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Isolation and characterization of a tetrasialoganglioside from mouse brain, containing 9-O-acetyl,N-acetylneuraminic acid
Neurochemistry International, Vol. 4, No. 6, pp. 531 to 539, 1982. Printed in Great Britain.
0197-0186/82/060531-09503.00/0 © 1982 Pergamon Press Ltd.
ISOLATION A N D CHARACTERIZATION OF A
TETRASIALOGANGLIOSIDE FROM MOUSE BRAIN, CONTAINING 9-O-ACETYL,N-ACETYLNEURAMINIC ACID VANNA CHIGORNO, SANDRO SONNINO, RICCARDO GHIDONI and G u m o TETTAMANTI Department of Biological Chemistry, The Medical School, University of Milan. Milan, Italy (Received 19 April 1982; accepted 28 May 1982) A~tract--A new ganglioside, containing an alkali-labile linkage, was extracted from mouse brain and purified. It represents 3.6% of total lipid-bound sialic acid in the tissue and was obtained in pure form with a yield of about 35%. It contains sphingosine, glucose, galactose, N-acetylgalactosamine and sialic acid in the molar ratio 1 : 1 : 2 : 1 : 4 and, upon exhaustive sialidase treatment gives the monosialoganglioside GM1. Partial acid hydrolysis, methylation analysis, gas-liquid chromatography-mass spectrometry and chromium trioxide oxidation studies showed its basic neutral glycosphingolipid core to be ganglio-N-tetraose-ceramide. Three of the four sialic acid residues are N-acetylneuraminic acid and one, as shown by gas-liquid chromatography-mass spectrometry, is 9-O-acetyI,N-acetylneuraminic acid, which contains the alkali labile linkage. 9-O-acetyl,Nacetylneuraminic acid is ~-ketosidically linked to position 8 of the N-acetylneuraminic acid residue bound to position 3 of the internal galactose. The other two N-acetylneuraminic acid residues form a disialosyl residue linked to position 3 of external galactose. The complete structure of the studied ganglioside is as follows: NeuAc~t2-8NeuAc~t2-3Galfll-3GalNAcfll-4(9-O-Ac-NeuAc~2-8NeuAcct2-3) Galfll-4 Glcfll- l'-N-acylsphingosine, and it can be considered as a derivative of the tetrasialoganglioside GQlb.
In the course of a systematic study on mouse brain gangliosides we observed that some minor components changed their chromatographic behaviour after exposure of the ganglioside mixture to alkaline conditions (Sonnino et al., 1978a). Since all the gangliosides known at that time were resistant to such treatment we concluded that the mixture contained some gangliosides with alkali labile groups. This chemical feature was an indication of a novel type of gangliosides or of natural ganglioside derivatives. One of the alkali labile gangliosides occurring in mouse brain, recently identified (Ghidoni et al., 1980) resulted to be a trisialoganglioside (GTlb) bearing 9-O-acetyi,N-acetylneuraminic acid, and was coded O-Ac-GT 1b. The present paper describes the isolation and structural characterization of a second alkali labile ganglioside of mouse brain. It is a tetrasialoganglioside,
namely G Q I b , containing 9-O-acetyl,N-acetylneuraminic acid. The 9-O-acetyl group represents the alkali labile group of this ganglioside. This compound is provisionally coded O-Ac-GQlb.* MATERIALS AND METHODS Materials Commercial chemicals were of analytical or of the highest available grade. Solvents were redistiUed before use. Water for routine use was freshly redistilled in a glass apparatus. Silica gel for column chromatography (Kieselgel 100, 0.063-0.2 mm; 70-230 mesh, ASTM, and high performance thin layer chromatography (HPTLC) plates were purchased from Merck GmbH (Darmstadt, F.R.G.); Sephadex G 25, fine, from Pharmacia (Uppsala, Sweden); reagents for gas chromatography (GLC) from C. Erba (Milan, Italy); N-acetylneuraminic acid from Sigma Chem. Co. (St. Louis, MO, U.S.A.); glucose, galactose, N-acetylgalactosamine and m-inositol from Fluka (Buchs, Switzerland); Vibrio Cholerae sialidase (EC 3.2.1.18) from Behfingwerke (Marburg, F.R.G.); 9-O-acetyl,N-acetylneuraminic acid was isolated from bovine submandibular glands and crystallized according to Buscber et al. (1974). Whole brains (total fresh tissue: 500g), removed from adult mice (C17BI/6J strain) immediately after slaughtering, were washed in cold saline isotonic solution, frozen and finally lyophilized. This material was stored at - 20°C.
Address correspondence to: Prof. Guido Tettamanti, Istituto di chimica Biologica, Via Saldini, 50 20133, Milano, Italy. * Abbreviation used: gangliosides are coded according to Svennerholm (1964). TLC, thin layer chromatography; GLC, gas liquid chromatography; MS, mass spectrometry. 531
q32
\ *~\",'~ ( I H l a ) R N ~ ) cl it/.
M e{ hod.~ l~olution and puri/ication of O-4c-GQlh. The extractitm and puritication of the total gangliosides fiom the lyophilized brains was performed according to the procedure of Fettamanti eta/. t19731. Ganglioside O-Ac-GQ lb was isolated from the total ganglioside extract b 3 column chromatography on Kieselgel 100. The experimental conditions v,ere the following: 4 × 2(X)cm column: temperature. Ig 20C: solvent, chloroform: methanol: 0.3",, aqueous CaCI 2 60:35:6 by volume: llow rate. 6 0 m l h ~:total w)l. for complete elution. 4.5 5.01: 20ml fi-actions automatically collected. The elution pattern was monitored by thin layer chromatography (TLC) on HPTLC plates using the solvent system chloroform:methanol:0.2'!i, aqueous CaCI 2, 50:40: 10, by volume. The O-Ac-GQIb containing fractions obtained after the first chromatography werc collected and rechromatographed until the isolated product was homogeneous. The material thus obtained was purified by column chromatography on Sephadex G 25 according to Ghidoni ct al. (1976). in order to remove salts, small molecular weight impurities and silica gel lines. The eluted fractions containing the product wrere combined and evaporated to dryness in t'acuo at 35 (7. The residue was further purified by acetone precipitation {Sonnino et ul., 1978b). The final white powder (about 12mg of O-Ac-GQlb) was stored al - 20 C. The purity of the isolated ganglioside was assessed by chemical analyses (see belowl and its homogeneity by TLC on HPTLC plates under the conditions specified below. The gangliosides GM3, GM2, GMI, Fuc-GMI, GDIa, GDIb. GTIb, G Q I b and O-Ac-GTIb were prepared and structurally identified as previously described (Tettamanti et al.. 1973: Ghidoni et al.. 1976: Sonnino et aI., 1978: Ghidoni el a/., 1980). Ganglioside G Q l c was isolated and purified from codfish brain as reported by Ando and Yu (1979). Chemical composition and structural .~tudics (71 O-,4cGQIh. The following analyses were performed: (a) determination of carbohydrate and lipid composition: (b) partial hydrolysis of ganglioside followed by isolation and chemical characterization of the neutral glycosphingolipids; It) cleavage by sialidase or mild acid treatment, isolation and chemical characterization of acylneuraminic acids from ganglioside: (d) isolation and chemical characterization of the gangliosides obtained by partial sialidase treatment of the original O-Ac-GQIb: {el alkaline treatment of O-AcG Q l b and of the gangliosides obtained after its hydrolysis by sialidase; (f} methanolysis of ganglioside and of glycosphingolipids obtained by its partial hydrolysis: (g)anomeric configuration analysis. The experimental details on the above analytical methods have been given in a previous paper (Ghidoni et al.. 1980). The isolation of the individual gangliosides obtained by partial sialidase hydrolysis of O-Ac-GQIb was attained using the procedure described by lwamori and Nagai (197gi.
RESULTS The ffactionation of the total ganglioside mixture obtained from mouse brain led to isolation of a compound with a c h r o m a t o g r a p h i c behaviour intermediate between O - A c - G T I b and G T I b (see Fig. 1).
This behaviour ~sas different trom that ol the gallg[iosides known so far. Since after alkaline trc~ttmcnt it behaved as G Q I b (see Fig. 2.) it was coded O-Ac:G Q I b . in terms of bound NeuAc gangliosidc O-AcG Q I b represents 3.6"o of the total gangliosidc bound NeuAc. The compound, after isolation and purification from the ganglioside mixture 112 mg trom 500 g of fresh bruin, with a 35",,yicldi is chromatographically homogeneous in different solvent s~stcms
Chemical composition ~# O-A c-GO lh The results of G L C and chemical analyses reveal that O - A c - G Q l b contains sphingosine, glucose, galactose, N-acetylgalactosamine and sialic acid in a molar ratio of 1.00:1.05 : 1.94:0.93:3.88 {theoretical t : 1 : 2 : 1 : 4 ) (see Table 1). The sialic acid value represents, in molar terms, the sum of N'euAc and 9-0acetylNeuAc. The nature of the sialic acids was established after release of these residues by mild acid hydrolysis. A pure sialic acid fraction of about 100/2g was obtained from 2.0mg of O - A c - G Q I b after this treatment. It was composed of N-acetylneuraminic acid and of 9-O-acetyl,N-acetylneuraminic acid in a molar ratio of about 4:1. The latter c o m p o u n d comigrates with authentic 9-O-acetyl,N-acetylneuraminic acid on both cellulose and silica gel thin layer plates. When analysed by G L C two peaks are detected (see Fig. 3.) with retention times of 1.00 and 1.14 relative to per-O-trimethylsilyI-N-acetyl neuraminic acid methyl ester. This chromatographic behaviour indicates the presence of two c o m p o u n d s in the ganglioside, N-acetylneuraminic acid (compound 1) and 9-O-acetyI,Nacetylneuraminic acid (compound 2). These results were supported by G L C MS. In fact the n u m b e r and mass units of the characteristic fragment ions obtained from c o m p o u n d s 1 and 2 (see Fig. 4.) are identical with the corresponding ones obtained in our previous investigation on O - A c - G T I b ganglioside (Ghidoni et al., 1980), and are consistent with those expected respectively for N-acetylneuraminic acid and 9-O-acetyl,N-acetylneuraminic acid (Kamerling et al., 1975: Kamerling et al., 1974; Kamerling et al., 1978). The fatty acid and long chain base composition of O - A c - G Q I b is shown in Table 1. The major fatty acid components are C 1 8 : 0 and C 2 2 : 0 (63.5",, of total), The long chain bases are predominantly C 18 : 1 and C 20 : 1 (801),i of total); the remainder is constituted by the corresponding saturated derivatives. Structure ~g O - A c-GQ lb ganglioside O-Ac-GQIb, when submitted to partial acid hydrolysis, gives rise to four different neutral glycosphingolipids which have the same TLC behaviour as
New ganglioside from mouse brain
533
GM3 GM2 GM1 FucGM1 GDla
GDlb 9-O- Ac -GT1 b GTlb GOlb GQlc
Fig. 1. Thin layer chromatographic behaviour of isolated and purified O-Ac-GQlb. Solvent system: chloroform: methanol: 0.2% aqueous CaCI2, 50:40 : 10, by volume. (A) mouse brain ganglioside mixture; (B) same as (A) after alkali treatment; (C) isolated O-Ac-GQIb; D: same as (C), after alkali treatment; (E) standard gangliosides,
GQlb GQIc
A B C D E Fig. 2. Thin layer chromatographic behaviour of alkali treated O-Ac-GQIb and standard G Q l b and GQlc. Solvent system as in Fig. 1. (A) standard gangliosides G Q I b and GQIc; (B) O-Ac-GQlb; (C) alkali treated GQIb; (D) alkali treated O-Ac-GQlb; (E) alkali treated G Q l b + GQIc.
\ \ ~ , \ , \ (. tll i ( * k \ l l
q~4
t'! ~1/
lablc t. (hcmlc'al composltioi~ ui ()-Ac-GQI b gangliosidc Sphi¢l¢lu.siitc ~md
(~omponent
"i,*
Molar ratio+
Sphingosinc Glucose Galactosc N-acetylgalactosammc Sialic acid++
11.70 7.05 12.1 I 7.71 46.83
1.00 1.05 1.94 0.93 3.88
Lipid moiety composition
t:atty acid
",,
Long chain base
°i,
14:0 16:0 16:1 18:0 18:1 20:0 20:1 22:0 24:0
2.1 3.8 5.0 24.0 6.1 4.5 2.3 39~5 12.4
18:0 18:1 20:0 20:1
12.0 46.1 8.4 33.5
* As dehydrated compound. + Sphingosine = I. * As NeuAc. + latter permethylated derivative of NeuAc is the only the corresponding products obtained from GM1 one observed when the product of exhaustive sialidase ganglioside, processed in parallel. The four neutral hydrolysis of O-Ac-GQlb was submitted to the same glycosphingolipids obtained have the following sugar treatment. It is noteworthy that the two derivatives of composition (expressed in molar basis): compound I (fastest moving), Glc; compound II, Glc and Gal (1.00 : 1.08); compound III, Glc :Gal : GalNAc 1 : DERIVATIVE OF Neu Ac (1.00:1.12:0.93); compound IV, Glc :Gal :GalNAc 2: DERIVATIVE OF 9.O.Ac Neu Ac (1.00:2.16:0,88). Thus the neutral gtycosphingolipid core of the analyzed ganglioside is: Gal G a l N A c Gal-4Slc-ceramide. Exhaustive treatment of O-AcG Q l b with sialidase gives rise to a compound having the same chemical and chromatographic characteristics as GM1 ganglioside. The results of the permethylation studies performed 1.00 on: (a) O-Ac-GQlb; (b) the ganglioside chromatographically behaving as GM1 obtained after exhaustive sialidase hydrolysis of O-Ac-GQlb; (c) the tetrahexosylceramide obtained by partial acid hydrolysis from the same O-Ac-GQlb, are summarized in Table 2. The data regarding the methylated derivatives of NeuAc and GalNAc were confirmed by 1 2 G L C - M S studies. The results of G L S - M S analyses of the derivative of N-acetylneuraminic acid present in O-Ac-GQlb ganglioside, in strict agreement with Haverkamp et al. (1977), are consistent with the pres- Fig. 3. Gas liquid chromatogram of the acetyl-neuraminic acids released from O-Ac-GQlb by mild acid hydrolysis. ence of 1, 2, 4, 7, 9-penta-O-methyl-8-O-trimethylsilyl- The retention times of the two compounds (1 and 2} relaN-acetyl, methylneuraminic acid and 1, 2,4, 7, 8, 9- tive to per-O-t rimethylsilyl-N-acetytneuraminic acid hexa-O-methyl-N-acetyl, methylneuraminic acid. This methyl ester are reported.
New ganglioside from mouse brain
535
i
...............
-u~'~O
B
-I'o,i
[0.
A.M+.mlnu s .CH3
..........
rF-. . . . . . . . . . . . . . . . . . . . .
~Tt-~ff¢-rMs'o.; rMs'o.
i
•
Hi
. . . . . . . . . . . . . . . . . . . . . .
TMS. SI (CX3)3
compound
R
1
TMS
1.00 668 624 478 ~
Ii t
A
B
C
D
E
F
G
H
2
Ac
1.14 638 594 478 298 317 175 173 400
317 L)05 173 400
Fig. 4. Formation of the characteristic fragment ions A-H of N-acetylneuraminic acid (compound 1) and 9-O-acetyl,N-acetylneuraminic acid (compund 2) derived from O-Ac-GQ 1b.
NeuAc were also obtained from G D I b and GTlb, processed in parallel, while only the second one could be recognized with G M I and G D l a treated in the same manner. The methylated derivative of GalNAc contained in O-Ac-GQIb, isolated by ion exchange chromatography and submitted to trimethylsilylation according to Yang and Hakomori (1971) gives by GLC one major peak. The spectrum of this peak is the same as that described in a previous work (Sonnino et al., 1978b) and as that obtained from GM1 when submitted to the same treatment; it is consistent with the following GalNAc derivative, 4,6-di-Omethyl-l,3-di-O-trimethylsilyl-2-methylacetamido-2deoxygalactose. The recovery of glucose, galactose,
and N-acetylgalactosamine after oxidation of O-AcG Q I b with chromium trioxide is extremely low (from 0 to 10~). O-Ac-GQlb, when submitted to alkali treatment, changes its chromatographic behaviour and, comigrating with pure standard G Q l b and G Q l c (see Fig. 2), behaves exactly as G Q l b ganglioside. The alkali treated O-Ac-GQlb contains Glc, Gal, GalNAc and NeuAc in the molar ratio of 1 : 2 : 1:4, the same composition and molar ratio as the untreated material. The position of 9-O-acetyl,N-acetylneuraminic acid in the O-Ac-GQlb molecule was established by gradual sialidase hydrolysis (see Fig. 5). The first products of the enzyme hydrolysis are N-acetylneuraminic acid
Table 2. Different sugar derivatives obtained from O-Ac-GQIb ganglioside and from the products of its enzymatic hydrolysis, GMI, and mild acid hydrolysis, tetrahexosylceramide
(THC). Sugar derivative 1,4,5-Tri-O-acetyl-2,3,6-tri-O-methylglucitol* 1,4,5-Tri-O-acetyl-2,3,6-tri-O-methylgalactitol* 1,3,4,5-Tetra-O-acetyl-2,6-di-O-methylgalactitol* 1,5-Di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol* 1,3,5-Tri-O-acetyl-2,4,6-tri-O-methylgalactitol* 4,6-Di-O-methyl- 1,3-di-O-trimethylsilyl-2-methylacetamido2-deoxygalactose 1,2,4,7,8,9-Hexa-O-methyl-N-acetyl, methylneuraminic acid~" 1,2,4,7,9-Penta-O-methyl-8-O-trimethylsilyl-N-acetyl, methylneuraminic acidt
Molar ratio O-Ac-GQlb GM1 THC 1.00 no 1.06 no 0.89 +
1.00 no 0.93 1.07 no +
1.00 0.89 no 1.03 no +
+
+
no
+
no
no
* Referred to glucose derivative taken as 1.00. t In O-Ac-GQIb the molar ratio of 1,2,4,7,8,9-Hexa-O-methyl-N-acetyl, methylneuraminic acid to 1,2,4,7,9-penta-O-methyl-8-O-trimethylsilyl-N-acetylneuraminic acid is 1.06/1.00.
\ ~,g", \ ('tlit,i)l,~"-,iJ V/ d /
5~('~
GM3 GM2 GM] Fuo-GMI GDIa GDIb O-Ac_GTIb CTlb GQIb
Fig. 5. Gradual hydrolysis of O-Ac-GQI b by Vibno cholerae sialidase. After incubation the mixtures were dialyzed (1 day) at 4°C against twice distilled water in order to remove liberated sialic acids. Solvent system as in Figure 1. A: standard gangliosides; B: O-Ac-GQIb: C: products of sialidase treatment of O-Ac-GQIb; D: standard O-Ac-GTIb: E: same as C. after alkali treatment. and a ganglioside behaving chromatographically as O-Ac-GTlb. Prolongation of the sialidase treatment leads to release of another residue of N-acetylneuraminic acid and a ganglioside which, after alkali treatment, behaves as G D l b . This compound releases. upon exhaustive sialidase hydrolysis, 9-O-acetyl,Nacetylneuraminic acid (which after alkali treatment behaves as NeuAc) and an alkali stable, sialidase resistant ganglioside which contains NeuAc and has been recognized to be (see above) G M I ganglioside. The products of gradual hydrolysis of O-Ac-GQlb by sialidase are schematically reported in Fig. 6. O-Ac-GQlb and G Q l b were submitted to Vibrio cholerae sialidase treatment (at 37~C) under identical conditions and the time course of sialic acid release followed (Fig. 7). The process is much slower in O-AcG Q l b than in G Q l b . The release of the first residue of sialic acid (which is NeuAc in either gangliosidel takes place in 60rain from O-Ac-GQIb and only 10min from G Q l b ; the release of all the residues of sialic acid takes place in 3 3½h from G Q I b and in 17 18h from O-Ac-GQIb.
already isolated and its structure elucidated (Ghidoni et al., 1980). A second alkali labile ganglioside of mouse brain was isolated in pure form and structurally characterized. It is a tetrasialoganglioside conraining sphingosine, glucose, galactose, N-acetylgalactosamine and sialic acid in the molar ratio 1:1:2:1:4. On exhaustive treatment with Vibrio cholerae sialidase it gives rise to a compound having the same characteristics as GM1 ganglioside. Partial acid hydrolysis provided the following saccharide sequence of its neutral glycosphingolipid core:Gal-GalNAcGal-Glc-ceramide. Methylation studies performed on this new ganglioside, coded O-Ac-GQIb, enabled us to draw the following conclusions about its structure: one galactose residue is substituted in positions 3 and 4. the second galactose residue in position 3, glucose
O-Ac-GQIb
1
--"~ NeuAc
O-Ac-GTIb I "--" NeuAc
DISCUSSION The occurrence in mouse brain of gangliosides characterized by the presence of an alkali labile linkage was reported in previous work (Sonnino et al. 1978a). One of these gangliosides, O - A c - G T I b , was
0 - A e - GDIb ~ ....-..-~ 9 - O - A c - NeuAc GMt
Fig. 6. Action of I/ihrio cho/erae sialidase on O-Ac-GQI b.
New ganglioside from mouse brain
I
537
&-.-& GQlb A--A 9--O-At GOlb 9,
2
4 6 INCUBATION TIME AT 3"~C
18
hours
Fig. 7. Time course of sialic acid release by Vibrio cholerae sialidase from GQlb and O-Ac-GQlb. Ganglioside concentration for the assay: 0.2 raM.
in position 4, N-acetylgalactosamine in position 3, two residues of sialic acid are substituted in position 8 and the other two, clearly in terminal position, are not substituted. Both galactose residues are substituted in position 3 with ~t-glycosidically linked sialic acid. The anomerity of the other glycosidic linkages present in O-Ac-GQIb was determined by the chromium trioxide procedure. Glucose, galactose and N-acetyigalactosamine, after treatment with chromium trioxide have an extremely low recovery, this indicating the presence of fl-linkages only. Thus O-AcGQ I b contains ganglio-N-tetraose. O-Ac-GQlb ganglioside when submitted to alkali treatment changes its chromatographic behaviour and behaves exactly as G Q l b ganglioside. The alkali treated O-Ac-GQlb contains glucose, galactose, N-acetylgalactosamine and sialic acid in the molar ratio 1:2:1:4, the same composition and molar ratio exhibited by the untreated material. The peculiar behaviour of O-Ac-GQlb under alkaline conditions drew our attention to the nature of the sialic acid contained therein. The GLC-MS studies performed on the sialic acids released from O-Ac-GQlb by mild acid hydrolysis or by sialidase treatment led unequivocally t o the conclusion that, besides N-acetylneuraminic acid, 9-O-acetyl,N-acetylneuraminic acid is present in O-Ac-GQlb. The position of 9-O-acetyl,Nacetylneuraminic acid in the molecule of O-Ac-GQ 1b
was established by gradual sialidase hydrolysis. The first products of the enzyme hydrolysis of O-Ac-GQlb were N-acetylneuraminic acid and O-Ac-GTlb. Alkali treatment of this ganglioside yields, as expected, GTlb. Further hydrolysis by sialidase releases an additional residue of N-acetylneuraminic acid and a ganglioside containing a disialosyl residue and still alkali labile. This disialoganglioside, treated with alkali, gives GDlb. Upon exhaustive sialidase hydrolysis the same ganglioside releases 9-O-acetyl,Nacetylneuraminic acid and GM1 ganglioside which contains N-acetylneuraminic acid. Therefore, of the four sialic acid residues present in O-Ac-GQlb three are N-acetylneuraminic acid (two of them are linked to positions 3 of both internal and external galactose; one to position 8 of the sialic acid residue linked to external galactose), and one is 9-O-acetyl,N-acetylneuraminic acid, ct, 2-8 linked to the NeuAc residue bound to the internal galactose. It is worth commenting that the release of sialic acid residues by the action of sialidase is much slower from O-Ac-GQlb than from GQlb. Particularly not only the rate of release of 9-O-Ac-NeuAc is reduced (which is expected, Schauer and Faillard, 1968) but also that of NeuAc. This indicates that the chemical difference between O-Ac-GQlb and G Q l b (that iS the 9-O-acetyl group on sialic acid) is recognized by Sialidase and changes its catalytic properties.
538
V~,N".A (. H,tiORNOel a/
In conclusion we suggest the following structure for O-Ac-GQ lb ganglioside : Galfll- 3GalNAcfll-4Gal/~?l-4GIcfll- 1'--ceramide 3
3
b
r
NeuAc~2
NeuAc72
8
8
I
NeuAcct2
P
9-O-Ac-NeuAcct2
According to the I U P A C IUB Commission on Biochemical Nomenclature (1977) it is designated as I I a ( 9 - O - A c - N e u A c c t 2 8NeuAc) IV 3 (NeuAcct2 .... 8NeuAc)GgOse4Cer. O - A c - G Q I b is the second individual ganglioside isolated from brain tissue which contains a 9-O-acetyl,N-acetylneuraminic acid. It adds to the hematoside (GM3) containing 4-O-acetyl,N-glycolylneuraminic acid, isolated from equine erythrocytes by Hakomori and Saito (1969). It is worth commenting that the fatty acid and long chain base composition of O-Ac-GT 1b and the here described O - A c - G Q l b is different, this making unlikely a metabolic relationship between them. Preliminary experiments, which are being developed, showed that O-acetylated gangliosides, including O - A c - G T l b and O - A c - G Q l b are present in the brain of many animals and constitute in some species a considerable portion of the total ganglioside content. Thus their recognition should be one of the goals in studies aimed at establishing brain ganglioside patterns at the phylogenetic, ontogenetic, tissue, cellular and subcellular level. The occurrence in brain as well as in other tissues of this type of ganglioside poses two important questions: (a) whether the O-acetylation of sialic acid occurs prior or not to the incorporation of sialic acid into the ganglioside molecule; (b) that would be the physiological meaning of sialic acid O-acetylation since, as we observed and reported in more detail elsewhere (Sonnino et al., 1982), O-acetylation of sialic acid appears to make sialosyl residues less sensitive to sialidase action. The availability of individual gangliosides of this type may facilitate and encourage studies aimed to approach the above problems. Acknowledgements--This work was supported by grants from the Consiglio Nazionale delle Ricerche (C.N.R.). Rome, Italy. The advice and help given by Prof. R. Schauer in the elucidation of the chemical nature of 9-O-acetyl,Nacetylneuraminic acid isolated from O-Ac-GQIb ganglioside is gratefully acknowledged.
REFERENCES Ando, S. and Yu, R. K. (1979). Isolation and characterization of two isomers of brain tetrasialogangliosides. J. biol. Chem., 254, 12,224 12,229. Buscher, H. P., Casals-Stenzel, J. and Schauer, R. (1974~. Identification of N-glycoloyl-O-acetylneuraminic acids and N-acetyl-O-glicoloylneuraminic acids by improved methods for detection of N-acyl and O-acyl groups and by gas-liquid chromatography. Eur. J. Biochem. 5{}, 71--82. Ghidoni, R., Sonnino, S., Tettamanti, G., Wiegandt, H. and Zambotti, V. (1976). On the structure of two new gangliosides from beef brain. J. Neurochem. 27, 511. 515. Ghidoni, R., Sonnino, S., Tettamanti, G., Baumann, N., Reuter, G. and Schauer R. (1980). Isolation and characterization of a trisialoganglioside from mouse brain, containing 9-O-acetyl-N-acetylneuraminic acid. J~ biol. Chem. 255, 6990-6995. Hakomori, S. and Saito, T. (1969). Isolation and characterization of a glycosphingolipid having a new siatic acid. Biochemistry 8, 5082-5088. Haverkamp, J., Kamerling, J. P., Vliegenthart, J. F. G.; Veh R. W. and Schauer R. (1977). Methylation analysis determination of acylneuraminic acid residue type 2-8 glycosidic linkages. Application to GTlb ganglioside and colominic acid. FEBS Lett. 73, 215-219. lwamori, M. and Nagai, Y. (1978). A new chromatographic approach to the resolution of individual gangliosides. Ganglioside mapping. Biochim. biophys. Acta 528, 257-267. IUPAC-IUB Commission on Biochemical Nomenclature (1977). The nomenclature of lipids. Lipids 12, 455-468. Kamerling J. P., Vliegenthart, J. F. G. and Vink, J. I1974). Mass spectrometry of pertrimethylsilyl neuraminic acid derivatives. Carbohyd. Res. 33, 297 406. Kamerling, J. P., Vliegenthart, J. F. G., Versluis, C. and Sehauer, R. (1975). Identification of O-acetylated N-awlneuraminic acids by mass spectrometry. Carbohyd. Res. 41, 7-17. Kamerling, J. P., Vliegenthart, J. F. G., Versluis; C. and Sehauer, R. (1978). In: Recent Developments in Ma.ss Spectrometry in Biochemistry and Medicine (Frigerio A., ed.) Vol. l, pp. 503-520, Plenum Press, New York. Schauer, R., and Faillard, H. (1968). Zur wirkungssPezifitat Neuraminidase. Das Verhalten isomerer N,O-Diacetylneuramins~iureglycoside im Submaxillarismucin yon Pferd und Rind bei Einwirkung bakterieller neuraminidase. Hoppe Seyler's Z. physiol. Chem. 349, 961-968.
New ganglioside from mouse brain Sonnino, S., Ghidoni, R., Baumann, N., Harpin, M. L. and Tettamanti, G. (1978a). Occurrence in mouse brain of a new ganglioside carrying an alkali labile linkage. Bull. Mol. Biol. and Med. 3, 170-178. Sonnino, S., Ghidoni, R., Chigorno, V. and Tettamanti, G. (1982). Chemistry of gangliosides carrying O-acetylated sialic acid. In: New Vista in Glycolipid Research (Makita A., Handa S., Taketomi T. and Nagai, Y. eds.) Plenum Publishing Corporation. In press. Sonnino, S., Ghidoni, R., Galli, G. and Tettamanti, G. (1978b). On the structure of a new fucose containing
539
ganglioside from pig cerebellum. J. Neurochem. 31, 947-956. Svennerholm, L. (1964). The gangliosides. J. Lipid Res. 5, 145-155. Tettamanti, G., Bonali, F., Marchesini, S. and Zambotti, V. (1973). A new procedure for the extraction, purification and fractionation of brain gangliosides. Biochim. biophys. Acta 296, 160-170. Yang, H. J. and Hakomori, S. (1971). A sphingolipid having a novel type of ceramide and lacto-N-fucopentaose III. J. biol. Chem. 246, 1192-1200.
0197-0186/82/060531-09503.00/0 © 1982 Pergamon Press Ltd.
ISOLATION A N D CHARACTERIZATION OF A
TETRASIALOGANGLIOSIDE FROM MOUSE BRAIN, CONTAINING 9-O-ACETYL,N-ACETYLNEURAMINIC ACID VANNA CHIGORNO, SANDRO SONNINO, RICCARDO GHIDONI and G u m o TETTAMANTI Department of Biological Chemistry, The Medical School, University of Milan. Milan, Italy (Received 19 April 1982; accepted 28 May 1982) A~tract--A new ganglioside, containing an alkali-labile linkage, was extracted from mouse brain and purified. It represents 3.6% of total lipid-bound sialic acid in the tissue and was obtained in pure form with a yield of about 35%. It contains sphingosine, glucose, galactose, N-acetylgalactosamine and sialic acid in the molar ratio 1 : 1 : 2 : 1 : 4 and, upon exhaustive sialidase treatment gives the monosialoganglioside GM1. Partial acid hydrolysis, methylation analysis, gas-liquid chromatography-mass spectrometry and chromium trioxide oxidation studies showed its basic neutral glycosphingolipid core to be ganglio-N-tetraose-ceramide. Three of the four sialic acid residues are N-acetylneuraminic acid and one, as shown by gas-liquid chromatography-mass spectrometry, is 9-O-acetyI,N-acetylneuraminic acid, which contains the alkali labile linkage. 9-O-acetyl,Nacetylneuraminic acid is ~-ketosidically linked to position 8 of the N-acetylneuraminic acid residue bound to position 3 of the internal galactose. The other two N-acetylneuraminic acid residues form a disialosyl residue linked to position 3 of external galactose. The complete structure of the studied ganglioside is as follows: NeuAc~t2-8NeuAc~t2-3Galfll-3GalNAcfll-4(9-O-Ac-NeuAc~2-8NeuAcct2-3) Galfll-4 Glcfll- l'-N-acylsphingosine, and it can be considered as a derivative of the tetrasialoganglioside GQlb.
In the course of a systematic study on mouse brain gangliosides we observed that some minor components changed their chromatographic behaviour after exposure of the ganglioside mixture to alkaline conditions (Sonnino et al., 1978a). Since all the gangliosides known at that time were resistant to such treatment we concluded that the mixture contained some gangliosides with alkali labile groups. This chemical feature was an indication of a novel type of gangliosides or of natural ganglioside derivatives. One of the alkali labile gangliosides occurring in mouse brain, recently identified (Ghidoni et al., 1980) resulted to be a trisialoganglioside (GTlb) bearing 9-O-acetyi,N-acetylneuraminic acid, and was coded O-Ac-GT 1b. The present paper describes the isolation and structural characterization of a second alkali labile ganglioside of mouse brain. It is a tetrasialoganglioside,
namely G Q I b , containing 9-O-acetyl,N-acetylneuraminic acid. The 9-O-acetyl group represents the alkali labile group of this ganglioside. This compound is provisionally coded O-Ac-GQlb.* MATERIALS AND METHODS Materials Commercial chemicals were of analytical or of the highest available grade. Solvents were redistiUed before use. Water for routine use was freshly redistilled in a glass apparatus. Silica gel for column chromatography (Kieselgel 100, 0.063-0.2 mm; 70-230 mesh, ASTM, and high performance thin layer chromatography (HPTLC) plates were purchased from Merck GmbH (Darmstadt, F.R.G.); Sephadex G 25, fine, from Pharmacia (Uppsala, Sweden); reagents for gas chromatography (GLC) from C. Erba (Milan, Italy); N-acetylneuraminic acid from Sigma Chem. Co. (St. Louis, MO, U.S.A.); glucose, galactose, N-acetylgalactosamine and m-inositol from Fluka (Buchs, Switzerland); Vibrio Cholerae sialidase (EC 3.2.1.18) from Behfingwerke (Marburg, F.R.G.); 9-O-acetyl,N-acetylneuraminic acid was isolated from bovine submandibular glands and crystallized according to Buscber et al. (1974). Whole brains (total fresh tissue: 500g), removed from adult mice (C17BI/6J strain) immediately after slaughtering, were washed in cold saline isotonic solution, frozen and finally lyophilized. This material was stored at - 20°C.
Address correspondence to: Prof. Guido Tettamanti, Istituto di chimica Biologica, Via Saldini, 50 20133, Milano, Italy. * Abbreviation used: gangliosides are coded according to Svennerholm (1964). TLC, thin layer chromatography; GLC, gas liquid chromatography; MS, mass spectrometry. 531
q32
\ *~\",'~ ( I H l a ) R N ~ ) cl it/.
M e{ hod.~ l~olution and puri/ication of O-4c-GQlh. The extractitm and puritication of the total gangliosides fiom the lyophilized brains was performed according to the procedure of Fettamanti eta/. t19731. Ganglioside O-Ac-GQ lb was isolated from the total ganglioside extract b 3 column chromatography on Kieselgel 100. The experimental conditions v,ere the following: 4 × 2(X)cm column: temperature. Ig 20C: solvent, chloroform: methanol: 0.3",, aqueous CaCI 2 60:35:6 by volume: llow rate. 6 0 m l h ~:total w)l. for complete elution. 4.5 5.01: 20ml fi-actions automatically collected. The elution pattern was monitored by thin layer chromatography (TLC) on HPTLC plates using the solvent system chloroform:methanol:0.2'!i, aqueous CaCI 2, 50:40: 10, by volume. The O-Ac-GQIb containing fractions obtained after the first chromatography werc collected and rechromatographed until the isolated product was homogeneous. The material thus obtained was purified by column chromatography on Sephadex G 25 according to Ghidoni ct al. (1976). in order to remove salts, small molecular weight impurities and silica gel lines. The eluted fractions containing the product wrere combined and evaporated to dryness in t'acuo at 35 (7. The residue was further purified by acetone precipitation {Sonnino et ul., 1978b). The final white powder (about 12mg of O-Ac-GQlb) was stored al - 20 C. The purity of the isolated ganglioside was assessed by chemical analyses (see belowl and its homogeneity by TLC on HPTLC plates under the conditions specified below. The gangliosides GM3, GM2, GMI, Fuc-GMI, GDIa, GDIb. GTIb, G Q I b and O-Ac-GTIb were prepared and structurally identified as previously described (Tettamanti et al.. 1973: Ghidoni et al.. 1976: Sonnino et aI., 1978: Ghidoni el a/., 1980). Ganglioside G Q l c was isolated and purified from codfish brain as reported by Ando and Yu (1979). Chemical composition and structural .~tudics (71 O-,4cGQIh. The following analyses were performed: (a) determination of carbohydrate and lipid composition: (b) partial hydrolysis of ganglioside followed by isolation and chemical characterization of the neutral glycosphingolipids; It) cleavage by sialidase or mild acid treatment, isolation and chemical characterization of acylneuraminic acids from ganglioside: (d) isolation and chemical characterization of the gangliosides obtained by partial sialidase treatment of the original O-Ac-GQIb: {el alkaline treatment of O-AcG Q l b and of the gangliosides obtained after its hydrolysis by sialidase; (f} methanolysis of ganglioside and of glycosphingolipids obtained by its partial hydrolysis: (g)anomeric configuration analysis. The experimental details on the above analytical methods have been given in a previous paper (Ghidoni et al.. 1980). The isolation of the individual gangliosides obtained by partial sialidase hydrolysis of O-Ac-GQIb was attained using the procedure described by lwamori and Nagai (197gi.
RESULTS The ffactionation of the total ganglioside mixture obtained from mouse brain led to isolation of a compound with a c h r o m a t o g r a p h i c behaviour intermediate between O - A c - G T I b and G T I b (see Fig. 1).
This behaviour ~sas different trom that ol the gallg[iosides known so far. Since after alkaline trc~ttmcnt it behaved as G Q I b (see Fig. 2.) it was coded O-Ac:G Q I b . in terms of bound NeuAc gangliosidc O-AcG Q I b represents 3.6"o of the total gangliosidc bound NeuAc. The compound, after isolation and purification from the ganglioside mixture 112 mg trom 500 g of fresh bruin, with a 35",,yicldi is chromatographically homogeneous in different solvent s~stcms
Chemical composition ~# O-A c-GO lh The results of G L C and chemical analyses reveal that O - A c - G Q l b contains sphingosine, glucose, galactose, N-acetylgalactosamine and sialic acid in a molar ratio of 1.00:1.05 : 1.94:0.93:3.88 {theoretical t : 1 : 2 : 1 : 4 ) (see Table 1). The sialic acid value represents, in molar terms, the sum of N'euAc and 9-0acetylNeuAc. The nature of the sialic acids was established after release of these residues by mild acid hydrolysis. A pure sialic acid fraction of about 100/2g was obtained from 2.0mg of O - A c - G Q I b after this treatment. It was composed of N-acetylneuraminic acid and of 9-O-acetyl,N-acetylneuraminic acid in a molar ratio of about 4:1. The latter c o m p o u n d comigrates with authentic 9-O-acetyl,N-acetylneuraminic acid on both cellulose and silica gel thin layer plates. When analysed by G L C two peaks are detected (see Fig. 3.) with retention times of 1.00 and 1.14 relative to per-O-trimethylsilyI-N-acetyl neuraminic acid methyl ester. This chromatographic behaviour indicates the presence of two c o m p o u n d s in the ganglioside, N-acetylneuraminic acid (compound 1) and 9-O-acetyI,Nacetylneuraminic acid (compound 2). These results were supported by G L C MS. In fact the n u m b e r and mass units of the characteristic fragment ions obtained from c o m p o u n d s 1 and 2 (see Fig. 4.) are identical with the corresponding ones obtained in our previous investigation on O - A c - G T I b ganglioside (Ghidoni et al., 1980), and are consistent with those expected respectively for N-acetylneuraminic acid and 9-O-acetyl,N-acetylneuraminic acid (Kamerling et al., 1975: Kamerling et al., 1974; Kamerling et al., 1978). The fatty acid and long chain base composition of O - A c - G Q I b is shown in Table 1. The major fatty acid components are C 1 8 : 0 and C 2 2 : 0 (63.5",, of total), The long chain bases are predominantly C 18 : 1 and C 20 : 1 (801),i of total); the remainder is constituted by the corresponding saturated derivatives. Structure ~g O - A c-GQ lb ganglioside O-Ac-GQIb, when submitted to partial acid hydrolysis, gives rise to four different neutral glycosphingolipids which have the same TLC behaviour as
New ganglioside from mouse brain
533
GM3 GM2 GM1 FucGM1 GDla
GDlb 9-O- Ac -GT1 b GTlb GOlb GQlc
Fig. 1. Thin layer chromatographic behaviour of isolated and purified O-Ac-GQlb. Solvent system: chloroform: methanol: 0.2% aqueous CaCI2, 50:40 : 10, by volume. (A) mouse brain ganglioside mixture; (B) same as (A) after alkali treatment; (C) isolated O-Ac-GQIb; D: same as (C), after alkali treatment; (E) standard gangliosides,
GQlb GQIc
A B C D E Fig. 2. Thin layer chromatographic behaviour of alkali treated O-Ac-GQIb and standard G Q l b and GQlc. Solvent system as in Fig. 1. (A) standard gangliosides G Q I b and GQIc; (B) O-Ac-GQlb; (C) alkali treated GQIb; (D) alkali treated O-Ac-GQlb; (E) alkali treated G Q l b + GQIc.
\ \ ~ , \ , \ (. tll i ( * k \ l l
q~4
t'! ~1/
lablc t. (hcmlc'al composltioi~ ui ()-Ac-GQI b gangliosidc Sphi¢l¢lu.siitc ~md
(~omponent
"i,*
Molar ratio+
Sphingosinc Glucose Galactosc N-acetylgalactosammc Sialic acid++
11.70 7.05 12.1 I 7.71 46.83
1.00 1.05 1.94 0.93 3.88
Lipid moiety composition
t:atty acid
",,
Long chain base
°i,
14:0 16:0 16:1 18:0 18:1 20:0 20:1 22:0 24:0
2.1 3.8 5.0 24.0 6.1 4.5 2.3 39~5 12.4
18:0 18:1 20:0 20:1
12.0 46.1 8.4 33.5
* As dehydrated compound. + Sphingosine = I. * As NeuAc. + latter permethylated derivative of NeuAc is the only the corresponding products obtained from GM1 one observed when the product of exhaustive sialidase ganglioside, processed in parallel. The four neutral hydrolysis of O-Ac-GQlb was submitted to the same glycosphingolipids obtained have the following sugar treatment. It is noteworthy that the two derivatives of composition (expressed in molar basis): compound I (fastest moving), Glc; compound II, Glc and Gal (1.00 : 1.08); compound III, Glc :Gal : GalNAc 1 : DERIVATIVE OF Neu Ac (1.00:1.12:0.93); compound IV, Glc :Gal :GalNAc 2: DERIVATIVE OF 9.O.Ac Neu Ac (1.00:2.16:0,88). Thus the neutral gtycosphingolipid core of the analyzed ganglioside is: Gal G a l N A c Gal-4Slc-ceramide. Exhaustive treatment of O-AcG Q l b with sialidase gives rise to a compound having the same chemical and chromatographic characteristics as GM1 ganglioside. The results of the permethylation studies performed 1.00 on: (a) O-Ac-GQlb; (b) the ganglioside chromatographically behaving as GM1 obtained after exhaustive sialidase hydrolysis of O-Ac-GQlb; (c) the tetrahexosylceramide obtained by partial acid hydrolysis from the same O-Ac-GQlb, are summarized in Table 2. The data regarding the methylated derivatives of NeuAc and GalNAc were confirmed by 1 2 G L C - M S studies. The results of G L S - M S analyses of the derivative of N-acetylneuraminic acid present in O-Ac-GQlb ganglioside, in strict agreement with Haverkamp et al. (1977), are consistent with the pres- Fig. 3. Gas liquid chromatogram of the acetyl-neuraminic acids released from O-Ac-GQlb by mild acid hydrolysis. ence of 1, 2, 4, 7, 9-penta-O-methyl-8-O-trimethylsilyl- The retention times of the two compounds (1 and 2} relaN-acetyl, methylneuraminic acid and 1, 2,4, 7, 8, 9- tive to per-O-t rimethylsilyl-N-acetytneuraminic acid hexa-O-methyl-N-acetyl, methylneuraminic acid. This methyl ester are reported.
New ganglioside from mouse brain
535
i
...............
-u~'~O
B
-I'o,i
[0.
A.M+.mlnu s .CH3
..........
rF-. . . . . . . . . . . . . . . . . . . . .
~Tt-~ff¢-rMs'o.; rMs'o.
i
•
Hi
. . . . . . . . . . . . . . . . . . . . . .
TMS. SI (CX3)3
compound
R
1
TMS
1.00 668 624 478 ~
Ii t
A
B
C
D
E
F
G
H
2
Ac
1.14 638 594 478 298 317 175 173 400
317 L)05 173 400
Fig. 4. Formation of the characteristic fragment ions A-H of N-acetylneuraminic acid (compound 1) and 9-O-acetyl,N-acetylneuraminic acid (compund 2) derived from O-Ac-GQ 1b.
NeuAc were also obtained from G D I b and GTlb, processed in parallel, while only the second one could be recognized with G M I and G D l a treated in the same manner. The methylated derivative of GalNAc contained in O-Ac-GQIb, isolated by ion exchange chromatography and submitted to trimethylsilylation according to Yang and Hakomori (1971) gives by GLC one major peak. The spectrum of this peak is the same as that described in a previous work (Sonnino et al., 1978b) and as that obtained from GM1 when submitted to the same treatment; it is consistent with the following GalNAc derivative, 4,6-di-Omethyl-l,3-di-O-trimethylsilyl-2-methylacetamido-2deoxygalactose. The recovery of glucose, galactose,
and N-acetylgalactosamine after oxidation of O-AcG Q I b with chromium trioxide is extremely low (from 0 to 10~). O-Ac-GQlb, when submitted to alkali treatment, changes its chromatographic behaviour and, comigrating with pure standard G Q l b and G Q l c (see Fig. 2), behaves exactly as G Q l b ganglioside. The alkali treated O-Ac-GQlb contains Glc, Gal, GalNAc and NeuAc in the molar ratio of 1 : 2 : 1:4, the same composition and molar ratio as the untreated material. The position of 9-O-acetyl,N-acetylneuraminic acid in the O-Ac-GQlb molecule was established by gradual sialidase hydrolysis (see Fig. 5). The first products of the enzyme hydrolysis are N-acetylneuraminic acid
Table 2. Different sugar derivatives obtained from O-Ac-GQIb ganglioside and from the products of its enzymatic hydrolysis, GMI, and mild acid hydrolysis, tetrahexosylceramide
(THC). Sugar derivative 1,4,5-Tri-O-acetyl-2,3,6-tri-O-methylglucitol* 1,4,5-Tri-O-acetyl-2,3,6-tri-O-methylgalactitol* 1,3,4,5-Tetra-O-acetyl-2,6-di-O-methylgalactitol* 1,5-Di-O-acetyl-2,3,4,6-tetra-O-methylgalactitol* 1,3,5-Tri-O-acetyl-2,4,6-tri-O-methylgalactitol* 4,6-Di-O-methyl- 1,3-di-O-trimethylsilyl-2-methylacetamido2-deoxygalactose 1,2,4,7,8,9-Hexa-O-methyl-N-acetyl, methylneuraminic acid~" 1,2,4,7,9-Penta-O-methyl-8-O-trimethylsilyl-N-acetyl, methylneuraminic acidt
Molar ratio O-Ac-GQlb GM1 THC 1.00 no 1.06 no 0.89 +
1.00 no 0.93 1.07 no +
1.00 0.89 no 1.03 no +
+
+
no
+
no
no
* Referred to glucose derivative taken as 1.00. t In O-Ac-GQIb the molar ratio of 1,2,4,7,8,9-Hexa-O-methyl-N-acetyl, methylneuraminic acid to 1,2,4,7,9-penta-O-methyl-8-O-trimethylsilyl-N-acetylneuraminic acid is 1.06/1.00.
\ ~,g", \ ('tlit,i)l,~"-,iJ V/ d /
5~('~
GM3 GM2 GM] Fuo-GMI GDIa GDIb O-Ac_GTIb CTlb GQIb
Fig. 5. Gradual hydrolysis of O-Ac-GQI b by Vibno cholerae sialidase. After incubation the mixtures were dialyzed (1 day) at 4°C against twice distilled water in order to remove liberated sialic acids. Solvent system as in Figure 1. A: standard gangliosides; B: O-Ac-GQIb: C: products of sialidase treatment of O-Ac-GQIb; D: standard O-Ac-GTIb: E: same as C. after alkali treatment. and a ganglioside behaving chromatographically as O-Ac-GTlb. Prolongation of the sialidase treatment leads to release of another residue of N-acetylneuraminic acid and a ganglioside which, after alkali treatment, behaves as G D l b . This compound releases. upon exhaustive sialidase hydrolysis, 9-O-acetyl,Nacetylneuraminic acid (which after alkali treatment behaves as NeuAc) and an alkali stable, sialidase resistant ganglioside which contains NeuAc and has been recognized to be (see above) G M I ganglioside. The products of gradual hydrolysis of O-Ac-GQlb by sialidase are schematically reported in Fig. 6. O-Ac-GQlb and G Q l b were submitted to Vibrio cholerae sialidase treatment (at 37~C) under identical conditions and the time course of sialic acid release followed (Fig. 7). The process is much slower in O-AcG Q l b than in G Q l b . The release of the first residue of sialic acid (which is NeuAc in either gangliosidel takes place in 60rain from O-Ac-GQIb and only 10min from G Q l b ; the release of all the residues of sialic acid takes place in 3 3½h from G Q I b and in 17 18h from O-Ac-GQIb.
already isolated and its structure elucidated (Ghidoni et al., 1980). A second alkali labile ganglioside of mouse brain was isolated in pure form and structurally characterized. It is a tetrasialoganglioside conraining sphingosine, glucose, galactose, N-acetylgalactosamine and sialic acid in the molar ratio 1:1:2:1:4. On exhaustive treatment with Vibrio cholerae sialidase it gives rise to a compound having the same characteristics as GM1 ganglioside. Partial acid hydrolysis provided the following saccharide sequence of its neutral glycosphingolipid core:Gal-GalNAcGal-Glc-ceramide. Methylation studies performed on this new ganglioside, coded O-Ac-GQIb, enabled us to draw the following conclusions about its structure: one galactose residue is substituted in positions 3 and 4. the second galactose residue in position 3, glucose
O-Ac-GQIb
1
--"~ NeuAc
O-Ac-GTIb I "--" NeuAc
DISCUSSION The occurrence in mouse brain of gangliosides characterized by the presence of an alkali labile linkage was reported in previous work (Sonnino et al. 1978a). One of these gangliosides, O - A c - G T I b , was
0 - A e - GDIb ~ ....-..-~ 9 - O - A c - NeuAc GMt
Fig. 6. Action of I/ihrio cho/erae sialidase on O-Ac-GQI b.
New ganglioside from mouse brain
I
537
&-.-& GQlb A--A 9--O-At GOlb 9,
2
4 6 INCUBATION TIME AT 3"~C
18
hours
Fig. 7. Time course of sialic acid release by Vibrio cholerae sialidase from GQlb and O-Ac-GQlb. Ganglioside concentration for the assay: 0.2 raM.
in position 4, N-acetylgalactosamine in position 3, two residues of sialic acid are substituted in position 8 and the other two, clearly in terminal position, are not substituted. Both galactose residues are substituted in position 3 with ~t-glycosidically linked sialic acid. The anomerity of the other glycosidic linkages present in O-Ac-GQIb was determined by the chromium trioxide procedure. Glucose, galactose and N-acetyigalactosamine, after treatment with chromium trioxide have an extremely low recovery, this indicating the presence of fl-linkages only. Thus O-AcGQ I b contains ganglio-N-tetraose. O-Ac-GQlb ganglioside when submitted to alkali treatment changes its chromatographic behaviour and behaves exactly as G Q l b ganglioside. The alkali treated O-Ac-GQlb contains glucose, galactose, N-acetylgalactosamine and sialic acid in the molar ratio 1:2:1:4, the same composition and molar ratio exhibited by the untreated material. The peculiar behaviour of O-Ac-GQlb under alkaline conditions drew our attention to the nature of the sialic acid contained therein. The GLC-MS studies performed on the sialic acids released from O-Ac-GQlb by mild acid hydrolysis or by sialidase treatment led unequivocally t o the conclusion that, besides N-acetylneuraminic acid, 9-O-acetyl,N-acetylneuraminic acid is present in O-Ac-GQlb. The position of 9-O-acetyl,Nacetylneuraminic acid in the molecule of O-Ac-GQ 1b
was established by gradual sialidase hydrolysis. The first products of the enzyme hydrolysis of O-Ac-GQlb were N-acetylneuraminic acid and O-Ac-GTlb. Alkali treatment of this ganglioside yields, as expected, GTlb. Further hydrolysis by sialidase releases an additional residue of N-acetylneuraminic acid and a ganglioside containing a disialosyl residue and still alkali labile. This disialoganglioside, treated with alkali, gives GDlb. Upon exhaustive sialidase hydrolysis the same ganglioside releases 9-O-acetyl,Nacetylneuraminic acid and GM1 ganglioside which contains N-acetylneuraminic acid. Therefore, of the four sialic acid residues present in O-Ac-GQlb three are N-acetylneuraminic acid (two of them are linked to positions 3 of both internal and external galactose; one to position 8 of the sialic acid residue linked to external galactose), and one is 9-O-acetyl,N-acetylneuraminic acid, ct, 2-8 linked to the NeuAc residue bound to the internal galactose. It is worth commenting that the release of sialic acid residues by the action of sialidase is much slower from O-Ac-GQlb than from GQlb. Particularly not only the rate of release of 9-O-Ac-NeuAc is reduced (which is expected, Schauer and Faillard, 1968) but also that of NeuAc. This indicates that the chemical difference between O-Ac-GQlb and G Q l b (that iS the 9-O-acetyl group on sialic acid) is recognized by Sialidase and changes its catalytic properties.
538
V~,N".A (. H,tiORNOel a/
In conclusion we suggest the following structure for O-Ac-GQ lb ganglioside : Galfll- 3GalNAcfll-4Gal/~?l-4GIcfll- 1'--ceramide 3
3
b
r
NeuAc~2
NeuAc72
8
8
I
NeuAcct2
P
9-O-Ac-NeuAcct2
According to the I U P A C IUB Commission on Biochemical Nomenclature (1977) it is designated as I I a ( 9 - O - A c - N e u A c c t 2 8NeuAc) IV 3 (NeuAcct2 .... 8NeuAc)GgOse4Cer. O - A c - G Q I b is the second individual ganglioside isolated from brain tissue which contains a 9-O-acetyl,N-acetylneuraminic acid. It adds to the hematoside (GM3) containing 4-O-acetyl,N-glycolylneuraminic acid, isolated from equine erythrocytes by Hakomori and Saito (1969). It is worth commenting that the fatty acid and long chain base composition of O-Ac-GT 1b and the here described O - A c - G Q l b is different, this making unlikely a metabolic relationship between them. Preliminary experiments, which are being developed, showed that O-acetylated gangliosides, including O - A c - G T l b and O - A c - G Q l b are present in the brain of many animals and constitute in some species a considerable portion of the total ganglioside content. Thus their recognition should be one of the goals in studies aimed at establishing brain ganglioside patterns at the phylogenetic, ontogenetic, tissue, cellular and subcellular level. The occurrence in brain as well as in other tissues of this type of ganglioside poses two important questions: (a) whether the O-acetylation of sialic acid occurs prior or not to the incorporation of sialic acid into the ganglioside molecule; (b) that would be the physiological meaning of sialic acid O-acetylation since, as we observed and reported in more detail elsewhere (Sonnino et al., 1982), O-acetylation of sialic acid appears to make sialosyl residues less sensitive to sialidase action. The availability of individual gangliosides of this type may facilitate and encourage studies aimed to approach the above problems. Acknowledgements--This work was supported by grants from the Consiglio Nazionale delle Ricerche (C.N.R.). Rome, Italy. The advice and help given by Prof. R. Schauer in the elucidation of the chemical nature of 9-O-acetyl,Nacetylneuraminic acid isolated from O-Ac-GQIb ganglioside is gratefully acknowledged.
REFERENCES Ando, S. and Yu, R. K. (1979). Isolation and characterization of two isomers of brain tetrasialogangliosides. J. biol. Chem., 254, 12,224 12,229. Buscher, H. P., Casals-Stenzel, J. and Schauer, R. (1974~. Identification of N-glycoloyl-O-acetylneuraminic acids and N-acetyl-O-glicoloylneuraminic acids by improved methods for detection of N-acyl and O-acyl groups and by gas-liquid chromatography. Eur. J. Biochem. 5{}, 71--82. Ghidoni, R., Sonnino, S., Tettamanti, G., Wiegandt, H. and Zambotti, V. (1976). On the structure of two new gangliosides from beef brain. J. Neurochem. 27, 511. 515. Ghidoni, R., Sonnino, S., Tettamanti, G., Baumann, N., Reuter, G. and Schauer R. (1980). Isolation and characterization of a trisialoganglioside from mouse brain, containing 9-O-acetyl-N-acetylneuraminic acid. J~ biol. Chem. 255, 6990-6995. Hakomori, S. and Saito, T. (1969). Isolation and characterization of a glycosphingolipid having a new siatic acid. Biochemistry 8, 5082-5088. Haverkamp, J., Kamerling, J. P., Vliegenthart, J. F. G.; Veh R. W. and Schauer R. (1977). Methylation analysis determination of acylneuraminic acid residue type 2-8 glycosidic linkages. Application to GTlb ganglioside and colominic acid. FEBS Lett. 73, 215-219. lwamori, M. and Nagai, Y. (1978). A new chromatographic approach to the resolution of individual gangliosides. Ganglioside mapping. Biochim. biophys. Acta 528, 257-267. IUPAC-IUB Commission on Biochemical Nomenclature (1977). The nomenclature of lipids. Lipids 12, 455-468. Kamerling J. P., Vliegenthart, J. F. G. and Vink, J. I1974). Mass spectrometry of pertrimethylsilyl neuraminic acid derivatives. Carbohyd. Res. 33, 297 406. Kamerling, J. P., Vliegenthart, J. F. G., Versluis, C. and Sehauer, R. (1975). Identification of O-acetylated N-awlneuraminic acids by mass spectrometry. Carbohyd. Res. 41, 7-17. Kamerling, J. P., Vliegenthart, J. F. G., Versluis; C. and Sehauer, R. (1978). In: Recent Developments in Ma.ss Spectrometry in Biochemistry and Medicine (Frigerio A., ed.) Vol. l, pp. 503-520, Plenum Press, New York. Schauer, R., and Faillard, H. (1968). Zur wirkungssPezifitat Neuraminidase. Das Verhalten isomerer N,O-Diacetylneuramins~iureglycoside im Submaxillarismucin yon Pferd und Rind bei Einwirkung bakterieller neuraminidase. Hoppe Seyler's Z. physiol. Chem. 349, 961-968.
New ganglioside from mouse brain Sonnino, S., Ghidoni, R., Baumann, N., Harpin, M. L. and Tettamanti, G. (1978a). Occurrence in mouse brain of a new ganglioside carrying an alkali labile linkage. Bull. Mol. Biol. and Med. 3, 170-178. Sonnino, S., Ghidoni, R., Chigorno, V. and Tettamanti, G. (1982). Chemistry of gangliosides carrying O-acetylated sialic acid. In: New Vista in Glycolipid Research (Makita A., Handa S., Taketomi T. and Nagai, Y. eds.) Plenum Publishing Corporation. In press. Sonnino, S., Ghidoni, R., Galli, G. and Tettamanti, G. (1978b). On the structure of a new fucose containing
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ganglioside from pig cerebellum. J. Neurochem. 31, 947-956. Svennerholm, L. (1964). The gangliosides. J. Lipid Res. 5, 145-155. Tettamanti, G., Bonali, F., Marchesini, S. and Zambotti, V. (1973). A new procedure for the extraction, purification and fractionation of brain gangliosides. Biochim. biophys. Acta 296, 160-170. Yang, H. J. and Hakomori, S. (1971). A sphingolipid having a novel type of ceramide and lacto-N-fucopentaose III. J. biol. Chem. 246, 1192-1200.