High Performance Liquid Chromatography of Membrane Glycolipids

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High Performance Liquid Chromatography of Membrane Glycolipids Assessment of Cerebrosides on the Surface of Myelin SHOJI YAHARA and YASUO KISHIMOTO The John F. Kennedy Institute and Department of Neurology, Johns Hopkins University School of Medicine, Baltimore,MD20205 JOSEPH PODUSLO Neuroimmunology Branch, National Institutes of Health, Bethesda, MD 20205 1

Carbohydrates occur on cells or plasma membranes primarily in the form of glycoproteins or glycolipids. Those accessible on the outer surface of the cell or membrane may have important biological functions as adhesion sites in terms of cell recognition, as receptors for hormones, toxins, or viruses, or as specific immunological determinants or antibody receptors. The presence of galactose or galactosamine as a terminal carbohydrate in these membrane surface glycolipids or glycoproteins has been determined by a procedure which utilizes the reaction with galactose oxidase (1). The enzyme converts the terminal primary alcohol group of these carbohydrates to an aldehyde. This aldehyde group can then be reduced by NaBH to the original alcoholic group in glycoproteins or glycolipids. By this series of reactions, part of the membrane galactolipids or galactoproteins are labeled with tritium (Chart 1). Since galactose oxidase is not permeable to membrane, the identification of H-labeled galactolipids or galactoproteins in extracts from cells or membranes has been considered acceptable evidence for locating these compounds on the surface of cells or membranes. This procedure has been useful for identifying a variety of carbohydrate-bearing macromolecules (in particular glycoproteins) on the surface of cell membranes (2, 3, 4). There are, however, two major disadvantages to studying glycolipids in this manner. First, many lipids other than galactolipids are also labeled by this procedure. The exact nature of the labeling has not been elucidated, but, at least some double bonds are reduced with tritium and some ester linkage is cleaved yielding radioactive saturated lipid and radioactive alkyl alcohols, respectively. The exchange of hydrogen with tritium may also be occurring. Pretreatment of cells or membranes with nonradioactive NaBH prior to galactose oxidase treatment helps to circumvent this problem to some extent but cannot make this procedure free from this complication. High levels of such non-specific reduction were observed Current address: Department of Neurology, Mayo Clinic, Mayo Foundation, Rochester, Minnesota 55901 3

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0-8412-0556-6/80/ 47-128-015S5.00/ 0 © 1980 American Chemical Society Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

C E L L SURFACE GLYCOLIPIDS

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when the i n t a c t myelin sheath p r e p a r a t i o n was t r e a t e d w i t h N a B ^ i n the absence of galactose oxidase (5j). A major r a d i o a c t i v e peak was observed near cerebroside a f t e r s e p a r a t i o n of the lower phase l i p i d s by TLC. Further e v a l u a t i o n of t h i s m a t e r i a l by hyd r o l y s i s showed no r a d i o a c t i v i t y i n g a l a c t o s e , f a t t y a c i d s or s p h i n g o s i n e . In a d d i t i o n , by v a r y i n g the s o l v e n t system, t h i s r a d i o a c t i v e peak could be separated from the c e r e b r o s i d e s . Cons e q u e n t l y , such n o n - s p e c i f i c r e d u c t i o n can e a s i l y r e s u l t i n e r r o neous i n t e r p r e t a t i o n of surface membrane c o n s t i t u e n t s . A second disadvantage of t h i s procedure i s t h a t i t does not have q u a n t i t a t i v e c a p a b i l i t i e s f o r determining surface g l y c o lipids. I t merely demonstrates whether a p o r t i o n o f a given g l y c o l i p i d i s on the surface but not the r a t i o o f surface l i p i d to i n a c c e s s i b l e l i p i d . C u s t o m a r i l y , a l a r g e amount of r a d i o a c t i v i t y i s used f o r such l a b e l i n g but o n l y a very small f r a c t i o n of i t i s incorporated i n t o the l i p i d . T h i s was p a r t i c u l a r l y the case f o r the nonhydroxycerebroside where the amount o f l a beled g a l a c t o s e observed a f t e r h y d r o l y s i s o f t h i s i s o l a t e d g l y c o l i p i d was o n l y a minor percentage o f the t o t a l l a b e l (5). Interp r e t a t i o n of such low l e v e l s of r a d i o a c t i v i t y may be u n r e l i a b l e f o r a s s e s s i n g surface membrane g l y c o l i p i d s , s i n c e i t may, i n f a c t , represent damage to the membrane b i l a y e r or s p l i t t i n g of the l a m e l l a r . We have r e c e n t l y developed a s e n s i t i v e and s p e c i f i c method f o r the q u a n t i t a t i v e and q u a l i t a t i v e determination of c e r e b r o s i d e s and s u l f a t i d e s using high performance l i q u i d chromatography (16, ]_). In order to q u a n t i t a t e cerebrosides on a s u r f a c e , we developed an a d d i t i o n a l new method t h a t s e p a r a t e l y compares the amount of surface cerebrosides w i t h the remaining cerebrosides by using high performance l i q u i d chromatography. T h i s method a l s o uses galactose o x i d a s e , but i n s t e a d o f reducing the aldehyde formed by N a B r U , the aldehyde i s converted to 2 , 4 - d i n i t r o p h e n y l hydrazone followed by p e r b e n z o y l a t i o n . The product produces separate peaks from t h a t of perbenzoylated c e r e b r o s i d e . Thus, the r a t i o of o x i d i z e d and unoxidized cerebrosides can be d i r e c t l y compared by high performance l i q u i d chromatography. In t h i s manuscript, we w i l l f i r s t d e s c r i b e the newly d e v e l oped high performance l i q u i d chromatography of c e r e b r o s i d e , s u l f a t i d e , and o t h e r minor g a l a c t o l i p i d s . T h i s method a l l o w s complete a n a l y s i s o f a very small amount of these g l y c o l i p i d s i n c e l l or membrane p r e p a r a t i o n s . T h i s w i l l be followed by a des c r i p t i o n of our new method of determining surface g a l a c t o l i p i d s and i t s a p p l i c a t i o n to myelin c e r e b r o s i d e s . 3

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Procedures M a t e r i a l s . Galactose oxidase was Biochemicals ( F r e e h o l d , N J ) . 1 was (Arlington Heights, I L ) . A l l solvents products of B u r d i c k - J a c k s o n (Muskegon, 125

purchased from Worthington obtained from Amersham ( g l a s s - d i s t i l l e d ) were the M I ) . P y r i d i n e was stored

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

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YAHARA ET A L .

Chromatography

of Membrane

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over KOH p e l l e t s and used without f u r t h e r p u r i f i c a t i o n . Benzoyl c h l o r i d e was obtained from A l d r i c h Chemicals (Milwaukee, WI) and 70% HClOu (double d i s t i l l e d from Vycor) from 6. F r e d e r i c k Smith Chemical (Columbus, OH). T r y p s i n was obtained from Worthington, w h i l e the t r y p s i n i n h i b i t o r (turkey egg w h i t e ) , phospholipase C ( C I . W e l c h i i type I ) , c a t a l a s e and phosphatidyl c h o l i n e (egg yolk) were a l l obtained from Sigma Chemical Co. ( S t . L o u i s , MO). T h i n l a y e r chromatographic p l a t e s precoated w i t h 0.25 mm t h i c k S i l i c a Gel GF were purchased from Anal tech (Newark, DE). M y e l i n was prepared e i t h e r from b r a i n s o f young Sprague-Dawley r a t s (Charles R i v e r CD, Charles R i v e r B r e e d i n g , Wilmington, MA) or from s p i n a l cords of a d u l t Osborn-Mendel r a t s ( V e t e r i n a r y Resources Branch at the National I n s t i t u t e s of Health) according to Norton and Poduslo ( 8 ) . Standard 6-dehydrocerebrosides were prepared by o x i d i z i n g with g a l a c t o s e oxidase according to Radin (9). Instrumentation. The HPLC equipment c o n s i s t e d of two Model 740 s o l v e n t d e l i v e r y systems combined w i t h a Model 744 s o l v e n t programmer, Model 714 pressure monitor and a Model 755 sample i n j e c t o r ( a l l from S p e c t r a - P h y s i c s , Santa C l a r a , CA). The column used was 25 cm x 3 mm i . d . s t a i n l e s s s t e e l tube packed w i t h either Spherisorb s i l i c a 5 y or Spherisorb ODS 5 y . Detection was made with a Schoeffel Instrument Corporation (Westwood, NJ) Model SF 770 spectromonitor. Peak areas were measured by the cut and weight method. R a d i o a c t i v i t y was measured by d i r e c t measurement i n a S e a r l e Model 1185 Automatic Gamma System. Determination o f c e r e b r o s i d e s , s u l f a t i d e s and o t h e r g a l a c t o 1 i p i d s i n myelin by HPLC. T o t a l l i p i d s were e x t r a c t e d w i t h chloroform/methanol ( 2 / 1 ) , washed according to Folch et al_. (10), and then subjected to b e n z o y l a t i o n - d e s u l f a t i o n as described p r e viously (6). The t o t a l l i p i d s were heated with 20 y l benzoyl c h l o r i d e and 100 y l p y r i d i n e and d e s u l f a t e d w i t h 0.2 M HCIO^ i n a c e t o n i t r i l e (prepared by mixing 0.17 ml 70% HCIO^ and 10 ml a c e t o n i t r i l e ) . With t h i s procedure, cerebrosides were converted to perbenzoyl d e r i v a t i v e s w h i l e s u l f a t i d e s were converted to p a r t i a l l y benzoylated cerebroside i n which the hydroxyl group a t g a l a c t o s e - 3 i s f r e e (Chart 2 ) . A p o r t i o n of the r e a c t i o n mixture d i s s o l v e d i n a known volume o f hexane was i n j e c t e d i n t o the HPLC equipped with Spherisorb s i l i c a 5 y column. The column was e l u t e d w i t h hexane/isopropanol ( 9 9 . 5 / 0 . 5 , v / v ) i s o c r a t i c a l l y f o r the f i r s t 3 min followed by g r a d i e n t e l u t i o n from 0.5 to 10% i s o p r o panol i n hexane i n 20 m i n . The flow r a t e was maintained a t 1.2 ml/min throughout. Peaks o f g l u c o c e r e b r o s i d e , nonhydroxycerebros i d e , hydroxycerebroside, monogalactosyl d i g l y c e r i d e , nonhydroxys u l f a t i d e , and h y d r o x y s u l f a t i d e were separated from each other under these c o n d i t i o n s , and c o n c e n t r a t i o n s o f these l i p i d s were determined from the peak s i z e . Peaks f o r minor nonpolar g a l a c t o 1 i p i d s , namely cerebroside e s t e r s and 1 - 0 - a l k y l isomers o f monogalactosyl d i g l y c e r i d e s overlap w i t h one of the above peaks.


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Products of Benzoylation-Desulfation CH2OBZ

H

Bz

0-CH2-C-N-CO-CH2-R' iBz

H

CH2OBZ H

C

H H

n

R

,

B

BzO-?^ H

z

CH2OBZ

NS

HS

H

R

Bz

BzQ/~C\p-CH2-C -N-CO-CH2 BzO-C^NR )Bz H CH2OBZ H H OBz BZO^°NX)-CH2-C-N-CO-CH-R'

OBz

BzO-C^

^

R

H

CH2OBZ

GC

OBz

Bz0y-0xJ)-CH2-C -N-C0-CH-R'

H

?z

^-CH -C-N-C0-CH2 2

B z O > ^ BzO-CV N ^ R OBz H CH2OBZ H GD Bz(^g^-CH2-C-0-C0-R' OBz

CH2-O-CO-R'

Bz= Benzoyl.

R

=

C 1 3 H 2 7

Rralkyl-

Chart 2


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YAHARA ET A L .

Chromatography

of Membrane

Glycolipids

19

E l u t i n g the column i s o c r a t i c a l l y w i t h hexane/tetrahydrofuran ( 9 0 . 2 5 / 9 . 7 5 , v / v ) provides s e p a r a t i o n of the above overlapped peaks (11). Determination o f homolog compositions of c e r e b r o s i d e s , s u l f a t i d e s , monogalactosyl d i g l y c e r i d e , and i t s 1 - 0 - a l k y l ether isomer by reverse phase HPLC. The above b e n z o y l a t i o n - d e s u l f a t i o n product i s placed on a TLC p l a t e coated with S i l i c a Gel GF. At l e a s t 1 nmol (approximately 1 yg) i s r e q u i r e d to o b t a i n s a t i s f a c t o r y r e s u l t s f o r each i n d i v i d u a l l i p i d . The p l a t e i s developed w i t h hexane/isopropanol ( 9 8 / 2 , v / v ) once or twice depending on the r e l a t i v e a c t i v i t y of the p l a t e . A f t e r the f i r s t development, the p l a t e was examined under UV l i g h t . I f each component i s s u f f i c i e n t l y separated, as shown i n F i g . 1, a second run i s not necessary. The spots were marked under the UV l i g h t a l l o w i n g 1/2 height of the spot on the top and bottom of each spot (or band) so t h a t any p a r t i c u l a r homolog was not s e l e c t i v e l y missed. The powder from the spot was scraped and mixed w i t h 0.5 ml of 95% e t h a n o l . The mixture was sonicated i n a sonic cleaner bath f o r 2 m i n . 1.5 ml Of ether was added to the mixture and then shaken v i g o r o u s l y f o r 30 min w i t h a W-8 Twist A c t i o n Shaker (New Brunswick Instrument, New Brunswick, N J ) . The mixture i s then c e n t r i f u g e d , and the supernatant i s evaporated to dryness. The residue i s d i s s o l v e d i n a known volume of cyclohexane, and a p o r t i o n i s i n j e c t e d to HPLC equipped w i t h Spherisorb ODS 5 y column. Although spots of perbenzoylated nonhydroxycerebroside, monogalactosyl d i g l y c e r i d e , and hydroxycerebroside, and p e r benzoyl ated-desul f a t e d nonhydroxy- and h y d r o x y s u l f a t i d e are w e l l separated from each o t h e r , the spot o f benzoylated d e r i v a t i v e of 1 - 0 - a l k y l etherisomer o f monogalactosyl d i g l y c e r i d e overlaps w i t h t h a t of benzoylated nonhydroxycerebroside. The amount of the 1 - a l k y l , 2 - a c y l , 3 - m o n o g a l a c t o s y l g l y c e r o l i s normally so small t h a t i t w i l l not i n t e r f e r e s i g n i f i c a n t l y with the a n a l y s i s of nonhydroxycerebroside. However, i f the a n a l y s i s of t h i s minor g l y c o l i p i d i s d e s i r e d , the m a t e r i a l e l u t e d from the band o f benzoylated nonhydroxycerebroside can be rechromatographed on another TLC system, such as the use of hexane/tetrahydrofuran on S i l i c a Gel GF p l a t e . These two benzoylated l i p i d s separate w e l l from each other under t h i s c o n d i t i o n . I f the examination of homolog composition of monogalactosyl d i g l y c e r i d e and i t s 1 - 0 - a l k y l ether isomers i s d e s i r e d , a l a r g e r amount of b r a i n sample i s r e q u i r e d , s i n c e t h e i r c o n c e n t r a t i o n i s much s m a l l e r than cerebrosides and s u l f a t i d e s . HPLC of g a l a c t o l i p i d s from a membrane t r e a t e d w i t h galactose o x i d a s e . The membrane t r e a t e d w i t h galactose oxidase i s extracted with chloroform/methanol as described above. The e x t r a c t cont a i n i n g up to 1 mg of t o t a l l i p i d s i s shaken w i t h a s o l u t i o n of 2 mg of d i n i t r o p h e n y l h y d r a z i n e HC1 i n 100 y l p y r i d i n e f o r 2 h at room temperature. The s o l v e n t i s evaporated to dryness under a


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n i t r o g e n f l o w , and the r e s i d u e i s f u r t h e r d r i e d i n an evacuated desiccator f o r 1 h r . To the d r i e d r e s i d u e , 30 y l of benzoyl c h l o r i d e and 150 y l of dry p y r i d i n e i s added, and the mixture i s heated at 60° f o r 1 h . The r e a c t i o n mixture i s evaporated to d r y ness under a n i t r o g e n f l o w , and the r e s i d u e i s f u r t h e r d r i e d i n an evacuated desiccator f o r 30 min. The residue i s d i s s o l v e d i n 2 ml hexane. The hexane s o l u t i o n i s washed once w i t h 1 ml o f 3% aqueous sodium carbonate followed twice by a c e t o n i t r i l e / w a t e r ( 4 / 1 , v / v ) and then evaporated to dryness. The residue i s d i s s o l v e d i n a known volume o f hexane and i n j e c t e d i n t o the HPLC system equipped with Spherisorb S i l i c a 5 y column. The column was f i r s t e l u t e d i s o c r a t i c a l l y f o r the f i r s t 5 min w i t h hexane/isopropanol ( 9 9 . 5 / 0 . 5 , v / v ) and then by i n c r e a s i n g l i n e a r l y the p r o p o r t i o n of isopropanol i n the next 20 min reaching the f i n a l c o n c e n t r a t i o n of hexane/isopropanol ( 9 6 / 4 , v / v ) . The flow r a t e was maintained a t 1.2 ml/min throughout. Two peaks due to perbenzoylated products o f 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e of o x i d a t i o n products from nonhydroxy- and hydroxycerebrosides appear a f t e r the peak of benzoylated hydroxycerebrosides under these c o n d i t i o n s . Treatment w i t h galactose o x i d a s e . O x i d a t i o n of m y e l i n with galactose oxidase was performed as described p r e v i o u s l y f o r s i m i l a r o x i d a t i o n of r a t s p i n a l cord preparations ( 4 ) . T y p i c a l l y , myelin c o n t a i n i n g 0.2-1.1 mg p r o t e i n i s incubated w i t h 100-500 u n i t s of galactose oxidase i n 1-3 ml of phosphate buffer (10-100 mM, pH 7 . 2 - 7 . 4 ) w i t h o r without c a t a l a s e . A f t e r the i n c u b a t i o n at room temperature to 30°C f o r the d u r a t i o n of 30 min to o v e r n i g h t , myelin i s recovered by c e n t r i f u g a t i o n , washed, and l y o p h i l i z e d . Total l i p i d s were e x t r a c t e d from the d r i e d r e s i d u e and the o x i d i z e d cerebroside as well as unaltered cerebrosides were analyzed as described above. A l t e r n a t i v e l y , the i n c u b a t i o n was stopped by the a d d i t i o n of 5 volumes of chloroform/methanol ( 2 / 1 , v / v ) and mixed. The lower l a y e r a f t e r c e n t r i f u g a t i o n of the mixture i s washed and then evaporated to d r y n e s s , and the t o t a l l i p i d s obt a i n e d were analyzed as described above. R a d i o i o d i n a t i o n of galactose o x i d a s e . The chloramine T procedure (12) was used f o r the r a d i o i o d i n a t i o n of galactose o x i d a s e . The enzyme, s o l u b i l i z e d i n 0.01 M sodium phosphate b u f f e r , pH 7 . 4 , was l a b e l e d using 1 mCi Na I (13-17 m C i / n g l ) , 0.42 mM chloramine T (Eastman), and 1.14 mM sodium m e t a b i s u l f i t e ( B a k e r ) . Unreacted i o d i d e was separated from the i o d i n a t i o n enzyme by d i a l y s i s , and the enzyme was d i l u t e d i n 0.1 M sodium phosphate b u f f e r , pH 7 . 4 , c o n t a i n i n g 0.001 M c u p r i c s u l f a t e . 1 2 5

P r e p a r a t i o n of liposomes. The method f o r the liposome prepa r a t i o n was a m o d i f i c a t i o n of C o s t a n t i n o - C e c c a r i n i , et a l ^ . , (13). A mixture o f 0.3 mg nonhydroxycerebroside, 0.2 mg of hydroxyc e r e b r o s i d e , and 5 mg o f egg y o l k l e c i t h i n was swollen i n 1 ml of


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Chromatography

of Membrane

Glycolipids

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a s o l u t i o n c o n t a i n i n g 130 mM KC1 and 10 mM T r i s - H C l pH 7.4 f o r 30 min. The tube was f l a s h e d w i t h n i t r o g e n and sonicated f o r 30 min. The sonicated mixture was c e n t r i f u g e d at 50,000 x£ f o r 15 min to remove s o l i d c e r e b r o s i d e s . The liposome s o l u t i o n obtained cont a i n e d 165 yg of nonhydroxycerebroside and 110 yg o f hydroxycerebroside i n 1 ml when measured by high performance l i q u i d chromatography. Results Myelin g a l a c t o l i p i d a n a l y s i s by HPLC. F i g . 2 and 3 show HPLC chromatograms obtained from myelin l i p i d s on S i l i c a column and reverse phase column, r e s p e c t i v e l y . Reverse phase HPLC of monog a l a c t o s y l d i g l y c e r i d e and i t s 1 - 0 - a l k y l ether homolog was not examined but t y p i c a l chromatograms of these l i p i d s obtained from c a l f b r a i n stem were presented p r e v i o u s l y (1J_). Myelin was obt a i n e d from 25 d a y - o l d r a t b r a i n s . O x i d a t i o n o f myelin surface cerebrosides by g a l a c t o s e oxidase. F i g . 4 shows s i l i c a HPLC of a mixture c o n t a i n i n g benzoylated-nonhydroxy and hydroxycerebroside and benzoylated d e r i v a t i v e s of 2 , 4 dinitrophenylhydrazone of o x i d a t i o n products from nonhydroxy- and hydroxycerebroside. Standard curves o f two 6 - d e h y d r o - d e r i v a t i v e s were shown i n F i g . 5. These standard curves demonstrate t h a t the response of the benzoylated dinitrophenylhydrazones are l i n e a r between 0.025 nmol and 0.6 nmol. Since cerebrosides c o n t a i n i n g 5 nmol can be determined without t a i l i n g to these peaks, t h i s method should a l l o w the determination of as l i t t l e as 0.5% of the o x i d a t i o n product. The f a c t t h a t each curve i n t e r s e c t s 0 p o i n t i n both the a b s c i s s a and o r d i n a t e i n d i c a t e s t h a t even s m a l l e r amounts of these compounds can be detected by t h i s technique. We obtained unexpected f i n d i n g s using t h i s method to study m y e l i n : the o x i d a t i o n by galactose oxidase of myelin-bound cerebrosides could not be d e t e c t e d . The o x i d a t i o n d i d not occur e i t h e r w i t h the i n t a c t s p i n a l cord p r e p a r a t i o n , w i t h i s o l a t e d m y e l i n , or even w i t h l y o p h i l i z e d m y e l i n . In one experiment, l y o p h i l i z e d myelin c o n t a i n i n g 5 mg each of dry weight was i n c u bated w i t h 100, 200, and 500 u n i t s of g a l a c t o s e oxidase f o r 60 min a t room temperature, and no cerebroside o x i d a t i o n o c c u r r e d . To examine whether the enzyme i s a c t i v e under the same c o n d i t i o n s , we coated 0.1 mg each o f nonhydroxy- and hydroxycerebrosides on 10 mg C e l i t e ( A n a l y t i c a l grade) and incubated i t w i t h 100 u n i t s of g a l a c t o s e oxidase f o r 60 min at room temperature. The r e s u l t i n d i c a t e d t h a t 5.6 nmol and 3.5 nmol each o f nonhydroxy and hydroxycerebrosides (approximately 4.6 and 3.0% each were o x i d i z e d . O x i d a t i o n of the same cerebrosides by the same galactose oxidase i n a t e t r a h y d r o f u r a n / w a t e r mixture as described by Radin {9) r e s u l t e d i n n e a r l y complete o x i d a t i o n . To f u r t h e r examine the i n a b i l i t y of galactose oxidase i n o x i d i z i n g myelin-bound c e r e b r o s i d e s , one mg each of l y o p h i l i z e d


CELL SURFACE GLYCOLIPIDS

22

Figure 1. TLC of myelin lipids after treatment with perbenzoylation-desulfation. Line S, standard; line M, derivatized myelin lipids. Spots 1 through 6 are benzoylated-desulfated derivatives of: (1) glucocerebroside, (2) nonhydroxycerebroside, (3) mono galactosyl diglyceride, (4) hydroxycerebroside, (5) nonhydroxysulfatide, and (6) hydroxysulfatide, respectively. See text for details of TLC conditions.

Figure 2. Silica HPLC of myelin lipids. NC, nonhydroxycerebroside; HC, hydroxycerebroside; NS, nonhydroxy sulfatide; HS, hydroxy sulfatide; and GD, monogalactosyl diglyceride. See text for details of TLC conditions.


2.

YAHARA ET A L .

Chromatography

of Membrane

Glycolipids

c

Figure 3. Reverse-phase HPLC of (A) perbenzoylated nonhydroxycerebroside, (B) hydroxycerebroside, (C) perbenzoylated-desulfated nonhydroxy sulfatide, and (D) hydroxy sulfatide. Each homolog peak was identified by fatty acid symbols, carbon numbers followed by number of double bonds.


23


0 Figure 4.

10

5

15

min

Silica HPLC of perbenzoylated derivative of dinitrophenylhydrazone 6-dehydrocerebrosides.

of

50 fig of nonhydroxycerebroside and 30 fig hydroxycerebroside is mixed with equal amounts of 6-dehydroderivatives of hydroxy- and nonhydroxycerebroside. These mixtures were subjected to dinitrophenylhydrazone-benzoylation as described above, and 1/20 of each reaction mixture was injected into silica-HPLC. NC, nonhydroxycerebroside; HC, hydroxycerebroside; NA, 6-dehydrononhydroxycerebroside; HA, 6-hydrohydroxycerebroside.

t

1

1

1

r

n mol Figure 5. Standard curve of perbenzoylated derivative of dinitrophenylhydrazone of 6-dehydrocerebrosides as analyzed by silica HPLC. Open and closed circles: derivatives from nonhydroxycerebroside and hydroxycerebroside, respectively.


2.

YAHARA ET A L .

Chromatography

of Membrane

Glycolipids

25

myelin was wetted w i t h 0.25 ml o f benzene o r benzene c o n t a i n i n g 167 nmols and 397 nmols each o f nonhydroxy and hydroxycerebrosides, r e s p e c t i v e l y . These were again l y o p h i l i z e d . Each d r i e d r e s i d u e was incubated with 150 u n i t s of g a l a c t o s e oxidase i n room temperature o v e r n i g h t . The r e s u l t s i n d i c a t e d t h a t the r e a c t i o n p r o d uct from the l y o p h i l i z e d myelin which was r e l y o p h i l i z e d w i t h benzene alone showed no d e t e c t a b l e o x i d a t i o n . On the other hand, the product of the same myelin p r e p a r a t i o n but "coated" with cerebrosides showed 12.5 nmol and 8.8 nmol o f nonhydroxy- and hydroxycerebroside o x i d i z e d by the enzyme r e a c t i o n , as shown i n F i g . 6 and F i g . 7. One p o s s i b l e e x p l a n a t i o n f o r the l a c k of o x i d a t i o n by g a l a c tose oxidase was thought to be s t e r i c hindrance. To i n v e s t i g a t e t h i s p o s s i b i l i t y , l y o p h i l i z e d m y e l i n c o n t a i n i n g 5.45 mg p r o t e i n i n 5 ml 0.1 M phosphate b u f f e r , pH 7 . 4 , was mixed with 0.5 ml of the same b u f f e r s o l u t i o n c o n t a i n i n g 1400 u n i t s of t r y p s i n and incubated f o r 1 h a t 37°C. 5 mg Of t r y p s i n i n h i b i t o r i n 1 ml of the same b u f f e r was added and the mixture was then incubated f o r 30 min at the same temperature. Galactose oxidase (942 u n i t s ) i n 1 ml of the same b u f f e r was next added to the above m i x t u r e , and the i n c u b a t i o n continued f o r 1 h at room temperature. This experiment, however, gave no evidence t h a t o x i d a t i o n by galactose o x i dase o c c u r r e d . In another experiment, 3.3 mg of l y o p h i l i z e d mye l i n was incubated w i t h 3.3 mg o f phospholipase C (6 units/mg) i n 10 ml of b u f f e r c o n t a i n i n g 10 mM T r i s - H C l pH 7 . 4 , 1 mM C a C l a t 37°C f o r 2 h (T4). The r e a c t i o n mixture was c e n t r i f u g e d at 44,000 x£ f o r 1 h and the p e l l e t s obtained were rehomogenized i n 1 ml o f 10 mM pH 7.2 phosphate b u f f e r . The homogenate was then incubated w i t h 200 u n i t s o f g a l a c t o s e oxidase at room temperature o v e r n i g h t . The i n c u b a t i o n product d i d not show any d e t e c t a b l e oxidation. The i n a b i l i t y of galactose oxidase to o x i d i z e myelin-bound cerebrosides may a l s o be due to the a b s o r p t i o n of the enzyme by m y e l i n . We examined t h i s p o s s i b i l i t y by l a b e l i n g g a l a c t o s e o x i dase w i t h I . In one experiment, f r e s h l y prepared myelin cont a i n i n g 2.09 mg p r o t e i n was incubated w i t h 3.77 u n i t s of Il a b e l e d g a l a c t o s e oxidase c o n t a i n i n g 226,500 cpm at room temperat u r e f o r v a r i o u s periods o f t i m e , and the mixture was c e n t r i f u g e d a t 16,000 rpm. The r a d i o a c t i v i t y i n the supernatant was counted by a y - c o u n t e r . In another experiment, the same amount o f myelin was incubated under i d e n t i c a l c o n d i t i o n s except t h a t 192.2 u n i t s of g a l a c t o s e oxidase c o n t a i n i n g the same amount o f r a d i o a c t i v i t y was used. The r e s u l t s of these experiments, shown i n Table 1, demonstrate t h a t the galactose oxidase indeed was bound to myelin. The binding appears to be s a t u r a t e d w i t h i n 5 min i n c u b a t i o n . With 3.77 u n i t s of g a l a c t o s e oxidase used, the average o f 1.67 u n i t s (44.3% of added enzyme) was bound to myelin c o n t a i n i n g 2.09 mg p r o t e i n . On the other hand, when 192.2 u n i t s of the enzyme was i n c u b a t e d , an average of 13.5% which i s 25.9 u n i t s , was bound to the same amount of m y e l i n . 2

1 2 5

1


2

5


26

Figure 6.

Silica HPLC of product from myelin, which was treated with benzene alone and lyophilized. See caption to Figure 4 for peak identification.

Figure 7. Silica HPLC of product from galactose oxidase-treated myelin which were "coated" by cerebrosides. See caption to Figure 4 for peak identification.


YAHARA ET A L .

Chromatography

of Membrane

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). About 20% of the t o t a l l i p i d c o n s i s t s of cerebroside and s u l f a t i d e . Because o f the l i p o p h i l i c nature of the ceramide moiety and the h y d r o p h i l i c nature of g a l a c t o s e , i t has been p o s t u l a t e d t h a t the galactose moiety of myelin c e r e b r o s i d e s i s f a c i n g the surface w h i l e the ceramide moiety i s buried w i t h i n the b i l a y e r . Even c o n s i d e r i n g the m u l t i l a m e l l a r s t r u c t u r e o f m y e l i n , a t l e a s t several percent of the cerebrosides should be present on the myelin s u r f a c e . The method described i n t h i s manuscript should a l l o w us to determine surface cerebrosides to as l i t t l e as 0.5% o f the t o t a l c e r e b r o s i d e s . Unexpectedly, we found t h a t myelin cerebrosides are not o x i d i z a b l e by g a l a c t o s e o x i d a s e , a t l e a s t not i n a d e t e c t a b l e degree. We have attempted to modify the myelin s t r u c t u r e so t h a t g a l a c t o s e oxidase would have a c c e s s i b i l i t y to the c e r e b r o s i d e s . These manipulations included l y o p h i l i z a t i o n , s o n i c a t i o n , hypotonic treatment, t r y p s i n d i g e s t i o n , and phospholipase C d i g e s t i o n . D i s r u p t i o n o f the myelin s t r u c t u r e u s i n g these treatments has been reported (17J. In f a c t , the e f f e c t of phospholipase C was obvious from the examination of l i p i d s by t h i n - l a y e r chromatography; n e a r l y a l l phosphatidyl c h o l i n e and ethanolamine were degraded. This i n a b i l i t y of g a l a c t o s e oxidase to o x i d i z e cerebrosides i s a d i r e c t c o n t r a d i c t i o n to a recent r e p o r t by L i n i n g t o n and Rumsby (18J. In t h e i r s t u d y , cerebrosides which were not o x i d i z e d by g a l a c t o s e oxidase were compared w i t h c h o l e s t e r o l by GLC. The cerebroside determination was made by measuring g a l a c t o s e a f t e r the m e t h a n o l y s i s ; o x i d i z e d cerebroside y i e l d e d 6-dehydrogalactose which was found unstable under methanolysis c o n d i t i o n s . By measuring the l o s s of g a l a c t o s e r e l a t i v e to the c h o l e s t e r o l c o n t e n t , they found t h a t approximately 36-50% of the cerebrosides i n myelin were attacked by g a l a c t o s e o x i d a s e . The reason f o r t h i s discrepancy between our present study and the f i n d i n g of L i n i n g t o n and Rumsby i s not c l e a r . Our enzyme was very a c t i v e . I t o x i d i z e d n e a r l y a l l cerebrosides when reacted i n a t e t r a h y d r o f u r a n / b u f f e r system. When cerebrosides were coated on c e l i t e or m y e l i n , galactose oxidase a t t a c k e d them. Two p o s s i b l e causes f o r the i n a b i l i t y of t h i s enzyme to o x i d i z e c e r e b r o s i d e i n i s o l a t e d myelin were c o n s i d e r e d . The f i r s t cause may be due to the absorption of galactose oxidase by myelin by e i t h e r i o n i c or hydrophobic i n t e r a c t i o n s . I f a p o r t i o n o f galactose oxidase i s hydrophobic, i t i s p o s s i b l e t h a t the enzyme can be incorporated i n t o the l i p i d m a t r i x . A c c o r d i n g l y , we l a b e l e d g a l a c t o s e oxidase with I and found that such a b s o r p t i o n was i n s i g n i f i c a n t 1 2 5


2.

Chromatography

YAHARA ET A L .

of Membrane

Glycolipids

31

compared to the t o t a l amount of enzyme present during the i n c u b a tion. The second cause may be due to s t e r i c hinderance of neighboring components w i t h i n the myelin sheath. However, we found t h a t d i g e s t i o n of t r y p s i n o r phospholipase C cannot a l l e v i a t e the problem. In a d d i t i o n , hypotonic treatment of myelin which a f f e c t s the i n t e g r i t y of the b i l a y e r and a l s o causes the s p l i t t i n g of the l a m e l l a e a t the e x t e r n a l a p p o s i t i o n (19, 2 0 ) , d i d not r e s u l t i n the o x i d a t i o n of the c e r e b r o s i d e s . Even cerebrosides i n liposomes made from pure phosphatidyl c h o l i n e could not be o x i d i z e d . I n c i d e n t a l l y , t h i s f i n d i n g a l s o c o n t r a d i c t s L i n i n g t o n and Rumsby who reported s i g n i f i c a n t o x i d a t i o n of cerebrosides i n liposomes made from myelin l i p i d s . At t h i s t i m e , our o n l y a l t e r n a t i v e e x p l a n a t i o n to our f i n d ings i s t h a t galactose oxidase may not be able to o x i d i z e the "bound" form of cerebrosides p o s s i b l y because of s i z e r e s t r i c t i o n s at the a c t i v e s i t e o f the enzyme. T h i s form i n c l u d e s cerebrosides i n membranes, lyposomes, or m i c e l l s . Matsubara and Hakomori a l s o r e c e n t l y determined the p r o p o r t i o n of 1actosylceramide and g l o b o s i d e l o c a t e d i n e r y t h r o c y t e surface membranes (T. Matsubura and S. Hakomori, personal communication). In t h i s s t u d y , they t r e a t e d e r y t h r o c y t e s w i t h galactose o x i d a s e , reduced i t by NaBDi* t r e a t ment, and measured the r a t i o of the deuterated l i p i d a g a i n s t undeuterated l i p i d w i t h mass spectrometry. They found somewhat more o x i d a t i o n ; 2-3% o f 1actosyl ceramide, and approximately 10% of globoside were l a b e l e d w i t h deuterium. T h e r e f o r e , i t i s l i k e l y t h a t the longer the saccharide chain to which galactose or galactosamine i s a t t a c h e d , the higher the r a t e of o x i d a t i o n t h a t can be achieved by galactose o x i d a s e . Although, as described above, L i n i n g t o n and Rumsby reported up to 50% of the o x i d a t i o n of myelin cerebrosides by g a l a c t o s e o x i d a s e , they a l s o reported very l i t t l e l a b e l i n g of myelin cerebrosides by the galactose oxidase — NaBH>4 method. They o x i d i z e d myelin (75 mg of dry w e i g h t ) , which presumably contained approximately 10 mg of cerebrosides i n 50 mg of t o t a l l i p i d s , w i t h 900 u n i t s of galactose oxidase and reduced i t w i t h 5 mCi of NaB Hi+. A f t e r 5 hrs of i n c u b a t i o n , they obtained 2,261,450 dpm (approximately 1 y C i ) of H i n c e r e b r o s i d e s . Although the s p e c i f i c a c t i v i t y of N a B ^ used was not g i v e n , the cerebrosides l a b e l e d could be about 0 . 1 - 0 . 2 y g , assuming the s p e c i f i c a c t i v i t y was i n the range o f 5-15 Ci/mmol as reported by Poduslo et a l _ . , (4j and a l s o assuming t h a t t h i s r a d i o a c t i v i t y represents s p e c i f i c l a b e l ing of the g a l a c t o s e moiety. T h i s amount of c e r e b r o s i d e , t h e r e f o r e , represents o n l y 0.001-0.002% o f the t o t a l c e r e b r o s i d e . A number of s i m i l a r s t u d i e s on c e l l surface g a l a c t o l i p i d s have been based on t h i s g a l a c t o s e oxidase-NaB Hn r e d u c t i o n procedure. However, i t i s now apparent t h a t o n l y a very small p o r t i o n , l e s s than 0.5% i f any, of the cerebrosides i n membranes are o x i d i z a b l e by galactose o x i d a s e . T h e r e f o r e , cerebrosides and p o s s i b l y o t h e r g a l a c t o l i p i d s p r e v i o u s l y i d e n t i f i e d by the surface l a b e l i n g 3

3

3

3


32


technique apparently represent only a small portion of the total surface galactolipids, and the results of such studies should be interpreted with caution. Abstract An HPLC method is described which determines the quantity and elucidates the homolog composition of cerebrosides and sulfatides in small tissue samples. Total lipids from the tissue were subjected to benzoylation-desulfation, and the product was analyzed quantitatively by silica HPLC. Another aliquot of the product was further fractionated by TLC. Spots of benzoylated cerebrosides and desulfated sulfatides were analyzed by reverse phase HPLC for the homolog compositions of these sphingolipids. Less than 1 mg of fresh brain or nerve tissue is sufficient for complete analysis. A new procedure has been developed which assesses the topographical distribution of cerebrosides in biological membranes. This method involves the treatment of cells or membrane fractions with galactose oxidase followed by extraction of the total lipids with chloroform-methanol. The lipids were then reacted with 2,4dinitrophenylhydrazine HCl in pyridine, and the reaction products were benzoylated and analyzed by silica HPLC. The cerebrosides which are oxidized by the enzyme resulted in perbenzoylated derivatives of 6-dehydrocerebrosides which yielded separate peaks behind the unoxidized perbenzoylated cerebrosides. Consequently this procedure would distinguish surface membrane cerebrosides from the unreactive "inaccessible" cerebrosides. This technique was applied to myelin from the central nervous system, and unexpectedly, myelin cerebrosides were found unoxidizable by galactose oxidase. Modifications of myelin, such as lyophilization, hypotonic treatment, trypsin digestion, and phospholipase C digestion, were not effective in exposing myelin-bound cerebrosides. Moreover, we also found that cerebrosides bound to brain microsomes, cytosol, or even in liposomes with lecithin were not oxidized by the enzyme. On the other hand, cerebrosides coated on Celite or myelin were oxidized by the enzyme. These results suggest that cerebrosides bound in a bilayer structure may not be available for oxidation by galactose oxidase. Acknowledgement This study was supported in part by Research Grants NS-13559, NS-13569 and HD-10891 from the National Institutes of Health, United States Public Health Service. Literature Cited 1.

Steck, T.L., "Membrane Research"; Academic Press, New York, 1972; pp. 71-93.


2. YAHARA ET AL. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Chromatography of Membrane Glycolipids 33

Steck, T.L. and Dawson, G. J. Biol. Chem. 1974, 249, 21352142. Gahmberg, C.G. and Hakomori, S. J. Biol. Chem. 1973, 248, 4311-4317. Poduslo, J.F.; Quarles, R.H. and Brady, R.O. J. Biol. Chem. 1976, 251, 153-158. Poduslo, J.F. Adv. Exp. Med. Biol. 1978, 100, 189-205. Nonaka, G. and Kishimoto, Y. Biochim. Biophys. Acta 1979, 572, 423-431. Yahara, S.; Moser, H.W.; Kolodny, E.H. and Kishimoto, Y. J. Neurochem. in press. Norton, W.T. and Poduslo, S.E. J. Neurochem. 1973, 21, 749757. Radin, N.S. Methods Enzymol. 1972, 28, 300-304. Folch, J.; Lees, M. and Sloane-Stanley, G.H. J. Biol. Chem. 1957, 226, 497-509. Yahara, S. and Kishimoto, Y. manuscript in preparation. Greenwood, F.C.; Hunter, W.M. and Glover, J.S. Biochem. J. 1963, 89, 114-123. Cestelli, A.; White, F.V. and Costantino-Ceccarini, E. Biochim. Biophys. Acta 1979, 572, 283-292. Feinstein, M.B. and Felsenfeld, H. Biochemistry 1975, 14, 3041-3048. Kawamura, N.; Yahara, S.; Kishimoto, Y. and Toutelotte, W.W. manuscript in preparation. Norton, W.T. and Poduslo, S.E. J. Neurochem. 1973, 21, 759773. Smith, M.E. and Benjamins, J.A. "Myelin"; Plenum Press, New York, 1977, pp. 447-488. Linington, C. and Rumsby, M.G. Adv. Exp. Med. Biol. 1978, 100, 263-273. Finean, J.B. and Bunge, R.E. J. Mol. Biol. 1963, 1, 672-682. McIntosh, T.J. and Robertson, J.D. J. Mol. Biol. 1976, 100, 213-217.

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December 10, 1979.


High Performance Liquid Chromatography of Membrane Glycolipids

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