THE OF CHEMISTRY 268, No. 10, IJ Novel Pentahexosyl ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY...
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Printed in IJ SA. Vol. 268, No. 17, Issue of September 10, pp. 10727-10730,1983 Novel Pentahexosyl Ganglioside of the Globo Series Purified from Chicken Muscle* (Receivedfor publication, March 14, 1983) Jaw-Long Chien and Edward L. Hogan From the Department of Neurology, Medical University of South Carolina, Charleston, South Carolina 29425 A monosialosyl ganglioside, sialosylpentaglycosyl- ceramide, has been isolated from chicken pectoral mus- cle. This ganglioside is the first tobefoundwhich contains the glycosphingolipid structure of the globo series. It was extracted from tissue with a mixture of tetrahydrofuran and aqueous KC1 and purified byan- ion exchange and silicic acid chromatography. En- zymic hydrolysis utilizing specific glycosidases and methylation technique were employed to establish the structure which is proposed to be: NeuAca2+3Gal/31+ 3GalNAc~1+3Gal~1+4Gal~1+4GlcCer. The long chain base was predominantly sphingosine and the fatty acids present were mainly stearate and oleate. Although approximately 40 ganglioside structures have been defined in vertebrate tissues none has been found con- taining the core oligosaccharide structure of the globoside series (1). In our studies of the glycosphingolipids of chicken skeletal muscle (2-4), a resorcinol-positive compound was resolved whose RF on TLC in three different solvent systems differed from the other glangliosides found which were of the lactosylceramide and ganglio series. Here we report the de- tailed structure of a monosialosylpentaglycosylceramide pu- rified from chicken skeletal muscle which contains a novel neutral core oligosaccharide chain incorporating the globoside sequence (globo series). EXPERIMENTAL PROCEDURES Materials-Pectoral muscles from adult leghorn chickens were obtained from a local supermarket or dissected from an inbred strain of chickens after death. Extraneous tissues were separated by gross dissection and the muscle ground and stored at -60 "C. DEAE- Sephadex A-50, N-acetyl- and N-glycolylneuraminic acids, and a- neuraminidase type IX were obtained from Sigma. Sphingosine, dihydrosphingosine, and fatty acid methyl esters were products of Supelco, Inc., as were GC packings of 10% DEGS-PS, 3% SP-2340, 3% OV-17, and 3% SE-30 (all on Supelcoport support). Sephadex LH-20 was purchased from Pharmacia and precoated silica gel plates (Silica Gel 60) were from Brinkmann. Bio-Si1 A was obtained from Bio-Rad. Ganglioside standards from human brain and neutral gly- cosphingolipid standards from bovine erythrocytes were prepared in our laboratory. @-Galactosidase and @-hexosaminidase were purified from jack bean meal (5, 6) and a-galactosidase from fig powder (7). Glycolipid Extraction-One kilogram of ground muscle was homog- enized in 10 volumes of tetrahydrofuran:O.Ol M KC1 (41, v/v) using a Waring blender. The mixture was stirred for 3 h, then filtered and the residue reextracted twice with 5 volumes of tetrahydrofuran:O.Ol M KC1 (81, v/v). The combined filtrates were concentratedina * This work was supported by a grant from the Muscular Dystrophy Association and by Grant NS-16120 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. rotary evaporator to a syrup; then 1 liter of chloroform:methanol(2:1, V/V) was added followed by 200 mlof Hz0 (8) in order to separate the gangliosides by partition. The lower layer was reextracted twice with theoretical upper phase containing 0.027% KC1. The combined upper layers were concentrated to dryness, dissolved in water, and dialyzed at 4 "C against four changes of distilled water. Zon Exchange Chromatography-DEAE-Sephadex A-50 (C- form) was converted to the acetate as previously described (9). The crude ganglioside fraction obtained after dialysis was taken to dryness in a rotary evaporator and dissolved in a small volume of chloro- form:methanol (2:1, v/v) for application to the column. The neutral lipids including neutral glycolipids were eluted with methanol and the gangliosides were then eluted with 5 volumes of methanol con- taining in succession 0.01, 0.02, and 0.2 M sodium acetate. The 0.01 M sodium acetate effluent contained only monosialogangliosides in- cluding the unusual galactosamine-containing ganglioside. Each of the fractions was concentrated to dryness and salt removed by di- alysis. Silicic Acid Chromatography-Bio-Si1 A was activated overnight at 110 "C, allowed to cool, suspended in chloroform, and packed into a glass column (1.5 X 45 cm) using a vibrator at full speed. The ganglioside fraction eluted by 0.01 M sodium acetate was dissolved in a small volume of ch1oroform:methanol (2:1, v/v) and applied to the column. Gangliosides were eluted first with 400 ml of solvent A (chloroform:methanol:H20, 1307012, v/v/v) and then with 500 ml of solvent B (chloroform:methanol:H,O, 1207014, v/v/v). Collection volumes were 6 ml of which 5O-pl aliquots from alternate tubes were taken to identify the gangliosides by TLC. Enzymic Sequence Analysis of the Oligosaccharide Chain-The saccharide chain sequence of glycosyl residues andthe anomeric configurations were determined by incubation of the purified ganglio- side with several specific glycosidases as previously described (9). Sialic acid elimination was effected with 40 milliunits of a-neuramin- idase from Clostridium perfringens (1 unit of enzyme will release 1 pmol ofNeuAc from bovine submaxillary mucin/min at 37 "C and pH 5.0) incubated overnight at 37 "C in a reaction mixture containing 30 pg of ganglioside in 200 p1 of 0.05 M sodium acetate buffer at pH 5.0. The reaction was stopped by addition of 1 ml of chloro- form:methanol (211, and theasialoglycolipidwas recovered fromthe lower layer. The hydrolysis of neutral glycosphingolipids was done with 0-galactosidase, 0-hexosaminidase, and a-galactosidase accord- ing to the procedures of Chien et al. (9). Permethylation Studies-The purified glycolipid (500 Kg) was methylated according to the procedure of Yang and Hakomori (10) and further purified on a column of Sephadex LH-20 before hydrolysis with 0.6 N HzSOI in 80% aqueous CH&OOH and acetylated accord- ing to the method of Bjorndal et al. (11). The partially methylated alditol acetate derivatives were analyzed on a Finnigan Model 3300 gas chromatograph-mass spectrometer. The partially methylated glu- citol and galactitol acetates were separated on 3% SP 2340 at 180 "C and the amino sugar derivative on3% OV 17 with programmed temperature rise from 180-200 "C (2 'C/min) (12-14), Other Methods-Fatty acid methyl esters were extracted with hexane after methanolysis (1.5 M anhydrous rnethanolic HC1, 80 "C, 24 h) and analyzed on a 10% DEGS column at 190 "C. Long chain bases were determined as trimethylsilyl derivatives on a 3% SE-30 column (15) after hydrolysis (16). Sialic acids were quantitated ac- cording to the method of Svennerholm (17) as modified by Miettinen and Takki-Luukainen (18). N-Acetyl- and N-glycolylneuraminic acids (19) and neutral and amino sugars (11) were determined by gas chromatography. 10727 by guest on November 17, 2020 http://www.jbc.org/ Downloaded from
Transcript of THE OF CHEMISTRY 268, No. 10, IJ Novel Pentahexosyl ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY...
THE JOURNAL OF BIOLOGICAL CHEMISTRY
Printed in I J S A . Vol. 268, No. 17, Issue of September 10, pp. 10727-10730,1983
Novel Pentahexosyl Ganglioside of the Globo Series Purified from Chicken Muscle*
(Received for publication, March 14, 1983)
Jaw-Long Chien and Edward L. Hogan From the Department of Neurology, Medical University of South Carolina, Charleston, South Carolina 29425
A monosialosyl ganglioside, sialosylpentaglycosyl- ceramide, has been isolated from chicken pectoral mus- cle. This ganglioside is the first to be found which contains the glycosphingolipid structure of the globo series. It was extracted from tissue with a mixture of tetrahydrofuran and aqueous KC1 and purified by an- ion exchange and silicic acid chromatography. En- zymic hydrolysis utilizing specific glycosidases and methylation technique were employed to establish the structure which is proposed to be: NeuAca2+3Gal/31+ 3GalNAc~1+3Gal~1+4Gal~1+4GlcCer. The long chain base was predominantly sphingosine and the fatty acids present were mainly stearate and oleate.
Although approximately 40 ganglioside structures have been defined in vertebrate tissues none has been found con- taining the core oligosaccharide structure of the globoside series (1). In our studies of the glycosphingolipids of chicken skeletal muscle (2-4), a resorcinol-positive compound was resolved whose RF on TLC in three different solvent systems differed from the other glangliosides found which were of the lactosylceramide and ganglio series. Here we report the de- tailed structure of a monosialosylpentaglycosylceramide pu- rified from chicken skeletal muscle which contains a novel neutral core oligosaccharide chain incorporating the globoside sequence (globo series).
EXPERIMENTAL PROCEDURES
Materials-Pectoral muscles from adult leghorn chickens were obtained from a local supermarket or dissected from an inbred strain of chickens after death. Extraneous tissues were separated by gross dissection and the muscle ground and stored at -60 "C. DEAE- Sephadex A-50, N-acetyl- and N-glycolylneuraminic acids, and a- neuraminidase type IX were obtained from Sigma. Sphingosine, dihydrosphingosine, and fatty acid methyl esters were products of Supelco, Inc., as were GC packings of 10% DEGS-PS, 3% SP-2340, 3% OV-17, and 3% SE-30 (all on Supelcoport support). Sephadex LH-20 was purchased from Pharmacia and precoated silica gel plates (Silica Gel 60) were from Brinkmann. Bio-Si1 A was obtained from Bio-Rad. Ganglioside standards from human brain and neutral gly- cosphingolipid standards from bovine erythrocytes were prepared in our laboratory. @-Galactosidase and @-hexosaminidase were purified from jack bean meal (5, 6) and a-galactosidase from fig powder (7).
Glycolipid Extraction-One kilogram of ground muscle was homog- enized in 10 volumes of tetrahydrofuran:O.Ol M KC1 (41, v/v) using a Waring blender. The mixture was stirred for 3 h, then filtered and the residue reextracted twice with 5 volumes of tetrahydrofuran:O.Ol M KC1 (81, v/v). The combined filtrates were concentrated in a
* This work was supported by a grant from the Muscular Dystrophy Association and by Grant NS-16120 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
rotary evaporator to a syrup; then 1 liter of chloroform:methanol(2:1, V/V) was added followed by 200 ml of Hz0 (8) in order to separate the gangliosides by partition. The lower layer was reextracted twice with theoretical upper phase containing 0.027% KC1. The combined upper layers were concentrated to dryness, dissolved in water, and dialyzed at 4 "C against four changes of distilled water.
Zon Exchange Chromatography-DEAE-Sephadex A-50 (C- form) was converted to the acetate as previously described (9). The crude ganglioside fraction obtained after dialysis was taken to dryness in a rotary evaporator and dissolved in a small volume of chloro- form:methanol (2:1, v/v) for application to the column. The neutral lipids including neutral glycolipids were eluted with methanol and the gangliosides were then eluted with 5 volumes of methanol con- taining in succession 0.01, 0.02, and 0.2 M sodium acetate. The 0.01 M sodium acetate effluent contained only monosialogangliosides in- cluding the unusual galactosamine-containing ganglioside. Each of the fractions was concentrated to dryness and salt removed by di- alysis.
Silicic Acid Chromatography-Bio-Si1 A was activated overnight at 110 "C, allowed to cool, suspended in chloroform, and packed into a glass column (1.5 X 45 cm) using a vibrator at full speed. The ganglioside fraction eluted by 0.01 M sodium acetate was dissolved in a small volume of ch1oroform:methanol (2:1, v/v) and applied to the column. Gangliosides were eluted first with 400 ml of solvent A (chloroform:methanol:H20, 1307012, v/v/v) and then with 500 ml of solvent B (chloroform:methanol:H,O, 1207014, v/v/v). Collection volumes were 6 ml of which 5O-pl aliquots from alternate tubes were taken to identify the gangliosides by TLC.
Enzymic Sequence Analysis of the Oligosaccharide Chain-The saccharide chain sequence of glycosyl residues and the anomeric configurations were determined by incubation of the purified ganglio- side with several specific glycosidases as previously described (9). Sialic acid elimination was effected with 40 milliunits of a-neuramin- idase from Clostridium perfringens (1 unit of enzyme will release 1 pmol of NeuAc from bovine submaxillary mucin/min at 37 "C and pH 5.0) incubated overnight at 37 "C in a reaction mixture containing 30 pg of ganglioside in 200 p1 of 0.05 M sodium acetate buffer at pH 5.0. The reaction was stopped by addition of 1 ml of chloro- form:methanol (211, and the asialoglycolipid was recovered from the lower layer. The hydrolysis of neutral glycosphingolipids was done with 0-galactosidase, 0-hexosaminidase, and a-galactosidase accord- ing to the procedures of Chien et al. (9).
Permethylation Studies-The purified glycolipid (500 Kg) was methylated according to the procedure of Yang and Hakomori (10) and further purified on a column of Sephadex LH-20 before hydrolysis with 0.6 N HzSOI in 80% aqueous CH&OOH and acetylated accord- ing to the method of Bjorndal et al. (11). The partially methylated alditol acetate derivatives were analyzed on a Finnigan Model 3300 gas chromatograph-mass spectrometer. The partially methylated glu- citol and galactitol acetates were separated on 3% SP 2340 at 180 "C and the amino sugar derivative on 3% OV 17 with programmed temperature rise from 180-200 "C (2 'C/min) (12-14),
Other Methods-Fatty acid methyl esters were extracted with hexane after methanolysis (1.5 M anhydrous rnethanolic HC1, 80 "C, 24 h) and analyzed on a 10% DEGS column at 190 "C. Long chain bases were determined as trimethylsilyl derivatives on a 3% SE-30 column (15) after hydrolysis (16). Sialic acids were quantitated ac- cording to the method of Svennerholm (17) as modified by Miettinen and Takki-Luukainen (18). N-Acetyl- and N-glycolylneuraminic acids (19) and neutral and amino sugars (11) were determined by gas chromatography.
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10728 Monosialosylpentaglycosylceramide
RESULTS
Thin Layer Chromatography of the Purified Ganglioside- The novel ganglioside had an RF close to GD3 (Fig. L4) in a neutral solvent system containing calcium (chloro- form:methanol:0.25% CaC12, 60:40:9) but a lesser RF in alka- line conditions (chloroform:methanol:O.25 M NH40H, 6040:9) where it migrated slightly behind GDla (Fig. 1B) from human brain. The best resolution of this ganglioside was achieved with tetrahydrofuran:O.l% KC1 (75:15). In this sol- vent system (Fig. IC) the novel ganglioside could be clearly separated from GD3 and GDla and had the highest RF of the three solvents.
Chemical Composition-The acyl groups of the ganglioside were mainly palmitic, stearic, and oleic acids (Table I). Sphin- gosine was the major long chain base and less than 15% was dihydrosphingosine. The ganglioside contained N-acetylneu- raminic acid, galactose, galactosamine, glucose, and sphingo- sine in a molar ratio of 1.01:3.12:0.95:1.00:0.98.
Hydrolysis of Saccharide Sequence by Specific Glycosi- dases-The purified ganglioside was not degraded by @-galac- tosidase, @-hexosaminidase, or a-galactosidase. When incu- bated with a-neuraminidase in the absence of detergent, it was hydrolyzed to a neutral glycosphingolipid with an RFclose to that of the pentaglycosylceramide purified from bovine erythrocytes (Lane 3, Fig. 2). Subsequent incubation with p- galactosidase converted it to a compound with the migration of globoside (Lane 4, Fig. 2). Further treatment of the glyco- lipid with @-hexosaminidase, a-galactosidase, and @-galacto- sidase yielded tri-, di-, and monohexosylceramides (Fig. 2).
A B C
GMI
GD3
GDlo
GDlb
G M I GD3
GDlo
GDI b
GMI
GD3
GDlo
GDI b
"
I 2 I 2 " "
1 2
FIG. 1. Thin layer chromatograms of novel ganglioside from chicken muscle. Lane I, human brain ganglioside standards; Lane 2, purified novel chicken monosialosylpentaglycosylceramide. Plates A, B, and C were developed with solvent systems as described in the text.
Linkages between Saccharide Units-The sites of the link- ages between the saccharide units have been established by identification of the partially methylated alditol acetates by gas chromatograph-mass spectrometer. Three peaks (Fig. 3a) were obtained upon analysis of derivatives from this ganglio- side and their mass spectra were those of 2,4,6-tri-O-methyl- galactitol-1,3,5-triacetate (Peak 1, Fig. 3a), 2,3,6-tri-O-meth- ylgalactitol-1,4,5-triacetate (Peak 2, Fig. 3a), and 2,3,6-tri-0- methylglucitol-1,4,5-triacetate (Peak 3, Fig. 3a). The peaks and mass spectra (Fig. 36) are identical with those derived from the Forssman hapten glycolipid (Fig. 3c). When the triglycosylceramide derived from the chicken muscle ganglio- side was analyzed, Peak 1 disappeared and a new peak (Peak 0) appeared (Fig. 4) which was identified as 2,3,4,6-tetra-O- methylgalactitol-l,5-diacetate, while Peaks 2 and 3 remained unchanged. This indicates substitution of the galactose resi- due of lactosylceramide at the C-4 position and the other two galactose residues at the C-3 position. The amino sugar deriv- ative was identified as 4,6-di-O-methy1-2-deoxy-2-N-meth- ylacetamidogalactitol-1,3,5-triacetate and was identical with that obtained from the penultimate N-acetylgalactosamide residue of Forssman glycolipid (Fig. 5).
G o l C e r
LocCer
G M 4
G M 3
G M 2
G M I
c LocCer
nLc4Cer
L. nlc5Cer
G D I o G D I b
""""
1 2 3 4 5 6 7 8
FIG. 2. Stepwise enzymatic hydrolysis of sialosylpentagly- cosylceramide from chicken breast muscle. Lane I, human brain glycosphingolipids from top: galactosylceramide (GalCer), lactosylcer- amide (LacCer), GM4, GM3, GM2, GM1, GDla, and GDlb. Lane 2, monosialosylpentaglycosylceramide. Lane 3, Lane 2 + a-neuramini- dase. Lane 4 , Lane 3 + @-galactosidase. Lane 5, Lane 4 + 8-hexos- aminidase. Lane 6, Lane 5 + a-galactosidase. Lane 7, Lane 6 + 8- galactosidase. Lane 8, neutral glycosphingolipid standards from bo- vine erythrocytes from top: lactosylceramide, globotriaosylceramide (GbaCer), neolactotetraosylceramide (nLc,Cer), and neolactopenta- glycosylceramide (nLc&er).
TABLE I Fatty acids and long chain bases of sialosylpentaglycosylcerarnide
Fatty acids C160 C161 C180 C181 C182 c200 c201 c21:o C22:l C261 96 15.4 3.0 39.2 31.8 1.2 2.2 0.5 1.0 1.7 4.0 Long chain bases 1 8 0 181 96 14.7 85.3
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Monosialosylpentaglycosylceramide 10729
B
A
m C 0 a is? c m c D
I
I
a
3
2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 Time (minutes)
Time (minutesl
FIG. 3. Gas chromatogram and mass spectra of partially methylated alditol acetates from chicken muscle mono- sialosylpentaglycosylceramide and Forssman hapten. a, gas chromatogram of partially methylated alditol acetates from monosialosylpentaglycosylceramide. b, mass spectra obtained from Peaks 1, 2, and 3, respectively. c, gas chromatogram of partially methylated alditol acetates from Forssman hapten.
I I 1 1 1 1 1 I I I ~
2 4 6 8 1 0 1 2 1 4 16 18 20 Time (minutes)
FIG. 4. Gas chromatogram of partially methylated alditol acetates of triglycosylceramide derived from monosialosylpentaglycosyl- ceramide by enzymatic hydrolysis.
Time (minutes)
A B I 3
Time (minutes)
FIG. 5. Gas chromatogram of methylated 2-deoxy-2-N- methylacetamidohexitol acetates from Forssman hapten (A) and chicken muscle monosialosylpentaglycosylceramide (B). Peak 1 was identified as 3,4,6-tri-O-methyl-2-deoxy-2-N-rnethylace- tamidogalactitol-1,5-diacetate and Peaks 2 and 3 were identified as 4-6-di-0-methyl-2-deoxy-2-N-methylacetamidogalactitol-1,3,5-tri- acetrate. The mass spectra ( C ) shown is obtained from Peak 3.
DISCUSSION
The structure of the novel ganglioside is NeuAcaB+ 3Galpl+ 3GalNAcPl-+ 3Galal+ 4GalPl- 4GlcCer and should be abbreviated as V3NeuAcIV3GalGbOse4Cer accord- ing to the short symbol designation of the IUPAC-IUB Com- mission on Lipid Nomenclature (1976). It constitutes 9 mol % of the total gangliosides of chicken muscle. The other species of gangliosides in this muscle are GM3 (38%) and GD3 (14%) the two gangliosides containing neolactotetraose, IV3NeuAcLcOse4Cer (ll%), V13NeuAcLcOseGCer (14%), GDla, and GT1 in smaller amounts. Hence, chicken skeletal muscle gangliosides have oligosaccharides of all four se- quences known to occur in glycolipids: lactose, gangliote- traose, neolactotetraose, and globotetraose.
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10730 Monosialosylpentaglycosylceramide
Although gangliosides have been studied in muscle of pig, rat (20), and rabbit (21), a similar detail of structural analysis has been reported only in human muscle where the major gangliosides in both skeletal (22) and cardiac muscle (23) are GM3, GM2, GD3, GDla, and IV3NeuAcnLcOse,Cer although their distribution in the two types of muscle differs. Recently, Iwamori and Nagai (24) characterized the monosialoganglio- sides of rabbit skeletal muscle and found gangliosides of lacto series containing oligosaccharides as large as octosaccharide length, but no ganglio or globo series. We have not observed the globo series ganglioside described here in our studies of human skeletal muscle (2) and suspect that there is species difference in muscle gangliosides.
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J L Chien and E L HoganNovel pentahexosyl ganglioside of the globo series purified from chicken muscle.
1983, 258:10727-10730.J. Biol. Chem.
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