Chemistry of gangliosides

Chem. Phys. Lipids 5 (1970) 220-234 © North-Holland Publ. Co., Amsterdam.

C H E M I S T R Y OF GANGLIOSIDES

R. H. McCLUER The Ohio State University Columbus, Ohio 43210, U.S.A.

Gangliosides are a group of glycosphingolipids which contain sialic acid. A large number of different gangliosides have now been reported and distribution studies seem to indicate that each organ may have a unique complement. In some instances certain gangliosides appear to be specific components of a given organ or cell type. Species specificity is also becoming evident. The gangliosides have been recently reviewed by Ledeenl), Wiegandt 2) and Klenk 3). I summarize here information on the different gangliosides which have been reported in reference to their occurrence in different organs and finally describe some of the recent studies of adrenal gland gangliosides conducted at Ohio State.

Brain gangliosides The nine brain gangliosides whose structures have been fairly well established are listed in table 1 along with structures and a summary of different notations and abbreviations to be used in this paper. Klenk and Naoi 4) recently established the linkage of the second NANA residue in GnSSLC. Several other brain gangliosides, not listed in table l and not thoroughly characterized, have been reported. A ganglioside which migrates below the major trisialoganglioside (SGGnSSLC) was reported by Kuhn and WiegandtS). Because the material upon treatment with neuraminidase gave rise to a component migrating as the trisialosylganglioside, it was designated as a tetrasialosylganglioside (Gv). Penick and McCluer 6) isolated a material with similar chromatographic properties but analytical data indicated it to be a trisialosylganglioside. Klenk et al. 7,s) have also reported a second trisialosylgangtioside (C2) which lost one galactose residue, when subjected to periodate oxidation, like the trisialosylganglioside examined by Johnson and McCluerg). A tetrasialosylganglioside (C4) was also reported which may be the same as Gv. Klenk and Gielen 10) have isolated a disialosyltrigalactosylceramide (D) from human brain. Tettamanti, Bertoza, Gualandi and Zambotti 11,1~) investigated the gangliosides of shark, frog, chicken, pig and 220

Sialosylgalactosyl ceramide Sialosyllactosyl ceramide Disialosyllactosyl ceramide N-acetylgalactosaminyl(sialesyl) lactosyl ceramide (Tay Sachs) N-acetylgalactosaminyl(disialosyl) lactosyl ceramide Galactosyl-N-acetylgalactosaminyl(sialosyl) lactosyl ceramide SialosylgalactosyI-N-acetylgalactosaminyl (sialosyl)lactosyl ceramide Galactosyl-N-acetylgalactosaminyl (disialosyl) lactosyl ceramide Sialosylgalactosyl-N-acetylgalactosaminyl (disialosyl)lactosyl ceramide

Trivial name

TABLE 1

GGAL GLAcl GLAC2 GGNTtlI1 (Go) GGnTrII2

N A N A ( 2 - + 3)Gal(l --+ 1)Cer NANA(2--~ 3)Gal(1 -+4)Glc(1 -+ l)Cer NANA(2--~ 8)NANA(2-~ 3)Gal(1 -+4)Glc(1 --~ 1)Cer GalNHAc(1 -+4)Gal(1 -+4)Glc(1 --> 1)Cer N A N A (2 -->3) -J GalNHAc(I ~ 4 ) G a l ( 1 ~4)Glc(1 ~ 1)Cer N A N A (2 --~8) N A N A (2 ~ 3) __1 Gal(l ---~3) GalNHAc (1 ---~4) Gal(1 ---~4) Glc(l ~ 1)Cer NANA(2 ~ 3) _J NANA(2---~ 3)Gal(1 ~ 3)GalNHAc(1 ---~4)Gal(1 --~4)Glc(1 -+ 1)Cer NANA(2-+3) __J

SGC SLC SSLC GnSLC

GnSSLC

Gal(1 ---~3)GalNHAc(I ~ 4 ) G a l ( 1 -+4)Glc(1 --~ l)Cer N A N A (2 ~ 8) N A N A (2 ~ 3) -J SGGnSSLC NANA(2---~ 3)Gal(1 ---~3)GalNHAc(1 ---~4)Gal(l---~4)GIc (1 --~ 1) Cer N A N A (2 ~ 8) N ANA (2 ~ 3) /

GGnSSLC

SGGnSLC

GGnSLC

GG~T2b (Gin) GGNT3a (G iv)

GGNT1 (GI) GG~T2a (GII)

Kuhn and Wiegandt

Abbreviation

Structure

Structure and nomenclature summary of brain gangliosides

A1

GM2

A2 B1

C1

C3

GMI GDIa

GDXb

GTI

B3

A0l Aoz GM3

G1

G2

G3

G4

G2A

Ge GaA G5

Korey Svennerholm Klenk and et al. et al. Gonatas

Notations

tJ

z r-

,.e

222

R.H.MCCLUER

monkey. They report the presence of a monosialosylganglioside which is apparently isomeric with GGnSLC, a disialosylhexosaminyltetrahexosylceramide (pig), a tetrasialosylganglioside which may be identical with Gv, a pentasialosylganglioside (frog and chicken), and a hexosamine-free trihexosytganglioside which may be identical with the trigalactosylganglioside reported by Klenk and Gielenl°). Booth, Goodwin and Cumings 13) noted two abnormal gangliosides in extracts of brain from gargoylism and Nieman-Pick's disease and reported that these materials analyzed as sialosyldigalactosylceramides. However, other workers14,15) did not detect these unusual gangliosides in gargoyle brain. The long chain bases (LCB) of brain gangliosides contain predominately C18 and C2o sphingosine with small amounts of the saturated bases16,~7). The C2o/C1 s ratio increases with age 18); C18 sphingosine being the predominant base in fetal brain ganglioside19). Klenk, Hof and Georgias s) reported evidence for the presence of C22-sphingosine in GGnSSLC (C1) and SGGnSSLC (C3). Stearic acid is the major fatty acid present, usually comprising about 90~o with 16:0, 20:0 and 22:0 making up the bulk of the remainder1). The minor brain gangliosides show deviations from this pattern. SGC contains long-chain and hydroxy fatty acids 20,21). Ledeen, Salsman and Cabrera 22) isolated nine gangliosides fractions from the brain of a boy with subacute sclerosing leukoencephalitis and determined fatty acid compositions. Seven contained stearate as the major component, while two (SSLC and GnSSLC) had relatively large proportions of oleate and palmitate. Gangfiosides of non-neural tissue

Erythrocyte gangliosides In 1951 Yamakawa and Suzuki 2a) isolated a glycolipid, from horse erythrocyte, which was subsequently identified as hematoside (SLC). This term was used to refer to a group of glycosphingolipids which contain sialic acid but no hexosamine and thus does not refer to SLC specificially 24). SLC from horse, cat and bovine erythrocytes contain only N G N A while dog SLC contains N G N A and NANA. Handa and Handa 25) isolated the main glycolipid from cat erythrocytes and identified it as SSLC which contains only NGNA. Kuhn and Wiegandt 5) identified a ganglioside from bovine erythrocytes which upon ozonolysis and mild hydrolysis gave lacto-Nneotetraose: Gal-(fl 1~ 3)GlcNAc(fl 1~ 4)Gal(fl I ~ 4)Glc. Gangliosides which contain galactosamine are also present in bovine erythrocytes. The SLC from erythrocytes seem to typically contain long-chain fatty acids with 24:0 and 24: lpredominating zn, 27).

CHEMISTRY OF GANGLIOSIDES

223

Spleen Gangliosides account for about 3.3~o of the total lipids of spleen2S). The major component is SLC 29) and is increased in amount in Gaucher's disease3O.31). Kenneway and Wolf 32) reported a case in which spleen ganglioside was increased twenty-fold. Svennerholm 33) mentioned the isolation of a monosialosyl trihexosylceramide from normal human spleen. The ganglioside giving rise to lacto-N-neotetraose mentioned above was also isolated from bovine spleenS). Wiegandt 2) has indicated that the spleen gangliosides show strong interspecies variation.

Other organs Svennerholm 29) investigated the gangliosides of human placenta and reported SLC to be the major glycolipid component. Small amounts of SSLC and more complex gangliosides were also detected. The total ganglioside concentration 020 rag/100 g dry weight)was reported to be greater than in kidney, liver, lung, lymph node and muscle. In kidney, chromatographic evidence indicates that SLC is the major ganglioside component present with smaller amounts of other more polar gangliosides a4). Mfirtensson 3~) (1966) reports that there are 0.3 mg of gang/g dry wt. Hakomori and Murakami 36) have reported on the glycolipids of hamster kidney fibroblasts and derived malignant transformed cell lines. These tissue culture cells contained SLC as the major ganglioside and smaller amounts of more complex gangliosides which exhibited interesting immunological properties. Vance, Shook and McKibbin 37) investigated dog intestine glycolipids and analyses of two ganglioside fractions were consistent with a ganglioside structure of sialosylgalactosyllactosylceramide which suggests they may be globoside derivatives. The glycolipids of porcine intestine 3s) contain SLCs with NANA and N G N A in about equal amounts. Small amounts of other acidic glycolipids were also detected. SLC has been detected in human and guinea pig lung 39) and in human liver40). The gangliosides of human lens were reported to consist of monosialosyldihexosylceramides and monosialosyltrihexosylceramides with dihydrosphingosine as the major LCB present and palmitate and nervonate as the predominant fatty acids 41). Ledeen, Salsman and Cabrera 42) have investigated the gangliosides of bovine adrenal medulla, about 90~o of which consist of two sialosylgalactosylglucosylceramides which contain NANA or NGNA. The remainder consists of at least four, more complex gangliosides. At Ohio State we (Siddiqui, Evans, Pagay and McCluer) have been

224

R . H . MCCLUER

studying the gangliosides of beef adrenal cortex and medulla. The following is a report on this work.

Isolation of adrenal gland gangliosides Fresh beef adrenal glands obtained from the slaughterhouse were carefully dissected into cortex and medulla and then lyophilized. These preparations were reconstituted with water to the original wet weights and extracted essentially according to the procedure of Suzuki4a), as shown in fig.i. The suspensions were homogenized with 19 vol of C : M (2: 1), filtered and the residue reextracted with 10 vol of C : M (1:2) containing 5% H20. The extracts were combined and chloroform added to make a final C: M ratio of 2:1 and 0.2 vol of 0.88% KCI added. After separation of the phases, the lower phase was extracted once with "Folch pure upper phase" with salt and once with "Folch pure upper phase" without salt. The combined upper phases were concentrated at 40°C in a rotary evaporator, dialyzed, passed over Dowex-50 x 2[H ÷-] and lyophilized to yield dark brown preparations. Crude gangliosides (880 mg) were placed on a 200 x 25 mm Anasil S column and eluted with 1.2 1 of C : M : H 2 0 (60:35:8), 900 ml of C : M : H 2 0 (60: 40: 10) and 800 ml of methanol. 10 ml fractions were collected. The fractions were assayed by TLC in p r o p a n o l : N H 3 (7:3) and pooled to make four preparations A a, B 1, C~ and DI as shown in fig. 2. The TLC behavior of these preparations are shown diagramatically in fig. 4. Components of fractions C-A 1 were purified by preparative TLC as shown in fig. 2. Component C-7 was obtained in only small quantities still contaminated with a nonganglioside material. Medulla gangliosides (330 mg) were similarly treated as shown in fig. 3. The TLC behavior of the first column preparations are shown diagrammatically in fig. 4. Note that cortex preparation C-A1 has a component not seen in the corresponding medulla preparation. The resorcinol-positive components were numbered according to their behavior in C: M: N H 3 (60 : 35 : 8) in a manner analogous to that used by Ledeen et al.32). Components from M-A~ (M-5 and M-6) were further purified by rechromatography on Anasil Columns as shown in fig. 3. Components M-5, M-6, C-5 and C-6 were chromatographically homogeneous in 3 solvent systems (fig. 5) and were used for further analysis.

Methods for structural analysis

Quantitation of carbohydrate moieties We determine the molar ratios and percent content of glucose and galactose by the methanolysis-silylation GLC procedure 44) as previously described 45).

J

Fig. l.

UPPER PHASE J

I

l

FINAL LOWER PHASE

WITHOUT KCI

I F.P.U.P.

LOWER PHASE

50% MeOH EXTRACT

~ LYOPHILIZE

RESIDUE

50% MeOH (3 Vols)

DIALYSIS I DOWEX 50 X-2 [H+]

EXTRACT

J

RESIDUE

I

I0 Vol C/M (2/1) + 5% H20

CORTEX (0.68% DRY WEIGHT), MEDULLA (0.91% DRY WEIGHT)

F.P.U .P. WITH KC1

J RESIDUE

Extraction of adrenal cortex and medulla gangliosides. F.P.U.P. refers to "Folch pure upper phase" (Eolch, Lees and Sloane-Stanley, 1957).

CRUDE ADRENAL GANGLIOSIDE

DIALYSIS DOWEX 50 X-2 LYOPHILIZE

J

UPPER PHASE

I

EXTHACT J

r

LOWER PHASE

0.2 Vol 0.88% KCI

COMBINED UPPER PHASES

UPPER PHASE

EXTRACT

19 Vol C/M (2/1)

CORTEX OR MEDULLA

226

R.H.MCCLUER

The following micromodification of the procedure has been used more recently. Approximately 0.05 ~mole of ganglioside and 50/~g of mannitol are placed in a small conical glass vial, dried and dissolved in 0.3 ml of 0.5N anhydrous methanol-HC1. The tube is flushed with N2, sealed and heated at 78 °C for 24 hr. The contents are diluted with an equal volume of methanol and extracted 3 times with n-heptane to remove fatty acid esters. The methanol phase is then passed over Dowex-50[H ÷] if hexosamine is present. The solution is taken to dryness under a stream of N2 in a small

CRUDE GANGLIOSIDES

ANASIL COLUMN 60/30/5, 60/40/10, MeOH

l

I

C-A 1 (5+6+7)

C-B 1 (pure C-5)

C-C 1 (2+3+4+5)

C-D 1 (1+2+3)

C-A I

Prep TLC Prop/NH 3 (7/3)

l

I

C-A 2

C-B 2 (C 5)

(C-6)

Prep TLC C/M/H20

C-C 2 (5+7)

Prep TLC Prep/H20 (7/3)

Prep TLC C/M/H20 (65/25/4)

borate

C-A 3

C-B 3

(C-6)

(C-5)

Fig. 2.

C-C 3 (impure C-7)

Chromatographic purification of adrenal cortex gangliosides.

CHEMISTRY

227

OF GANGLIOSIDES

conical vial and silylated with 20 ml of HMDS:TMCS:pyridine (5:1 : 10) at 60°C for 1 hr. (These silylating conditions are necessary for complete solubilization and reaction of the mannitol.) The reaction mixture is evaporated to dryness and products dissolved in 20/11 of CH2C12 or freshly distilled CS 2. It is usually necessary to evaporate the sample to dryness 3 times to remove residual pyridine. Hexosamine and sialic are detected by the silylationGLC procedure but we prefer to utilize micromodification of the resorci-

CRUDE GANGLIOSIDES

ANASIL COLUMN 60/30/5, 60/40/10, MeOH

I

I

M-A I (5+6)

M-B I (pure M-5)

M-C I

M-D 1

(4+3+2)

(1+2+?)

M-A 1

ANASIL COLUMN 65/25/4

f M-A 2 (6+?) L

M-B 2 (pure M-6)

M-A 2

M-C 2

M-D 2

(6+5) I

(5)

+ M-C 2

ANASIL COLUMN 70/25/3

f

I

M-A 3

M-B 3

M-C 3

(M-6)

(5+6)

¢'I-5)

Fig. 3. Chromatographicpurification of adrenal medulla gangliosides.

228

R.H. MCCLUER

no146) a n d M o r g a n - E l s o n m e t h o d s 47) for q u a n t i t a t i o n o f these c o m p o n e n t s .

Ozonolys& W e have utilized a m i c r o o z o n o l y s i s p r o c e d u r e to examine the sphingosine moiety o f the gangliosides. A n a p p a r a t u s similar to that described by

C/M/N H3 (60/35/8)

C-7

6 5

M-6

m

~

m

m~-~ m m m m m

2 3

mm m m m m

m

5 4 3 2 1

C - A 1 C - B 1 C-C 1 C-D 1 M-A 1 M-B 1 M-C1 M-D 1

Propano[/NFI 3 C-7 5 6

m m ~ mm

m

m m ~

m m m n m

(7/3)

m m m m m m

M-6 5

C-A~ C-B 1 C-C~ C-D1 M-A 1 M-B1M-C 1M-D 1

P r o p a n o [ / FI2 0 C-6 7 5

n m m m ~ m m m m m m

(7/3)

M-6 M-5

m m ~ m mm m m m

m

C-A 1 C-B1 C-C 1C-D 1 M-A 1M-B1M-C 1 M-D1

Fig. 4. Diagrammatic representation of adrenal gland TLC mobilities. Fractions are those obtained from Unasil S column as shown in figs 2. and 3. Spots were developed with resorcinol reagent.

229

C H E M I S T R Y OF G A N G L I O S I D E S

(a)

(b)

i ~,i~!i~~!~,i~ii,, i~:!,~!¸¸i i!i~i!ii~iii~iii ~i~ .......... ~~ ~'~'~~'i~I,'~ . . :~ . . . . . ~i~/i ~

~li~i~i!~m!, !i!!~ ~,'/i~i, '~,~¸~i'~i,~' i~!'~! i~~i~!~i~i!!~i~:~l i~i~i~iii~,~i~~i,~i~~i~ i ,i~~,

1 ~'i~~ ~i

i! 'IT ¸¸¸¸¸~~ ~":~ !

i!ii I

IN:

i

~ (c)

Fig. 5. T L C plates o f gangliosides T.S. ( G n S L C ) , C-5, C-6, M-5 a n d S T D ( s t a n d a r d mixture c o n t a i n i n g G n S L C , G G n S L C , S G G n S L C , G n S S L C a n d S G G n S S L C ) . A d s o r b a n t was Silica gel G a n d spots were developed with resorcinol reagent.

230

R.H.MCCLUER

Beroza and Bierl 4s) is used. Ozone is passed through 0.4 ml of ethanol containing 0.05 to 0.2 mg of ganglioside for 5 rnin at 70°C. The reaction mixture is allowed to warm to room temperature, mixed, flushed with N2 and the residue dissolved in 0.4 ml of 9 7 ~ MeOH. The aldehydes are extracted with 0.6 ml of hexane and the hexane is washed once with water. The aldehydes are identified on a 15~ EGS column (6 ft. × ¼ in.) at 150 °C with a carrier gas flow rate of 80 ml/min. Results indicate the presence of C 1s sphingosine in cerebrosides and approximately equal amounts of CI s and C2o sphingosine in brain gangliosides (fig. 6) in agreement with results

c ©

3

(D

-~

Time

Fig. 6. GLC (on 15 ~ EGS) of aldehydes obtained by ozonolysis of brain gangliosides GnSLC. Peaks identified as: (1) tetradecanal, (2) unidentified, (3) hexadecanal, (4) unidentified.

obtained by independent methods49). This ozonolysis procedure is rapid and sensitive, and will provide data for the monounsaturated LCB but of course will not detect any saturated or diene LCB which may be present.

Permethylation studtes We have utilized the permethylation procedure of Adams and Gray 50) for the analysis of gangliosides. The procedure is applicable to 0.2 to 1.0 mg of sample and involves rnethylation with methyl iodide in dimethylformamide in the presence of finely ground barium oxide and barium hydroxide. In some instances we have purified the methylated product by preparative T L C [Silica gel G developed with benzene:methanol (85: 15)]. The methylated product is subjected to methanolysis on 0.2 ml of 0.5 N anhydrous methanolic HC1 for 4 hr at 90 °C. The solution is evaporated to dryness, the residue is dissolved in 0.5 ml of hexane and placed on a small "Unisil" column (silicic acid). Liberated lipids are eluted with 10 ml of petroleum ether-

231

CHEMISTRY OF GANGLIOSIDES

diethyl ether (9: 1) and permethylated sugars are eluted with 10 ml of absolute methanol (in some cases, we have found it necessary to r e c h r o m a t o g r a p h the samples on Unisil in order to remove all traces of interfering lipids). The methyl ethers are examined directly by G L C on 3~o E C N S S - M column (6 ft x ¼ in glass coil) at 125°C and are also silylated with H M D S : T M C S : pyridine (5:1:10) and analyzed on the 3 ~ E C N S S - M column at 110°C and on a 15~o E G C column (6 ft x ¼ in) at 150 °C. Conditions for permethylation were checked and identification of methyl ethers was accomplished by methylation of methylglucoside, methylgalactoside, glucosyl ceramide, lactosyl ceramide, lactose, melibiose, and 3'neuraminosyllactose. Analysis of standard gangliosides, sialosyl-(1 ~3)-lactosyl ceramide (SLC), N-acetylgalactosaminyl-(1 ~4)-[sialosyl-(l--*3)]-lactosyl ceramide (GnSLC), galactosyl-(1--*3)-N-acetylgalactosaminosyl-(1 ~4)-[sialosyl-(1--*3)]-lactosyl ceramide ( G G n S L C ) , and the asialo c o m p o u n d galactosyl-(l~3)-N-acetylgalactosarninyl-(l ~4)-lactosyl ceramide ( G G n L C ) , have allowed us to identify the 2,6-dimethyl galactosides and 2,3,6-trimethylgalactosides. The relative retention times o f the methyl ethers are presented in table 2. The hexoTABLE2 Relative retention values of methylated hexoses Compounds Methyl-2,3,4,6-tetra-Omethylgalactosides Methyl-2,3,4,6-tetra-Omethylglucosides Methyl-2,3,6-tri-Omethylgalactosides Methyl-2,3,6-tri-Omethylglucosides Methyl-2,4,6-tri-Omethylgalactosides Methyl-2,3,4-tri-Omethylglucosides Methyl-2,6-di-O-methylgalactosides

3 ~ ECNSS-M 125°C

3 ~ ECNSS-M I10°C (TMSi)

1.00 0.92

1.00

0.74 0.47

0.76 0.48

-

-

2.74? 1.95

0.50 0.44

-

-

2.95 2.00

0.60 0.35

0.49 0.31

3.00 2.50

0.52

0.40

2.20 1.35

0.43 0.26

0.34 0.23

0.41 0.25

0.21 0.14

-

-

-

15 ~ EGS 150°C (TMSi)

-

1.00 0.93

-

samine derivatives formed under these methylation conditions do not interfere and are eluted from the E C N S S - M column as multiple peaks at 170°C.

Adrenal medulla gangliosides The two major gangliosides (Ms and M6) o f adrenal medulla were shown

232

R . H . MCCLUER

to contain N-glycolyl- and N-acetyl-neuraminic acids, respectively, as well as equimolar amounts of glucose and galactose and thus correspond to AG5 and A G 6 previously reported by Ledeen et al.42). Permethylation of these materials led to the production of 2,4,6-tri-O-methylgalactosides and 2,3, 6-tri-O-methylglucosides which were identified by GLC with and without silylation. Thus, the structure sialosyl-(2~3)-galactosyl-(1--,4)-glucosylceramide can be assigned to these gangliosides. The sphingosine moiety of M 5 and M 6 w a s examined by the microozonolysis procedure. In both samples tetradecanal was the major product obtained; no hexadecanal was detected. The major fatty acid components of these two gangliosides were found to be 16: 0, 18 : 0, 22: 0, 23 : 0, 24: 0 and 24:1. We have not examined the more complex gangliosides (M-1 through M-4 as yet).

Adrenal cortex gangliosides Gangliosides C-5, C-6 and C-7 from the adrenal cortex have been examined. Small amounts of C-7 still contaminated with non-ganglioside material showed chromatographic properties similar to M-6 and is presumably N-acetylneuraminosyllactosyl ceramide, but no further studies have been conducted on this trace component. Ganglioside C-5, which is the major ganglioside component of bovine adrenal cortex (80~o-90~o), was obtained chromatographically pure and crystallized from 95~ methanol. Its chromatographic behavior in four solvent systems was identical with that of M-5. It contained N-glycolylneuraminic acid, glucose and galactose in equimolar amounts. Permethylation led to the production of 2,3,6-tri-O-methylglucosides and 2,4,6-tri-Omethyl-galactosides. Thus C-5 is assigned the structure N-glycolylneuraminosyl-(2~3)-lactosyl ceramide. Evidence that C~s sphingosine was the major LCB present was obtained by ozonolysis. Fatty acid composition was similar to that of the medulla gangliosides. Ganglioside C-6 was obtained chromatographically homogeneous in several solvent systems but showed three resorcinol-positive spots in C: M :W (60:35 : 8). It showed unusual chromatographic characteristics-in C: M : H 2 0 (60: 35: 8) it migrated ahead of M-6 while in propanol : N H 3 (7 : 3) it migrated below GnSLC (Tay-Sachs) ganglioside (see fig. 5). The glucose-to-galactose ratio of C-6, as determined by GLC, was 1.3. No hexosamine was detected. Ganglioside C-6 was resistant to hydrolysis by CI. perfringens neuraminidase under conditions (pH 5.0, 37 °C for 72 hr) which completely hydrolyzed the sialosyllactosyl ceramides (M-5, M-6 and C-5). For mild acid hydrolysis the samples were heated in 0.05 N H2SO4 at 80 °C for 90 rain, neutralized with a small amount of Na2CO3, and 4 vol

CHEMISTRY OF GANGLIOSIDES

233

of C:M(2:I) a d d e d . The u p p e r phase was analyzed for sialic acid in p r o p . : N H 3 : H 2 0 ( 6 : 2 : 1 ) a n d the lower phase was e x a m i n e d by T L C in C : M : H 2 0 (65:25:4). U n d e r these conditions, gangliosides M-5 a n d M-6 were c o m p l e t e l y h y d r o l y z e d a n d yielded N - g l y c o l y l - a n d N - a c e t y l - n e u r a m i nic acid, respectively, as well as a p r o d u c t m i g r a t i n g like lactosyl ceramide. G a n g l i o s i d e C-6 a p p e a r e d to be only a b o u t 10~o h y d r o l y z e d a n d the u p p e r a q u e o u s phase yielded two resorcinol spots with mobilities the same as N-glycolyl- a n d N-acetyl n e u r a m i n i c acids. G a n g l i o s i d e C-6 was also resistant to hydrolysis by 0.03 N HCI at 80 °C for one h o u r which liberated m o s t o f the sialic acid f r o m M-5 a n d M-6 (SLCs). G a n g l i o s i d e C-6 was treated with 1.0 N N a O H at 37°C for 16 hr, neutralized a n d e x a m i n e d by T L C in 6 0 : 3 5 : 8 (C: M :W). A p r o d u c t f o r m e d which m i g r a t e d as N - g l y c o l y l n e u r a m i n o s y l l a c t o s y l ceramide. This ganglioside (C-6) gave a positive h y d r o x a m i c acid test a n d is p r o b a b l y a lactone or ester derivative o f SLC. Ozonolysis o f C-6 indicated t h a t sphingosine was the m a j o r L C B c o m p o n e n t a n d the fatty acid c o m p o s i t i o n was similar to that o f o t h e r a d r e n a l ga ngliosides.

References 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21) 22) 23) 24) 25)

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