Glycosphingolipids of Skeletal Muscle - ACS Symposium Series (ACS

9 Glycosphingolipids of Skeletal Muscle

Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 23, 2018 | https://pubs.acs.org Publication Date: July 31, 1980 | doi: 10.1021/bk-1980-0128.ch009

JAW-LONG CHIEN and EDWARD L. HOGAN Department of Neurology, Medical University of South Carolina, Charleston, SC 29403

It is currently held that glycosphingolipids are enriched in cell surface membranes and possible participants in such events as receptor interactions (1,2), permeability change (3), cellular adhesion (4) and cellular recognition (5). The likelihood of their localization in sarcolemma and possible role in myogenesis including cell fusion or in conduction of the action potential prompted us to begin their study with isolation and characterization of the gangliosides and neutral glycosphingolipids in chicken and human skeletal muscle. Although muscle comprises approximately 40% of the body weight, there have been only a few studies of glycosphingolipids of muscle. Puro and coworkers (6) studied the qualitative and quantitative patterns of gangliosides in several extraneural tissues including skeletal and cardiac muscles of rat, rabbit and pig, but did not purify the individual gangliosides. Lassaga et al. (7) isolated four gangliosides from the hind leg and back muscle of the rabbit. One had the molar composition of hematoside (GM3) but the structures of the others - two disialo- and one trisialoganglioside - were not fully clarified. Svennerholm et al. (8) did a more complete study of human skeletal muscle. They isolated four major gangliosides and determined their composition by gas chromatography to be consistent with GM3, GM2, GDla and a sialosyltetraglycosylceramide. Recently, Levis and coworkers (9) examined the glycosphingolipids in human heart and found that human cardiac muscle contains the same gangliosides as those of human skeletal muscle. However, the distribution of gangliosides was quite different. In heart, GM3 (23%), GD3 (22%) andGM1(16%) are the major ganlgiosides while in skeletal muscle GM3 (67%) predominates. Neutral glycosphingolipids have also been studied in human skeletal (8) and cardiac (9) muscle. In skeletal muscle, lactosylceramide is the predominant glycolipid (38.4%) followed by globotriaosylceramide (26.3%) and globoside (12.4%); while in heart, globoside predominates (43.0%) followed by globotriaosylceramide (32.0%). 0-8412-0556-6/ 80/47-128-13555.00/ 0 © 1980 American Chemical Society Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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The r e s u l t s presented here show that the g l y c o s p h i n g o l i p i d s of chicken p e c t o r a l muscle d i f f e r from those o f human s k e l e t a l and c a r d i a c muscles. In a d d i t i o n , we are p r e s e n t i n g the s t r u c tures o f two glucosamine-containing g a n g l i o s i d e s which were c h a r a c t e r i z e d by enzymatic h y d r o l y s i s and methylation s t u d i e s .


Materials P e c t o r a l muscle from a d u l t Leghorn chickens was obtained from a l o c a l supermarket (the main source) o r d i s s e c t e d immedia t e l y a f t e r s a c r i f i c e . Human s k e l e t a l muscle was obtained four hours post mortem from the p e c t o r a l and i l i o p s o s a s muscles of a 70-year-old b l a c k male who d i e d o f a gunshot wound to the head. Precoated s i l i c a g e l p l a t e s ( s i l i c a g e l 60) were purchased from S c i e n t i f i c Products. B i o - S i l A (200 - 400 Mesh) was obtained from Bio-Rad L a b o r a t o r i e s . Fatty a c i d methyl e s t e r s , sphingosine and dihydrosphingosine were products o f Supelco, Inc. as were 10% DEGS-PS, 3% SP-2340 and 3% OV-17 ( a l l on Supelcoport support). N-acetyl and N - g l y c o l y l neuraminic a c i d , DEAE-Sephadex A50 and neuraminidase type IX were obtained from Sigma Company. Ganglios i d e standards from human b r a i n and n e u t r a l g l y c o s p h i n g o l i p i d standards from bovine erythrocytes were prepared i n t h i s l a b o r a tory. 3-galactosidase was i s o l a t e d from papaya and $-hexosaminidase was prepared from j a c k bean meal (10). a-N-acetylgalactosaminidase was a generous g i f t of Dr. Y.-T. L i of Tulane U n i v e r s i t y . E x t r a c t i o n of g l y c o s p h i n g o l i p i d s The muscles were f r e e d by gross d i s s e c t i o n of extraneous t i s s u e which was mainly f a t and p e r i p h e r a l nerves, and then stored at -40°C. For an experiment, approximately 1 kg t i s s u e was macerated by a meat g r i n d e r and homogenized i n ten volumes of tetrahydrofuran:0.01 M KC1 (4:1, v / v ) , s t i r r e d f o r 3 hours, and f i l t e r e d through a Buchner f u n n e l . The e x t r a c t i o n was repeated twice and the f i l t r a t e s then combined and concentrated i n a r o t a r y evaporator. One l i t e r of chloroform-methanol (2:1, v/v) was added to the l i p i d e x t r a c t which has the appearance and consistency of syrup. Gangliosides were p a r t i t i o n e d i n t o the upper l a y e r by the a d d i t i o n o f 200 ml of water (11) and the lower layer extracted two a d d i t i o n a l times with t h e o r e t i c a l upper phase c o n t a i n i n g 0.027% KC1. The combined upper l a y e r s were then concentrated and d i a l y z e d e x h a u s t i v e l y a t 4°C with f i v e changes o f d i s t i l l e d water. DEAE-Sephadex Column Chromatography DEAE-Sephadex A-50 (Cl-form) was converted to the acetate form by the f o l l o w i n g procedure: The g e l was washed f i r s t with f i v e volumes of 0.1 N NaOH, and a f t e r r i n s i n g with d i s t i l l e d

Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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water, i t was converted i n t o the acetate form by washing with IN a c e t i c a c i d . The sedimented g e l was then r i n s e d repeatedly with water u n t i l n e u t r a l , washed with methanol and packed i n t o a column (1.8 x 21 cm). The l i p i d e x t r a c t obtained f o l l o w i n g d i a l y s i s was d i s s o l v e d i n C:M (2:8, v/v) and a p p l i e d to t h i s column which had been p r e v i o u s l y e q u i l i b r a t e d i n the same s o l v e n t . The n e u t r a l l i p i d s were eluted by the C:M (2:8, v/v) and gangliosides then e l u t e d by methanol containing sodium acetate i n the f o l l o w i n g concentrations: 0.01 M ( f r a c t i o n I ) , 0.02 M ( f r a c t i o n I I ) , and 0.2 M ( f r a c t i o n I I I ) . The f r a c t i o n s e l u t e d were concentrated and s a l t removed by d i a l y s i s . B i o - S i l A Column Chromatography of Ganglioside

Fractions

B i o - S i l A was a c t i v a t e d a t 110°C overnight, suspended i n chloroform and packed i n t o a column (1.5 x 45 cm). F r a c t i o n I (eluted from DEAE-Sephadex with 0.01 M sodium acetate was d i s solved i n C:M (2:1, v/v) and a p p l i e d to the column. Gangliosides were e l u t e d with a C:M:H20 solvent system of i n c r e a s i n g p o l a r i t y . We have been using the f o l l o w i n g mixtures: Solvent I - C:M:H Q (130:70:12, v / v ) , 0.4 C:M:H 0 (120:70:14, v / v ) , 0.5 l i t e r . 2

l i t e r and solvent 2 -

2

F r a c t i o n s o f 6 ml volume were c o l l e c t e d and 50 u l a l i q u o t s used to i d e n t i f y the gangliosides by TLC. Four gangliosides have been p u r i f i e d from f r a c t i o n I of chicken s k e l e t a l muscle d i r e c t l y from the column. S i l i c a - g e l G Column Chromatography of Neutral

Glycosphingolipids

The n e u t r a l l i p i d f r a c t i o n from the DEAE-Sephadex A-50 column was combined with the lower phase obtained a f t e r Folch p a r t i t i o n of the t o t a l l i p i d e x t r a c t and the combined l i p i d s d r i e d . To the same f l a s k , 100 ml of 0.6 M NaOH i n methanol was added. The mixture was incubated at 37°C f o r 5 hours. Five v o l umes of acetone were then added and stored overnight a t 4°C. The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n a t 4°C and d i s s o l v e d i n C:M (4:1, v / v ) . A f t e r a p p l i c a t i o n to the column (2.0 x 25 cm), the column was washed with chloroform. Neutral g l y c o l i p i d s were then e l u t e d with tetrahydrofuran: H2O (10:1). F r a c t i o n s c o n t a i n ing n e u t r a l g l y c o s p h i n g o l i p i d s were pooled and t h e i r g l y c o l i p i d content examined by t h i n - l a y e r chromatography. Enzymatic Hydrolysis Employing

Glycosidases

The sequence and anomeric c o n f i g u r a t i o n of the o l i g o s a c c h a r i d e chain was determined by step-wise h y d r o l y s i s with s p e c i f i c glycosidases. The conditions of incubation f o r h y d r o l y s i s are the same as those p r e v i o u s l y described (12). For the hydroly-


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s i s o f s i a l i c a c i d from g a n g l i o s i d e s , 30 yg of the g a n g l i o s i d e was d i s s o l v e d i n 150 y l of 0.05 M sodium acetate b u f f e r at pH 5.0, and incubated overnight a t 37°C with 4 m i n i u n i t s of neuraminidase from C l o s t r i d i u m p e r f r i n g e n s . For h y d r o l y s i s of a s i a l o gangliosides and n e u t r a l g l y c o s p h i n g o l i p i d s , 30 yg of g l y c o l i p i d was incubated overnight with 0.3 - 1.0 u n i t of $-galactosidase or 3-hexosaminidase at 37°C. A f t e r the r e a c t i o n was complete, the product was p a r t i t i o n e d t o the lower l a y e r by the a d d i t i o n of 5 v o l . of C:M(2:1, v / v ) , and the upper phase washed twice with t h e o r e t i c a l lower phase. The combined lower l a y e r s were then resolved by TLC. Permethylation methods The g l y c o s y l linkages were determined using methylation technique. In b r i e f , the p u r i f i e d g l y c o l i p i d s were exposed to d i m e t h y l s u l f i n y l i o n and then methylated with methyl i o d i d e (13). The methylated d e r i v a t i v e was a p p l i e d to an LH-20 column (0.5 x 24 cm) which had been packed and e l u t e d with acetone (14). The combined methylated g l y c o l i p i d s were hydrolyzed with 0.6N H2SO4 i n 80% aqueous a c e t i c a c i d a t 80°C f o r 18 hours, reduced and a c e t y l a t e d according to Bjorndal e t a l . (15). P a r t i a l l y methylated g a l a c t i t o l and g l u c i t o l acetates were separated i s o t h e r m a l l y at 180°C u s i n g a column packed with 3% OV-275 Supelcoport (100-120 Mesh). Amino sugar d e r i v a t i v e s are separated by a 3% 0V-17 Supelcoport (100-120 Mesh) column over a range of 180° - 200°C with a temperature increment r a t e of 2°/min. (16, 17, 18). Other Methods F a t t y a c i d methyl e s t e r s were extracted from the methanolysate with hexane and analyzed a t 190°C by GC using a 10% DEGS column. Sphingosine bases were determined a f t e r h y d r o l y s i s (19) as trime t h y l s i l y l d e r i v a t i v e s by GC u s i n g a 3% SE-30 column (20). S i a l i c a c i d was determined by the r e s o r c i n o l method (21) as modified by M i e t t i n e n and Takki-Luukainen (22). Species of s i a l i c a c i d (NANA, NGNA, etc.) were analyzed by TLC (23) and gas chromatography (24). Sugar composition and hexosamines were determined as a l d i t o l acetates using GC (15). Results Comparison of g l y c o s p h i n g o l i p i d s from human and chicken s k e l e t a l muscle. The e l u t i o n of g a n g l i o s i d e s from DEAE-Sephadex A 50 column with these 0.01, 0.02 and 0.2 M sodium acetate conc e n t r a t i o n s separated the g a n g l i o s i d e s i n t o mono-, d i - and p o l y sialo- fractions. The g a n g l i o s i d e s of human muscle are shown i n F i g . 1A. The monosialogangliosides GM3, GM2 and GM1 were e l u t e d with 0.01 M sodium acetate i n methanol (lane 2), GD3 and GDla with the 0.02 M s o l v e n t (lane 4) and others with 0.2 M acetate



9.

CHIEN AND

Figure 1A.

HOGAN

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139

Thin-layer chromatogram of human and chicken muscle ganglioside fractions eluted from DEAE-A

50 column

Lane 1, standard gangliosides from human brain. Lanes 2 and 3, gangliosides eluted by 0.01M, lanes 4 and 5 by 0.02M, and lanes 6 and 7 by 0.2M sodium acetate in methanol. Lanes 2, 4, and 6 from human muscle; lanes 3, 5, and 7 from chicken muscle. Solvent system: C:M:0.25% CaCl (60:40:9) 2

Figure IB.

Thin-layer chromatogram of

human and chicken neutral glycolipids Lane 1 contains (from the top) standard lactosylceramide, globotriaosylceramide, neolactotetraosylceramide, and lactopentaosylceramide prepared from bovine erythrocytes. Lane 2 contains the neutral glycosphingolipids of human skeletal muscle, and lane 3 contains those of the chicken. Solvent system: C:M:H 0 (60:40:9, v/v). 2


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(lane 6). When the upper phase g a n g l i o s i d e f r a c t i o n from chicken p e c t o r a l muscle was resolved i n a s i m i l a r way, four bands were e l u t e d with the 0.01 M acetate i n methanol. The major one had a m o b i l i t y corresponding to GM3. The others had Rf values c l o s e to those of GM1, GD3 and GDla r e s p e c t i v e l y ( F i g . 1A, lane 3 ) . However, a l l contained one s i a l i c a c i d per mole. The d i s i a l o g a n g l i o s i d e f r a c t i o n o f chicken muscle was a l s o considerably d i f f e r e n t from that o f human. Both contained GD3, but the other major d i s i a l o g a n g l i o s i d e s i n chicken migrated between GDla and GDlb; and GDlb and GT1 r e s p e c t i v e l y . Human muscle a l s o contained appreciable t r i s i a l o g a n g l i o s i d e of which very l i t t l e was found i n the chicken. The n e u t r a l g l y c o s p h i n g o l i p i d s o f human and chicken s k e l e t a l muscle were a l s o remarkably d i f f e r e n t ( F i g . IB). Human muscle contained l a c t o s y l c e r a m i d e as the major g l y c o l i p i d followed by globotriaosylceramide and globoside (17), while i n chicken muscle the major n e u t r a l g l y c o s p h i n g o l i p i d (48%) migrates between nLcOse^Cer and IV^Gal-nLcOse^er. I t contained two moles each of galactose and N-acetylgalactosamine and one mole of glucose, and was converted to globoside by the a-N-acetylgalactosaminidase from limpet (25). Thus, i t appears t o be a Forssman-active g l y c o l i p i d . G a s - l i q u i d chromatographic a n a l y s i s of the other n e u t r a l g l y c o l i p i d s was c o n s i s t e n t with the molar composition of galactosylceramide (20%), lactosylceramide (12%), g l u c o s y l c e r a mide (9%), globoside (8%) and globotriaosylceramide (3%). B i o - S i l A column chromatography and g l y c o s y l composition of the g a n g l i o s i d e s o f chicken muscle. The e l u t i o n p a t t e r n of monosialogangliosides from a B i o - S i l A column i s shown i n F i g . 2. Under these c o n d i t i o n s (see t e x t ) , the four g a n g l i o s i d e s separated w e l l . The f r a c t i o n s c o n t a i n i n g the same g a n g l i o s i d e were pooled and the p u r i t y confirmed by repeat TLC. When developed with C:M:0.25% C a C l (60:40:9, v / v ) , the four monosialoganglios i d e s comigrated with GM3, GM1, GD3 and GDla g a n g l i o s i d e s t a n dards i s o l a t e d from human b r a i n ( F i g . 3A). But i n another and a l k a l i n e solvent C:M:0.25N NaOH (60:40:9, v / v ) , three o r a l l except g a n g l i o s i d e I (lane 2) were obviously d i f f e r e n t i n m o b i l i ty ( F i g . 3B). G a n g l i o s i d e I I (lane 3) moved w e l l ahead of b r a i n GM1, g a n g l i o s i d e I I I (lane 4) was behind GD3, and the Rf of g a n g l i o s i d e IV (lane 6) was s l i g h t l y l e s s than that of the GDla. These d i f f e r e n c e s i n Rf must d e r i v e from the d i f f e r e n c e s i n sugar composition i n comparison to the standards from b r a i n (Table I ) . The composition of g a n g l i o s i d e I was the same as b r a i n GM3. Ganglioside I I d i f f e r e d from GM1 i n c o n t a i n i n g N-acetylglucosamine r a t h e r than N-acetylgalactosamine. Ganglioside I I I was a n o v e l s i a l o g l y c o l i p i d with a molar composition of s i a l i c a c i d : N-acetylgalactosamine: galactose: glucose: sphingosine of 1:1:3:1:1. Ganglioside IV had the same sugar composition as that of s i a l o s y l h e x a g l y c o s y l c e r a m i d e from human spleen (26) and bovine e r y t h r o c y t e s (12) with a molar r a t i o of s i a l i c a c i d : N-acetylglucosamine: g a l a c t o s e : glucose: sphingosine of 1:2:3:1:1. 2



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Figure 2. Elution pattern from Bio-Sil A column of monosialogangliosides prepared from chicken pectoral muscle. Column was eluted with C:M:H 0 (130:70:12, v/v) and changed to C:M:H 0 (120:70:14, v/v) at the arrow. 2

2

Figure 3.

Thin layer chromatograms of monosialogangliosides purified from chicken muscle

Lanes 1 and 5, human brain ganglioside standards; lanes 2, 3, 4, and 6 fractions from a Bio-Sil A colume (Figure 2). Solvent systems: (A) CHCl :MeOH:0.25 CaCl (60:49:9, v/v); (B) CHCl :MeOH:0.25M NHfiH (60:40:9, v/v). 3

2

3



Gal 1.07 2.16 2.80 2.96

1 1 1 1

I

II

III

IV

GANGLIOSIDE

GLc

1.83

1.18

GlcNAc

0.94

GalNAc

1.04

1.1

1.08

1.09

NANA

SUGAR COMPOSITION OF CHICKEN MUSCLE MONOSIALOGANGLIOSIDES

Table I


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Characterization of saccharide u n i t . G a n g l i o s i d e I was h y d r o l y z e d by n e u r a m i n i d a s e t o a n e u t r a l g l y c o l i p i d w h i c h was f u r t h e r c l e a v e d by ( 3 - g a l a c t o s i d a s e t o become g l u c o s y l c e r a m i d e . The s e q u e n c e o f t h e two g l u c o s a m i n e - c o n t a i n i n g g a n g l i o s i d e s ( I I and I V ) a r e shown i n F i g . 4 a n d 5 r e s p e c t i v e l y . G a n g l i o s i d e I I was h y d r o l y z e d b y n e u r a m i n i d a s e w i t h no d e t e r g e n t t o a compound with R corresponding to that of Neolactotetraosylceramide which was s u b s e q u e n t l y c o n v e r t e d t o l a c t r i a o s y l - , l a c t o s y l - , a n d g l u cosylceramide by the consecutive a c t i o n s of 3-galactosidase, ^ - h e x o s a m i n i d a s e and 3 - g a l a c t o s i d a s e ( F i g . 4 ) . G a n g l i o s i d e I V was h y d r o l y z e d t o become a n e u t r a l h e x a g l y c o s y l c e r a m i d e when i n c u b a t ed w i t h n e u r a m i n i d a s e f r o m C I . p e r f r i n g e n s w i t h o u t d e t e r g e n t . The a s i a l o g l y c o l i p i d was i n t u r n c l e a v e d b y a l t e r n a t e t r e a t m e n t w i t h 3-galactosidase and 3-hexosaminidase to y i e l d g l u c o s y l c e r a mide ( F i g . 5 ) . G a s - l i q u i d chromatographic a n a l y s i s of the p a r t i a l l y methylated h e x i t o l acetate d e r i v a t i v e s produced 2 , 4 , 6 tri-O-methyl-galactitol 1,3,5-tri-acetates; 2,3,6-tri-O-methylg l u c i t o l - l , 4 , 5 - t r i a c e t a t e s and 3 , 6 - d i - 0 - m e t h y l - 2 - d e o x y - 2 - N methyl-acetamidoglucitol-l,5-diacetate. There was no 2 , 3 , 4 , 6 tetra-O-methyl g a l a c t i c o l - 1 , 5-diacetate produced i n d i c a t i n g t h a t t h e s i a l i c a c i d i s a t t a c h e d a t the t e r m i n a l n o n r e d u c i n g end of t h e s a c c h a r i d e u n i t .


f

F a t t y acids and sphingosines. The l i p i d c o m p o s i t i o n of t h e f o u r m o n o s i a l o g a n g l i o s i d e s i s shown i n T a b l e I I . The major f a t t y a c i d s a r e p a l m i t i c , s t e a r i c and o l e i c a c i d s . The l o n g c h a i n base i s composed m a i n l y o f C - 1 8 s p h i n g o s i n e w i t h l e s s t h a n 20% o f d i h y d r o s p h i n g o s i n e . Discussion The s t r u c t u r e s o f t h e g l y c o s p h i n g o l i p i d s o f s k e l e t a l m u s c l e h a v e b e e n s t u d i e d i n human (8) a n d r a b b i t s k e l e t a l m u s c l e (7) and i n human c a r d i a c m u s c l e ( 9 ) . Q u a l i t a t i v e l y , human s k e l e t a l and c a r d i a c m u s c l e c o n t a i n t h e same n e u t r a l g l y c o s p h i n g o l i p i d s and g a n g l i o s i d e s though t h e g a n g l i o s i d e s o f r a b b i t s k e l e t a l muscle are q u i t e d i f f e r e n t . R a b b i t d o e s n o t c o n t a i n GM2 o r t h e g l u c o s a m i n e - c o n t a i n i n g g a n g l i o s i d e r e p o r t e d i n human s k e l e t a l muscle. We h a v e u s e d a D E A E - S e p h a d e x c o l u m n t o s e p a r a t e g a n g l i o s i d e s i n t o three groups, the mono-, d i - and p o l y s i a l o g a n g l i o s i d e s and t h i s e n a b l e d a more d e t a i l e d c o m p a r i s o n o f g a n g l i o s i d e s . Both h u m a n a n d c h i c k e n s k e l e t a l m u s c l e c o n t a i n GM3 a s t h e m a j o r g a n g lioside. The o t h e r m o n o s i a l o g a n g l i o s i d e s o f human m u s c l e a r e GM2 a n d GM1 b u t t h e s e t w o g a n g l i o s i d e s w e r e n o t d e t e c t e d i n c h i c k e n . I n s t e a d , g a n g l i o s i d e s c o n t a i n i n g glucosamine and a s i a losylpentaglycosylceramide constitute the other monosialoglycol i p i d s . C h i c k e n m u s c l e a l s o d i f f e r s f r o m human i n c o n t a i n i n g a n o v e l d i s i a l o g a n g l i o s i d e m i g r a t i n g between GDla and GDlb i n a l k a l i n e c o n d i t i o n s ( F i g . 3 B ) . The major n e u t r a l g l y c o s p h i n g o l i p i d of c h i c k e n muscle i s a Forssman-hapten p e n t a g l y c o s y l c e r a m i d e



CELL SURFACE

Figure 4.

GLYCOLIPIDS

Enzymatic hydrolysis of ganglioside II (lane 3 in Figure 3)

Lane 1, standard brain gangliosides. Lane 7, standard neutral glycolipids from bovine erythrocytes. Lane 2, ganglioside II. Lane 3, 2+ neuraminidase. Lane 4, 5+ f3-galactosidase. Lane 5, 4+ ^-hexosaminidase. Lane 6, 5+ j3-galactosidase.

Figure 5.

Enzymatic hydrolysis of ganglioside IV (lane 6 in Figure 3)

Lanes 1 and 9, ganglioside and neutral glycosphingolipid standards as in Figure 4. Lane 2, ganglioside IV. Lane 3, 2+ neuraminidase. Lane 4, 3+ j3-galactosidase. Lane 5, 4+ ^-hexosaminidase. Lane 6, 5+ fi-galactosidase. Lane 7, 6-f ft-hexosaminidase. Lane 8, 7+ /3-galactosidase.


Skeletal

CHIEN A N D HOGAN

Muscle

Table I I


FATTY ACIDS AND LONG CHAIN BASES OF CHICKEN MUSCLE MONOSIALOGANGLIOSIDES

Gangliosides Fatty Acids

I

II

III

IV

C 16:0

23.2

18.1

15.4

16.6

C 16:1

1.8

2.2

3.0

2.5

C 18:0

33.5

24.7

39.2

33.4

C 18:1

21.5

38.5

31.8

24.2

C 18:2

5.2

2.1

1.2

3.7

C 20:0

2.5

1.2

2.2

3.0

C 20:1

1.5

1.8

0.5

1.4

C 21:0

1.0

—

1.0

3.5

C 22:0

2.4

1.0

—

1.1

C 22:1

5.2

2.0

1.7

2.1

C 24:1

2.2

8.4

4.0

8.5

d 18:0

18.0

16.3

14.7

10.7

d 18:1

82.0

83.7

85.3

89.3

Sphingosines


CELL SURFACE


146

GLYCOLIPIDS

i d e n t i c a l to that p r e v i o u s l y described i n horse kidney and spleen (27), sheep e r y t h r o c y t e s , (28), and canine kidney and i n t e s t i n e (18). In c o n t r a s t , lactosylceramide predominates i n human muscle and c o n s t i t u t e s 38% of the t o t a l n e u t r a l g l y c o l i p i d s . Such s t r i k i n g d i f f e r e n c e s i n these compounds - both n e u t r a l and a c i d i c - suggests that species s p e c i f i c i t y p r e v a i l s over organ or t i s s u e c o n t r a i n t s upon the p a t t e r n or r a t i o of these l i p i d s . The use o f ion-exchange chromatography enabled the subsequent greater r e s o l u t i o n of i n d i v i d u a l gangliosides by s i l i c i c a c i d chromatography with B i o - S i l A and c h a r a c t e r i z a t i o n of the major monosialogangliosides i n chicken p e c t o r a l muscle by g l y c o s y l composition, enzymatic sequencing and methylation a n a l y s i s . The s t r u c t u r e s i d e n t i f i e d included GM3 (ganglioside I , NeuAca2-K3Gal$l->-4Glc-»Cer), s i a l o s y l - l a c t o - N - n e o t e t r a o s y l c e r a m i d e or I V aNeuAc-nLc0se4Cer (ganglioside I I , NeuAca2-*3Gal$l-*4GlcNAc 31->3Gal31->4Glc->Cer), s i a l o s y l lacto-N-neohexaglycosylceramide or Vl3 aNeuAc-nLc0se6Cer (ganglioside IV or NeuAca2->3Gal$l-* 4GlcNAc31->3Gal31->4GlcNAc31^3Gal31->4Glc->Cer), and a novel s i a l o sylpentaglycosylceramide (ganglioside I I I ) . The sequence of t h i s ganglioside has been determined by step-wise h y d r o l y s i s using s p e c i f i c glycosidases to be NeuAca-»Gal3- GalNAc3-*Gala->Gal 3Glc->Cer (V NeuAc, IV Gal-Gg0se4Cer) and i s to our knowledge the f i r s t g l y c o l i p i d o f the globo-series c o n t a i n i n g s i a l i c a c i d . There are evidences f o r i m p l i c a t i n g glycoconjugates on the muscle c e l l surface i n myogenesis (29, 30). Whatley e t a l , (29) for example found a t h r e e - f o l d increase i n GDla concentration j u s t p r i o r to f u s i o n i n a r a t myoblast c e l l l i n e while GM3, GM2 and GM1 d i d not change. McEvoy and E l l i s (30) found an increased b i o s y n t h e s i s o f s e v e r a l n e u t r a l g l y c o s p h i n g o l i p i d s and g a n g l i o s i d e s j u s t p r i o r to f u s i o n i n primary c u l t u r e s of chick embryo myoblasts. A r o l e f o r g l y c o l i p i d s i n such i n t e r c e l l u l a r regul a t i o n i s a l s o c o n s i s t e n t with the reported promotion of c e l l adhesion i n Hela c e l l s i n the presence of g l y c o l i p i d s p a r t i c u l a r l y those with tetraose chain length and a terminal galactose (5). I t would seem important to consider the s t r u c t u r e s of chicken muscle g l y c o s p h i n g o l i p i d s i n r e l a t i o n to the study of myogenesis i n view of the wide usage o f embryonic chick muscle as a model system. 3

>

Acknowledgment This work was supported i n part by the Muscular Dystrophy A s s o c i a t i o n . We thank Mrs. Eve Thrasher f o r s e c r e t a r i a l support and typing t h i s manuscript.


9. CHIEN AND HOGAN

Skeletal Muscle

147

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2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

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RECEIVED

December 10, 1979.


Glycosphingolipids of Skeletal Muscle - ACS Symposium Series (ACS

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