks identical in the retention times and the ratios of the peak areas to those obtained from fucose were found in ganglioside III. The mass soectral characteristics were also identical to those of fucose. Ganglioside.111 was sprayed with (NH,), SO, /(NH,)HSO 4 solution in addition to recorcinol HCl, to detect any organic impurities which could convey fucose to the fraction. Ganglioside III was homogeneous also by this detection method. N-acetylneuraminic acid was found to be the only sialic acid by thin-layer chromatography and gas-liquid chromatography after mild acid hydrolysis. The ratio of galactose : glucosamine : glucose : neuraminic acid was for ganglioside II (in parentheses as alditol acetates) 2.0 : 1.1 : 0.8 : 1.0 (2.0 : 0.9 ; 0.8). The ratio of fucose : galactose : glucose : glucosamine : neuraminic acid in ganglioside III was 0.6 : 2.0 : 0.9 : 0.7 : 0.9 (0.8 : 2.0 : 1.0 : 0.9). Gangliosides II and III accounted together for 5% of human kidney gangliosides (about equal amounts of both). Fraction 3. This fraction contained ganglioside IV, which was the other major ganglioside in human kidney. Ganglioside IV had the characteristic thinlayer chromatographic properties reported by Puro for di-N-acetylneuraminyllactosylceramide [ 91. On the basis of the Elson-Morgan reaction and gas-liquid chromatography this ganglioside does not contain amino sugars. Only N-acetylneuraminic acid was found in thin-layer chromatography and gas-liquid chromatography. As Me3Si derivatives in gas-liquid chromatography the ratio of galactose : glucose : neuraminic acid was 1.0 : 0.8 : 1.9. The carbohydrate structure of this ganglioside is therefore the same as that of di-hr-acetylneuraminyllactosylceramide isolated by Puro from bovine kidney [ 21. Ganglioside IV comprised 19% of human kidney gangliosides. Fraction 4. Gangliosides IV, V and VI were found in this fraction. Gan-
290
glioside V was purified to give a homogeneous band on thin-layer chromatography. The thin-layer chromatographic properties of V resembled to those of ganglioside GD, b. The Elson-Morgan reaction was positive. The presence of galactosamine was verified as described for glucosamine in gangliosides II and III. As before, N-acetylneuraminic acid was the only sialic acid. The ratio of galactose : glucose : galactosamine : neuraminic acid (as Me,Si derivatives) was 2.0 : 1.0 : 1.0 : 1.9. These data suggest that ganglioside V could have the same kind of carbohydrate structure as the brain disialoganglioside. Ganglioside V comprised 1% of human kidney gangliosides. Fraction 5. Gangliosides VI, VII and VIII were found in this fraction (and a small amount of ganglioside V). Two fractions were well separated in solvent D. Ganglioside VI was purified from the slower moving fraction to give a homogeneous band in solvents A-D. The slower band, though quite homogeneous in solvent D, revealed two recorcinol positive spots close to each other in solvents A-C. These were named gangliosides VII and VIII in order of decreasing thin-layer chromatographic mobility. Because of lack of material, gangliosides VII and VIII could not be purified to homogeneous fractions. The thinlayer chromatographic behaviour of ganglioside VI in the solvents used was near to gangliosides GD, b and GT 1, gangliosides VII and VIII moved near the trisi~ogangliosides of brain on thin-layer chromatography. The Elson-Morgan reaction was positive for ganglioside VI and for the mixture of VI1 and VIII. By gas-liquid chromatography-mass spectrometry, ganglioside VI and the mixture of gangliosides VII and VIII contain both glucosamine and galactosamine. The only sialic acid found on thin-layer chromatography after mild acid hydrolysis was N-acetylneuram.inic acid. The ratio of galactose : glucose : galactosamine : glucosamine : neuraminic acid in ganglioside VI was (as Me,Si ethers) 2.0 : 1.3 : 0.5 : 0.5 : 1.9. Therefore, this fraction contains either a ganglioside with galactosamine and glucosamine in the same molecule or it is still a mixture of two gangliosides. The carbohydrate ratios in the mixture of VII and VIII (as Me,Si derivatives) were galactose : glucose : galactosamine : glucosamine : neuraminic acid, 2.0 : 1.0 : 0.9 : 0.2 : 1.3. About 0.5% of human kidney gangliosides was due to ganglioside VI and about 0.5% to the mixture of VII and VIII. Composition of long-chain bases After methanolysis in dry HCl . methanol, acetylation and trimethylsilylation, long-chain basis were converted mainfy to 1,3-di-0-Me,Si-NAc and 1,3,4tri-O-Me~Si-NAc derivatives, which were eluted in gas-liquid chromato~aphy after neuraminic acid (Fig. 3). Because of by-product formation in dry HCl methanol [27] (mainly 0-methylation), dry methanolysis is not usually used for long-chain base determinations. However, by adding the peaks having m/e 174 in their mass spectra (m/e 174 is common to various species of long-chain bases, Fig. 4), the response of long-chain bases was fairly constant in gas-liquid chromatography (the peak area of long-chain bases as related to glucose was 0.5 in ganglioside I of human kidney). For various species of long-chain bases, a diagnostic peak [28] arises from the cleavage of the molecule between carbon atoms 2 and 3 (m/e 311 for 1,3-di-O-Me 3Si-NAc derivative of sphingosine and m/e 253 for l-0-Me,Si-3-0-Me-NAc derivative of sphingosine, Fig. 4). For tri-
291
Long-than
bases
III
30
50 TIME
(min)
10
Fig. 3. Gas-liquid chromatograms of trimethylsilylated monosaccharides and long-chain bases obtained in dry methanolysis (0.75 M HCl * methanol, 20 h, 80°C). A, hematoside GM3a from bovine kidney; B. ganglioside III from human kidney. I,II,l-O-MesSi-3-0-Me-NAc derivatives of long-chain bases (mainly from sphingosine); lII.IV.1.3-di-0-Me3Si-NAc derivatives of long-chain bases (III, mainly from sphingosine; IV, mainly from dihvdrosphingosine and sphingosine): V.1.3,4-tri-O-Me~Si-NAr derivatives of long-chain bases (mainly from phytosphingosine). Conditions: 2.2% SE-30, temperature programmed at 4”Cimin from 140 to 240°C. inositol as internal standard.
hydroxybases [ 291, a similar cleavage was also observed, but the cleavage between carbon atoms 3 and 4 favoured (for 1,3,4-tri-0Me,Si-NAc derivative of phytosphingosine m/e 401 and m/e 174 from the cleavage between carbons 2 and 3 and m/e 299 and m/e 276 from the cleavage between carbons 3 and 4, Fig. 4). In the conditions used, IV-acetylation was complete. The most prominent by-product was the 30methylated 4-sphingenine (Fig. 4). The long-chain bases of hematoslde GMja from bovine kidney were used as reference in gasliquid chromatography and gas-liquid chromatography-mass spectrometry. The main the long-chain bases in hematoside GM3, were by this method phytosphingosine and sphingosine, which is in agreement with the analysis of the oxidation products by lead tetraacetate [23]. In all fractions of human kidney gangliosides the main long-chain base was spingosine. Trihydroxybases were found in much smaller amounts than in bovine kidney hematosides (Fig. 3 and Fig. 4). Because of lack of material, identification of the minor fractions of long-chain bases was not attempted in this study.
0
50
50.
lLdu,,,l.l.l
/’
13
73
,73
100
!
132
132
‘P
$63 116 \
91 103
116
of long-chain bases. Above, derivative of sphingosine);
L
106~
Fig. 4. Mass spectra 1-0-MejSi-3-0-Me-NAc
F
?.
2
z iii
YD
50.
100
114
,,
!,l
200
&IL.&
167
.l,,,, 185
216
1,
2p .L
276
mle
.._...
241 /
I
253(t$1741
300
]31,,-,74~9o,
299 W2761
1
,:;‘“:‘-“““,,
M-575
L
401 : 174
: tOCH3
; 0-CH2
253 :174 CH=;Wi 3
:
NH : COCH3
OSiMea
400
.L
:
441
426 (r~9o~59)
I 276
500
: COW3
470(M~g0~,5) 1
299
CH3-(CH2),2-CH2-$H; CtifCH-CHpOStMe3 Me3St0 v+lO: NH
KH2),Z
401 (M-1741
366 (y-591;
360lM-32-151 1
CH3-
M=427
Me+O/
560 Ct$ 151
1
peak III/A of Fig. 3 (mainly 1,3-di-0-MejSi-NAc derivative of sphingosine); middle, peak I/B of Fig. 3 (mainly below, peak V/A of Fig. 3 (mainly 1,3,4-tri-O-Me3Si-NAc derivative of phytosphingosine).
147 I=’
141
174
211
311 NM.174) 1 292W90-103)
311 174
+ CH3~(CH2),iCH=CH-“iCH-CH20S,Me3
M=465
293
Discussion Isolation of gangliosides from extraneural tissues in reasonable yields for structural study is laborious and time consuming. In the handling of relatively large amounts of starting material, it is desirable to lessen the number of chromatographic runs. Therefore, an ethanol precipitation step was introduced after the Folch partition. This step gave an 86% recovery of neuraminic acid, and removed small molecular substances which leak into the upper phase during the Folch partition. Dialysis may be used to remove small molecular organic substances, but it was partially ineffective in this case, perhaps because gangliosides tend to form micelles with these [30]. When precipitated in ethanol after the modified Folch partition and dialysis, the gangliosides were fractionated fairly well on silicic acid columns, and showed minimal or no contamination on thin-layer chromatography. Recently, phosphotungstic acid precipitation has been recommended to purify the ganglioside preparation from small molecular organic contaminants in the water layer of chloroform/methanol/water partition [31]. The amount of neuraminic acid extracted from human kidneys by chloroform/methanol 2/l and l/2 mixtures was considerably lower than that reported for rat, rabbit, pig and bovine kidneys [1,9], i.e. 5.6 mg/lOO g dry weight compared with 13, 13, 16 and 16 mg/lOO g dry weight, respectively. This is a real difference, because there were no essential differences in experimental procedures. According to Martensson [ 32,331, the ganglioside content of human kidney is 0.24 pmol/g dry weight, which is somewhat more than the value calculated from the data of this work (0.17 pmol ganglioside/g dry weight). The dominating ganglioside in human kidney was N-acetylneuraminyllactosylceramide (hermatoside), which is in agreement with reports concerning several extraneural tissues [ 341. The corresponding hematoside containing N-glycolylneuraminic acid was another major ganglioside in bovine kidney [2], but could not be found in human kidney. The absence of N-glycolylneuraminic acid was confirmed for all ganglioside fractions of human kidney, which is in accordance with previous studies on extraneural human gangliosides [3] . The hematoside containing N-acetylneuraminic acid constituted about the same percentage of gangliosides in human kidney as the sum of hematosides GM3 a and GMs,, in bovine kidney [9]. The other major fraction was di-N-acetylneuraminyllactosylceramide, which also was the major ganglioside, after hematosides, in bovine kidney [2,9]. A glucosamine-containing ganglioside was first described in bovine erythrocytes and spleen [ 351, Glucosamine-containing oligosaccharides liberated from ganglioside preparations have been found in human spleen, kidney and liver and in bovine extraneural tissues [ 3,34,36]. Glucosamine has also been found in the gangliosides of human erythrocytes [37], human platelets [38], bovine platelets [ 391, human, bovine and rabbit plasma [40], human newborn brain, peripheral nerve, skeletal muscle, serum and red blood cells [41]. In this work, two gangliosides containing glucosamine as their only hexosamine were isolated. The other of these gangliosides contains fucose, which was unequivocally identified by gas-liquid chromatography-mass spectrometry. Fucose could
294
not be separated from this ganglioside by preparative thin-layer chromatography, and the homogeneity could also be confirmed by analytical thin-layer chromatography in four quite different solvents. These facts suggest that fucose exists in the same glycolipid molecule as neuraminic acid, which is an unusual structural feature. Fucose, galactose, glucose, glucosamine and neuraminic acid have also been found in A and B active glycolipid preparations [42,43]. However, the highly purified A and B glycolipids do not contain neuraminic acid [ 441. Wiegandt has found an H active oligosaccharide containing fucose, galactose, glucose, galactosamine and neuraminic acid from gangliosides of bovine liver [36]. In addition to these two glucosamine-containing gangliosides, a disialoganglioside containing galactosamine and a homogeneous ganglioside fraction containing both galactosamine and glucosamine were isolated. Gangliosides with two hexosamine moieties are not known to exist, and the possibility remains that the fraction containing both galactosamine and glucosamine is still a mixture. Ganglioside structures are often inferred from their thin-layer chromatographic mobilities using brain gangliosides as standards. This work shows quite clearly that these kinds of interpretations can lead to erroneous conclusions. For example, ganglioside III was near to ganglioside G, , a in its thin-layer chromatographi~ behaviour, although it, contains only one sialic acid, and gangliosides VII and VIII moved with brain trisialog~~liosides, although the mixture of these gangliosides contains only 1.32 neuraminic acids per molecule. The dominant long-chain base in all ganglioside fractions was 4sphingosine, as in neutral glycolipids of human kidney [45]. Compared with bovine kidney gangliosides, clearly less trihydroxybases were found in all fractions of human kidney gangliosides. Acknowledgements The technical assistance of Mrs. Liisa Kuivalainen and Mrs. Hilkka Rijnkkij is greatly appreciated. This work was supported by the Sigrid Juselius Foundation and the National Research Council for Medical Sciences, Finland. References 1
Pure,
K..
2
Pure,
K.
3
Wiegandt.
4
Svennerholm.
5
IUPAC-IUB Eur.
6
P. and
H. and
Biicking,
7
Huttunen.
8
Folch,
9
Pure,
21.
Svennerholm. Miettineo.
12
Svennerholm,
Eur.
of
10,
Biochim.
Biophys.
Acta
187,
‘23~-235
401-413 J. Biochem.
15.
287.-292
613-623
Biochemical
Nomenclature
The
(CBN)
Nomenclature
the
Nomenclature (CBN),
of
Organic
Tentative
Chemistry
Rules
for
(CNOC)
Carbohydrate
and
(1966)
Ann.
M. and
SIoane
Acta
I3
Miettrnen,
T.A.
Zimiriski.
T. and
Exp.
Stanley, Sand.
Biochim.
24.
(1961)
Acta Sand.
Borowski.
BioI.
Fenniae
G.H.
(1957) Acta
I.-T.
(1959)
Chem. J. Clin.
E. (1966)
44.
Supul.
J. Bioi.
12
Chem.
226,
497-509
13-22
Biophys.
Takki-Luukkainen.
L. (1958)
14
Med.
Chem.
I,. (1957) ‘I’. and
of
IUPAC-IUB
Nomenclature
455--477
(1970)
11
(1970)
187,
Lipids
(1967)
31
Nomenclature
Lees,
10
on
2, 127--l
J.K.
J.. K.
W.W.
(1969)
Acta
J. Neurochem.
Commission
Commission
J.K.
Biophys.
I,. (lV63)
Biochemical
Biochem.
Hut&men,
Biochxm.
J. Riochem.
IUPAC on
Maury. (196V)
Stand. Lab.
12,
24.
604-611
Acta
Chem.
Stand.
547-554
Invest.
J. Chromatog.
13,
Suppl.
23.
13
480.-482
13,
856-858
Commission (1971)
Eur.
J.
295
15
Penick.
16
Sawarderker,
R.J..
17
Kim,
18
Bjiirndal. Young.
20
Sweeley. Bhatti. Pun),
25
T.,
Rouser.
G.. ed.),
R.E.
and
A.
l,edeen. Kntchevsky.
Chem.
J.R.
(1970)
New
Gaver.
R.C.
and
Sweeley.
C.C.
(1965)
J. .4m.
011 Chem.
28
Gaver,
R.C.
and
Sweeley.
C.C.
(1966)
J. Am.
Chem.
29
Thorpe,
Svennerholm. Dunn.
32
MPrtensson,
E..
Percy,
33
MBrtensson.
E.
(1969)
in Progress
in the
367-407.
Pergamon
Press.
X,
Part
4, pp.
34
Wiegandt.
35
Kuhn.
36
Wiegandt,
37
Wherrett.
38
Marcus,
39
Etzrodt.
H. (1968)
40
Yu.
and
41
MPnsson.
and
Adv.
Wiegandt.
H. (1973) J.R. A.J.,
R.K.
Pullman,
Lipid
Res.
II. (1964)
222.
339-347
393-400 Chromatographic
Analysis
(Marinetti.
10,
113-120 Sot.
42.
88,
294-298
3643.
3647
6. 887-897
239~-250
I,. (1966) Chetmstry
of
11.1,. and
&Tier.
Acta L.B.
Univrrslty
(1972)
J. Lipid
J.,
Y.-T..
Li,
Acta
Paedlatr.
Fats
and
Stand.
other
Llpids
55.
l-9
(Holman,
R.T..
ed.),
Vol.
Oxford 19b, Chem.
%. Physiol. Biophys.
R.W.
Acta
9, 249-289
Biochim.
Holmgren.
187.
Sot.
Z. Naturforsch.
Dissertation.
Lcdern.
17.
Svennerholm.
IIoppe-Seyler’s
(1973)
J.-E..
Sand.
258-274
1801-1804
2.3, 293m-295
A. and
H. (1971)
R.
Chem.
J. Neurochem.
Res.
Biochemistry
30
(1974)
Acta
(1967)
31
A.
I,. (1963)
C.C.
Biophys.
in I.ipid
Klrkkiilnen.
Sweeley.
21.
Yorh
26
and
Carbohyd.
20,
Sand.
279.-287
506--516
.4. (1967)
Dekker.
Bmchem.
116.
1602-1607
1009~~1018
Acta
11.
37.
1192-1200
Biochim.
Res.
Actd
Chem.
1461-1466
J. 125.
Biophvs.
Yamamoto.
246.
36.
Blochem.
Marcel
1~. (19ti9)
Chem.
Chem.
27
S.R.
Vihko,
Anal.
Acta
J. Lipid
G. and 99-m162.
Biophys.
Anal.
(1967)
Biochim.
(1970)
Blochim.
(1965)
J.G.
J. Biol.
(1971)
(1969)
A.
S. (1967)
Anal.
Clamp.
J.R.
R.W.
1’01. I. pp. J. and
and
Clamp.
P~ercr.
(1971)
B. (1964)
(1966)
Jeanes,
Svensson.
S.-I.
Walker.
R.H.
and
T.11. and
B. and
Kerlnen.
and
McClucr. J.H.
Liao.
Chambers,
K. nnd
and
Hakomori.
and
R.E.
R.K.
G.\‘..
B..
and
C.C.
Chambers, Yu.
Shame.
H.J.
22 23
M.H.
Sloanecker.
11.. Lindberg.
21
24
d.S.,
J.H.,
19
Melsler.
326.
(1972) of
80~.-81 354.
1049~~1056
63-73 J. Clin.
Invest.
51.
2602--2612
Kdln
Res.
13.
\‘amer.
680-686
M.-T.
and
Svennrrholm,
L.
(1974)
Med.
Biol.
52.
240-243 42
Ilakomori,
S.-l.
and
43
Hakomori.
S.-l.
(1970)
44
Hakomori.
S.-l.
and
Press,
York
45
New
Martensson,
I?. (1965)
Strycharz. Chem. Kobata,
G.D.
(1968)
Phys.
Lipids
A.
Biochim.
(1974) Biophys.
Biochemistry
7. 12’79
-1286
5. 96-115
in The Acta
Antigens 116.
(Sela.
296-308
M..
ed.),
Vol.
Il.
pp.
79-140,
Academic