Cell Surface Glycolipids - American Chemical Society

7 Galactoglycerolipids of Mammalian Testis, Spermatozoa, and Nervous Tissue

Downloaded by RUTGERS UNIV on December 30, 2017 | http://pubs.acs.org Publication Date: July 31, 1980 | doi: 10.1021/bk-1980-0128.ch007

ROBERT K. MURRAY, RAJAGOPOLIAN NARASIMHAN, MARK LEVINE, and LES PINTERIC Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8 MARGARET SHIRLEY, CLIFFORD LINGWOOD, and HARRY SCHACTER Department of Biochemistry, Hospital for Sick Children, Research Institute, Toronto, Ontario, Canada M5G 1X8 The two major classes of glycolipids present in mammalian cells are glycosphingolipids and glycoglycerolipids (1). It is with certain members of the latter class that this article is concerned. Glycoglycerolipids are well established constituents of plant and bacterial cells (2,3,4). Galactosyl- and digalactosyldiacylglycerols are the major glycoglycerolipids found in plant cells, although trigalactosyldiacylglycerol, 6-O-acylgalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol have also been described (4). In bacteria, mono- and di- glycosyldiacylglycerols occur most frequently, with the latter generally being the major species. Glucose, galactose and mannose are the usual sugars present in these compounds. Certain of these lipids also contain uronic acids. Halobacterium cutirubrum contains a glycolipid with galactose-sulfate, mannose and glucose linked to a phytanyl diether glyceride (5). Acyl substitutions on the sugar residues of diglycosylglycerolipids have also been described, as have phosphoglycoglycerolipids (4). The presence of glycoglycerolipids in mammalian tissues, specifically nervous tissue, has been known since 1963 (6). Most of the mammalian glycoglycerolipids have been found to contain galactose as their sole sugar; however, the presence in gastric juice and saliva of a novel series of glucoglycerolipids has been described recently (7,8,9). Of the galactoglycerolipids, galactosyl- and digalactosyldiacylglycerols have received especial attention. An analog of galactosyldiacylglycerol, galactosylalkylacylglycerol, was also found in brain (10) shortly after the initial report of the presence of the diacyl compound in that organ (6). Interest in mammalian galactoglycerolipids accelerated when i t was discovered that the sulfated derivative of the lipid described by Norton and Brotz (10) was the major glycolipid of rat (11) and boar (12) testis. This sulfated galactolipid was partially characterized in a number of studies (e.g. 13 and 14) and has subsequently been fully characterized as l-O-alkyl-2-0acyl-3-0-$-D-(3'-sulfo)-galactopyranosyl-sn-glycerol (15). A variety of topics emerging from the study of this particular glycolipid have been reviewed previously (16). The present article will 0-8412-0556-6/ 80/ 47-128-10555.25/ 0 © 1980 American Chemical Society Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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C E L L SURFACE GLYCOLIPIDS

concentrate p r i m a r i l y on features concerning t h i s and s e v e r a l c l o s e l y r e l a t e d g a l a c t o g l y c e r o l i p i d s that have a r i s e n s i n c e the above mentioned review was w r i t t e n i n mid 1975. Many aspects of the biochemistry of the v a r i o u s s u l f a t e - c o n t a i n i n g g l y c o l i p i d s found i n mammalian t i s s u e s have r e c e n t l y been reviewed by Sweeley and S i d d i q u i (1), Dulaney and Moser (17) and Farooqui (18). Nomenclature, C l a s s i f i c a t i o n and

Tissue D i s t r i b u t i o n


,

Both l - 0 - a l k y l - 2 - 0 - a c y l - 3 - 0 - g - D - ( 3 - s u l f o ) - g a l a c t o p y r a n o s y l s n - g l y c e r o l and i t s non-sulfated species are major g l y c o l i p i d s of the t e s t i s and spermatozoa of a number of higher animals, i n c l u d ing humans (16). Despite the previous usage of names such as s e m i n o l i p i d (12), s u l f o g l y c e r o g a l a c t o l i p i d (19) and s u l f o g a l a c t o g l y c e r o l i p i d (20) to describe the s u l f a t e d species, i t now appears that s u l f o g a l a c t o s y l a l k y l a c y l g l y c e r o l i s the most chemically informative t r i v i a l name with which to r e f e r to t h i s compound. This a r i s e s from the f a c t that a c l o s e l y r e l a t e d compound, almost certainly l-0-acyl-2-0-acyl-3-0-3-D-(3 -sulfo)-galactopyranosyls n - g l y c e r o l , has been i s o l a t e d from b r a i n (21,22). The term s u l f o g a l a c t o g l y c e r o l i p i d would not d i s t i n g u i s h between these two compounds, p a r t i c u l a r l y when r e f e r r i n g to an organ such as r a t brain, i n which they c o - e x i s t (22,23). Hence, i t i s more p r e c i s e to r e f e r to the ether-containing l i p i d as s u l f o g a l a c t o s y l a l k y l a c y l g l y c e r o l (SGG) and to the d i a c y l - c o n t a i n i n g l i p i d as s u l f o g a l a c t o s y l d i a c y l g l y c e r o l (22). The non-sulfated species of these two l i p i d s w i l l be r e f e r r e d to as g a l a c t o s y l a l k y l a c y l g l y c e r o l (GG) and galactosyldiacylglycerol respectively. f

A c l a s s i f i c a t i o n of mammalian g a l a c t o g l y c e r o l i p i d s below. Table I. C l a s s i f i c a t i o n of Mammalian D i a c y l Sub-Class (A) G a l a c t o s y l d i a c y l g l y c e r o l (B) S u l f o g a l a c t o s y l d i a c y l g l y c e r o l (C) D i g a l a c t o s y l d i a c y l g l y c e r o l

i s given

Galactoglycerolipids A l k y l a c y l Sub-Class

(D)

Galactosylalkylacylg l y c e r o l (GG) (E) S u l f o g a l a c t o s y l a l k y l a c y l glycerol (SGG) (F) D i g a l a c t o s y l a l k y l a c y l glycerol

Several features of t h i s c l a s s i f i c a t i o n merit comment. Six l i p i d s have been included i n the Table, but the i d e n t i f i c a t i o n of two of them [(C) and (F)] i s not f i r m l y e s t a b l i s h e d . L i p i d (C) was t e n t a t i v e l y i d e n t i f i e d i n human b r a i n (24); e x t r a c t s of r a t b r a i n appear to be able to c a t a l y s e i t s formation when incubated under appropriate c o n d i t i o n s (25) ( t h i s i s discussed i n more det a i l subsequently). L i p i d (F) was detected i n human t e s t i s and sperm (26), and e x h i b i t e d chromatographic and other p r o p e r t i e s corresponding to what would be expected from a d i g a l a c t o s y l -

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


7.

MURRAY ET A L .

Galactoglycerolip

ids

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containing a l k y l a c y l g l y c e r o l . The systematic names f o r l i p i d s (A) and (C) are l-0-acyl-2-0-acyl-3-0-$-D-galactopyranosyl-sn-glycero l and l-0-acyl-2-0-acy1-3-0-[a-D-galactopyranosyl(l-*?)B-D-galact o p y r a n o s y l ] - s n - g l y c e r o l ; these l i p i d s are s t i l l widely r e f e r r e d to as monogalactosyl and d i g a l a c t o s y l d i g l y c e r i d e r e s p e c t i v e l y . Systematic names f o r l i p i d s (B), (D) and (E) were i n d i c a t e d above. I t i s premature to assign a systematic name to l i p i d ( F ) ; i t w i l l be of i n t e r e s t to determine whether the anomeric natures of the two g a l a c t o s y l residues are s i m i l a r to those i n l i p i d (C). With regard t o t h e i r d i s t r i b u t i o n i n mammalian t i s s u e s , compounds (A), (B) and (C) have been detected only i n nervous t i s s u e , compounds (D) and (E) i n both nervous t i s s u e and t e s t i s and spermatozoa, and compound (F) only i n human t e s t i s and spermatozoa. However, p r e l i m i n a r y evidence has been obtained (M. Levine and R.K. Murray, unpublished observations), suggesting that small amounts of compounds (A) and (B) may be present i n dog t e s t i s along with l a r g e r amounts of compounds (D) and (E). E x t r a c t i o n of

Galactoglycerolipids

We have found the method of Suzuki (27) to be s a t i s f a c t o r y f o r e x t r a c t i n g these l i p i d s from t e s t i s , sperm and b r a i n . A moderate l o s s of s u l f a t e - c o n t a i n i n g g a l a c t o g l y c e r o l i p i d s i n t o the upper phase of t h e F o l c h e x t r a c t employed i n t h i s method occurs. Using the method of column chromatography on s i l i c i c a c i d d e v e l oped by Vance and Sweeley (28), the g a l a c t o g l y c e r o l i p i d s shown i n Table I are a l l e l u t e d by acetone subsequent to i n i t i a l e l u t i o n of the column by chloroform. A f t e r evaporation of the acetone, i n d i v i d u a l g l y c o l i p i d s can be p u r i f i e d by p r e p a r a t i v e t h i n l a y e r chromatography. I f the g l y c o l i p i d composition of the t i s s u e under study i s complex ( c f . human t e s t i s (26)), f r a c t i o n a t i o n of these l i p i d s by chromatography using DEAE-cellulose (29) i s u s e f u l . Chemical C h a r a c t e r i z a t i o n

of G a l a c t o s y l a l k y l a c y l g l y c e r o l s

Table I I l i s t s the main procedures that have been used to q u a n t i t a t e the amounts of these l i p i d s present i n t e s t i s , sperm and b r a i n and to determine t h e i r chemical s t r u c t u r e s . Reference to some of the techniques a p p l i e d to the c h a r a c t e r i z a t i o n of s u l f o g a l a c t o s y l d i a c y l g l y c e r o l are a l s o included. One technique that we have found u s e f u l i n p e r m i t t i n g an i n i t i a l d i s t i n c t i o n between g l y c o s p h i n g o l i p i d s , g a l a c t o s y l a l k y l a c y l g l y c e r o l s and g a l a c t o s y l d i a c y l g l y c e r o l s i s the use of b r i e f h y d r o l y s i s i n m i l d a l k a l i ( c f . 20). T h i s can be a p p l i e d to e i t h e r the t o t a l g l y c o l i p i d e x t r a c t or to p u r i f i e d g l y c o l i p i d s . T y p i c a l r e s u l t s of t h i s procedure using a member of each of the above three c l a s s e s of g l y c o l i p i d s are shown i n F i g u r e 1. I t should be apparent that the use of t h i s treatment to remove a l k a l i - l a b i l e contaminating l i p i d s (e.g. phospholipids) from a g l y c o l i p i d ext r a c t i s unwise, u n t i l a f t e r a p r e l i m i n a r y a n a l y s i s has been


108


Table I I . Procedures Used to Quantitate and C h a r a c t e r i z e Galactoglycerolipids Procedure


General Analyses: Determination of sugar, g l y c e r y l ethers and f a t t y a c i d s by GLC S i m i l a r analyses by GLC-MS Elemental a n a l y s i s Q u a n t i t a t i o n by HPLC Analyses of the S u l f a t e Moiety: Detection of [35s] s u l f a t e Benzidine method Sodium r h o d i z i n a t e method Estimation of l i p i d - b o u n d sulfate IR spectroscopy Removal of s u l f a t e by hydrol y s i s i n mild acid Removal of s u l f a t e by s o l v o l y s i s i n dioxane Removal of s u l f a t e by a r y l sulfatase A Elution i n salts fraction during DEAE-cellulose chromatography Permethylation * Determination of attachment to galactose by r e s i s t a n c e to treatment with p e r i o d a t e Analyses of the Galactose Moieties: Determination of anomeric l i n k age by IR and NMR Determination of anomeric l i n k age by use o f (S-galactosidase E s t i m a t i o n of amount u s i n g galactose dehydrogenase Estimation of amount u s i n g fluorimetry Analyses of G l y c e r y l E t h e r s : Determination of isomers by TLC Stereochemical a n a l y s i s by optical rotatory dispersion

Compound Studied

Reference

Rat t e s t i s SGG Boar t e s t i s SGG

(11) (12)

Human t e s t i s SGG Rat b r a i n SGG Boar t e s t i s SGG Rat t e s t i s SGG

(15) (23) (12) (30)

Rat t e s t i s SGG Rat t e s t i s SGG Boar t e s t i s SGG Rat t e s t i s SGG

(11) (11) (12) (11)

Boar t e s t i s SGG Rat t e s t i s SGG

(12) (11)

Rat t e s t i s SGG

(19)

Rat t e s t i s SGG

(31)

Rat b r a i n SGG

(20)

Boar t e s t i s SGG Rat b r a i n s u l f o galactosyldiacylglycerol

(12) (21)

Boar t e s t i s SGG

(12)

Rat b r a i n SGG

(23)

Guinea p i g t e s t i s SGG

(13)

Human t e s t i s SGG

(15)

Rat t e s t i s SGG

(14)

Human t e s t i s SGG

(15)


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Galactoglycero lip ids


Table I I (continued) Procedure

Compound Studied

S u s c e p t i b i l i t y to d e - a c y l a t i o n by m i l d a l k a l i Other A n a l y s i s : I s o l a t i o n of g a l a c t o s y l glycerol

Guinea p i g t e s t i s SGG Rat b r a i n SGG

(13) (20)

Rat b r a i n s u l f o galactosyldiacyl glycerol

(21)

Boar t e s t i s SGG Rat t e s t i s SGG Rat t e s t i s SGG

(12) (11) (11)

Boar t e s t i s SGG

(12)

Rat t e s t i s SGG

(11)

Use of Spray Reagents to Exclude Other C o n s t i t u e n t s : Ninhydrin (free amino group) Benzidine (sphingosine) 2,4-dinitrophenylhydrazine (plasmalogenic linkage) B i a l ' s o r c i n o l reagent ( s i a l i c acid) A c i d molybdate (phosphate)

Reference

*This procedure n a t u r a l l y a l s o y i e l d s information on the nature of the galactose m o i e t i e s . The methods r e f e r r e d to i n t h i s Table have been used to q u a n t i tate and c h a r a c t e r i z e the t e s t i c u l a r SGG and GG species and a l s o the corresponding l i p i d s and s u l f o g a l a c t o s y l d i a c y l g l y c e r o l from b r a i n . References to s t u d i e s performed p r i o r to 1972 that c h a r a c t e r i z e d the g a l a c t o g l y c e r o l i p i d s of nervous t i s s u e have not been i n c l u d e d . Abbreviations: GLC, g a s - l i q u i d chromatography; MS, mass spectrometry; HPLC, high performance l i q u i d chromatography; IR, i n f r a - r e d ; DEAE, d i e t h y l a m i n o e t h y l ; NMR, nuclear magnetic resonance; TLC, t h i n l a y e r chromatography.


C E L L SURFACE

GLYCOLIPIDS


FR.

OR.

1

2 3 4

5

6

Figure 1. Schematic of the effects of brief treatment with mild alkali on the thinlayer chromatographic migrations of three types of glycolipids. 1,2: control and alkali-treated neutral glycosphingolipid; 3, 4: control and alkali-treated galactosylalky lacy Iglycerol; 5, 6: control and alkali-treated galactosyldiacylglycerol. OR: origin; FR: solvent front. The neutral glycosphingolipid is represented as a characteristic double band. We have not observed galactoglycerolipids to migrate as double bands on thin layer chromatography. Glucosyl- and lactosyl-ceramides exhibit the behavior (i.e., lack of effect of mild alkali on their migrations) of the compound shown in channels 1 and 2. SGG and GG behave in the same way as the compound represented in channels 3 and 4; the slower migrating product in channel 4 in the case of these two compounds would correspond to lyso-SGG and lyso-GG, respectively. Both galactosyl- and digalactosyldiacylgycerols show the behavior of the compound in channels 5 and 6; the product migrating at the origin in channel 6 in the case of these two compounds would correspond to galactosyIglycerol and digalactosylglycerol, respectively. Cerebroside esters are one type of glycosphingolipid whose migration would be affected by the above treatment. Conversely, if galactosyIdialkyglycerols exist in mammalian ceils, their chromatographic migrations would not be affected by treatment with mild alkali.


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111

performed on c o n t r o l and a l k a l i - t r e a t e d samples to determine i f the chromatographic migrations of any of the g l y c o l i p i d s present are a f f e c t e d by i t . The i n i t i a l c h a r a c t e r i z a t i o n s t u d i e s of the SGG derived from r a t (11) and boar (12) t e s t i s revealed the presence of approximately s t o i c h i o m e t r i c amounts of s u l f a t e , galactose, f a t t y a c i d and g l y c e r y l ether. Using NMR spectroscopy, the study of Ishizuka et a l . (12) a l s o suggested the 3 nature of the g a l a c t o s i d i c l i n k age to g l y c e r o l and the p o s i t i o n of the a c y l chain on carbon 2 of g l y c e r o l . In a d d i t i o n , analyses of the products of permethylation i n d i c a t e d that the s u l f a t e was attached to the 3 p o s i t i o n of gal a c t o s e . Measurement of the o p t i c a l r o t a t o r y d i s p e r s i o n of the g l y c e r y l ether moiety (15) e s t a b l i s h e d the d e f i n i t i v e s t r u c t u r e of SGG. Perhaps the most remarkable f e a t u r e of the SGG derived from t e s t i s i s i t s extremely r e s t r i c t e d a l k y l and a c y l composition. In the case of the SGG of r a t (11,14), boar (12), guinea p i g (13) and human (15,26) t e s t i s , over 80% of the a l k y l and a c y l composit i o n i s comprised of saturated 16 carbon moieties [ g l y c e r y l - 1 hexadecyl ether (chimyl a l c o h o l ) and hexadecanoic a c i d ( p a l m i t i c acid) r e s p e c t i v e l y ] . The SGG present i n r a t b r a i n appears to have a l e s s r e s t r i c t e d a l k y l and a c y l composition (20).


1

Biosynthesis

of T e s t i c u l a r and

Other G a l a c t o g l y c e r o l i p i d s

35 A number of s t u d i e s (11,13,14,32) have shown that [ S] s u l f a t e i s incorporated i n v i v o i n t o t e s t i c u l a r SGG. With regard to the mechanism i n v o l v e d , both Knapp et_ a l . (19) and Handa et a l . (32) have demonstrated formation of t h i s l i p i d i n v i t r o from GG by t r a n s f e r of s u l f a t e from 3 -phosphoadenosine-5'-phosphosulfate (PAPS), i n analogy with the pathway of b i o s y n t h e s i s of s u l f o g a l a c tosylceramide from galactosylceramide (reviewed i n 17). Other g l y c o l i p i d s with a terminal 3 - g a l a c t o s y l residue ( g a l a c t o s y l - and l a c t o s y l - ceramides and g a l a c t o s y l d i a c y l g l y c e r o l ) were found to be s u l f a t e d by the enzyme preparations employed, whereas compounds with a terminal ot-galactosyl residue ( g a l a c t o s y l g a l a c t o s y l g l u c o sylceramide and d i g a l a c t o s y l d i a c y l g l y c e r o l ) were not. Both of these s t u d i e s suggested that p r i m a r i l y one s u l f o t r a n s f e r a s e was involved i n the s u l f a t i o n of the v a r i o u s g l y c o l i p i d substrates; however, t h i s i s s u e i s not s e t t l e d c o n c l u s i v e l y . The sulf©transferase a c t i v i t y i n r a t t e s t i s (19) was markedly enriched i n a G o l g i apparatus f r a c t i o n of that organ, confirming the i n v o l v e ment of that o r g a n e l l e i n both s u l f a t i o n processes (33) and i n the b i o s y n t h e s i s of g l y c o l i p i d s (34,35). W e l l before the above s t u d i e s were performed, the biosynthes i s of g a l a c t o s y l d i a c y l g l y c e r o l i n r a t b r a i n had been examined by Wenger et_ a l . (36). These workers found a 3 - g a l a c t o s y l t r a n s f e r a s e a c t i v i t y capable of c a t a l y s i n g the f o l l o w i n g r e a c t i o n : f

1 , 2 - d i a c y l g l y c e r o l + UDP-gal •> G a l a c t o s y l d i a c y l g l y c e r o l +


UDP

112

CELL SURFACE GLYCOLIPIDS

Wenger £t a l . (25) l a t e r described the presence i n r a t b r a i n of an a - g a l a c t o s y l t r a n s f e r a s e a c t i v i t y , that used the product of the above r e a c t i o n as substrate and UDP-gal as donor to c a t a l y s e the formation of a second l i p i d , t e n t a t i v e l y assigned the s t r u c t u r e of d i g a l a c t o s y l d i a c y l g l y c e r o l (see e a r l i e r d i s c u s s i o n ) . Subsequently, Flynn et^ a l . (21) demonstrated the presence i n r a t b r a i n of a s u l f o t r a n s f e r a s e a c t i v i t y capable of s u l f a t i n g g a l a c t o s y l d i a c y l g l y c e r o l . The p r o p e r t i e s of t h i s enzyme a c t i v i t y were d e s c r i b ed i n more d e t a i l by Subba Rao et a l . (37). I n t e r e s t i n g l y , s i g n i f i c a n t d i f f e r e n c e s were observed between the formation of s u l f o g a l a c t o s y l d i a c y l g l y c e r o l and s u l f o g a l a c t o s y l c e r a m i d e , when c a t a l ysed by the enzyme preparation used. The data d i d not n e c e s s a r i l y lead to the conclusion that two sulf©transferases were present, but they d i d i n d i c a t e how c e r t a i n f a c t o r s (e.g. ATP and M g conc e n t r a t i o n s ) could c o n t r o l the r e l a t i v e amounts of these two l i p i d s that were synthesized. In analogy with the r e a c t i o n shown f o r formation of g a l a c t o s y l d i a c y l g l y c e r o l i n b r a i n , Levine e_t a l . (38) have examined the a b i l i t y of r a t t e s t i c u l a r e x t r a c t s to c a t a l y s e the f o l l o w i n g r e action:


2 +

1 , 2 - a l k y l a c y l g l y c e r o l + UDP-gal •> GG +

UDP

So f a r , although a v a r i e t y of c o n d i t i o n s of incubation have been i n v e s t i g a t e d , convincing evidence f o r the occurrence of t h i s r e a c t i o n i n r a t t e s t i s has not been obtained. The s i g n i f i c a n c e of a negative f i n d i n g of t h i s nature i s l i m i t e d , as i t may only r e f l e c t a f a i l u r e to s e l e c t appropriate c o n d i t i o n s . A l t e r n a t i v e l y , the p u t a t i v e g a l a c t o s y l t r a n s f e r a s e may be extremely l a b i l e or present i n very low a c t i v i t y . However, i t i s a l s o p o s s i b l e that another pathway, using a d i f f e r e n t acceptor molecule and/or a d i f f e r e n t g a l a c t o s y l donor, may be i n v o l v e d . The u t i l i z a t i o n of galactose f o r the i n v i v o b i o s y n t h e s i s of GG and SGG by r a t t e s t i s has a l s o been examined (38,39). [ C ] galactose was i n j e c t e d i n t o the t e s t e s of adult r a t s and the speci f i c a c t i v i t i e s of the g a l a c t o s y l moieties of these two l i p i d s determined at v a r i o u s time i n t e r v a l s . L a b e l l e d galactose appeared i n GG by 10 minutes, the peak s p e c i f i c a c t i v i t y o c c u r r i n g by 2 h a f t e r i n j e c t i o n , and d e c l i n i n g t h e r e a f t e r r e l a t i v e l y r a p i d l y . In c o n t r a s t , the appearance of r a d i o a c t i v e galactose i n the SGG was much slower (detectable by 1 h ) , i t s peak s p e c i f i c a c t i v i t y occurr i n g by 72 h a f t e r i n j e c t i o n . Moreover, the s p e c i f i c a c t i v i t y of the SGG subsequently d e c l i n e d very slowly over the f o l l o w i n g 2 weeks. These r e s u l t s are c o n s i s t e n t with the hypothesis that GG i s the precursor of SGG i n v i v o ; however, they do not prove t h i s , nor do they i n d i c a t e from which precursor GG i t s e l f i s s y n t h e s i z e d Thus, although i t appears reasonable to assume that s u l f a t i o n i s the f i n a l step i n the b i o s y n t h e s i s of t e s t i c u l a r SGG, l i t t l e i s known of the e a r l i e r steps. The pathway of b i o s y n t h e s i s of the g l y c e r y l ether backbone of 1 4


7.

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113

the t e s t i c u l a r SGG a l s o remains unexplored; i n view of the h i g h l y r e s t r i c t e d a l k y l and a c y l composition of the l i p i d , i t would be of i n t e r e s t to determine the substrate s p e c i f i c i t i e s of the enzymes involved i n formation and t r a n s f e r of these moieties.


Catabolism of

SGG

Yamato et a l . (31) p u r i f i e d a r y l s u l f a t a s e A from boar t e s t i s . The s p e c i f i c a c t i v i t i e s of the enzyme preparation towards three substrates - 4 - n i t r o c a t e c h o l s u l f a t e , SGG and s u l f o g a l a c t o s y l c e r amide - increased at almost the same r a t e through the v a r i o u s p u r i f i c a t i o n steps employed. The optimal pH f o r a c t i o n on both of the g l y c o l i p i d substrates was 4.5. The a c t i v i t y of the enzyme was somewhat greater using the s p h i n g o l i p i d as s u b s t r a t e , as compared with SGG. A v a r i e t y of procedures i n d i c a t e d that the two glycol i p i d s were both substrates f o r the enzyme. I t was suggested that SGG may be the p h y s i o l o g i c a l substrate for arylsulfatase A i n t e s t i s . E s s e n t i a l l y s i m i l a r r e s u l t s were obtained by F l u h a r t y et_ a l . (40), who examined the a c t i o n of the same enzyme, but p u r i f i e d from human u r i n e , on r a t t e s t i c u l a r SGG and on s u l f o g a l a c t o s y l c e r amide. Neither SGG nor c l a s s i c a l s u l f a t i d e was a substrate f o r a r y l s u l f a t a s e B. Again, these workers concluded that SGG appears to be a p h y s i o l o g i c a l substrate f o r a r y l s u l f a t a s e A. F l u h a r t y et_ a l . a l s o pointed out that a r y l s u l f a t a s e A has been found i n r a b b i t sperm acrosomes, i n which i t was suggested that i t might be i n volved i n the p e n e t r a t i o n of spermatozoa through the investments of the ovum (41). An i n t e r e s t i n g extension of the above work was performed by Yamaguchi et a l . (42). They compared the a c t i v i t i e s towards n i t r o catechol s u l f a t e , SGG and s u l f o g a l a c t o s y l c e r a m i d e of enzyme ext r a c t s from normal human b r a i n and from two cases of a l a t e i n f a n t i l e form of metachromatic leukodystrophy (MLD). The a c t i v i t i e s towards a l l three substrates were markedly d e f i c i e n t (1-5% of cont r o l a c t i v i t i e s ) i n the e x t r a c t s from the diseased b r a i n s . The authors concluded that the enzyme d e f i c i e n c y i n the type of MLD studied was due to a s i n g l e s u l f a t a s e , c a t a l y s i n g the degradation of a l l three substrates used. I t has so f a r not proven p o s s i b l e to determine whether SGG accumulates i n the t e s t e s of a d u l t s with l a t e developing forms of MLD. Nor has i t been e s t a b l i s h e d whether SGG can accumulate i n human b r a i n i n t h i s c o n d i t i o n ; indeed, two s t u d i e s have f a i l e d to demonstrate i t s presence i n that organ (_20,23). However, i t i s p o s s i b l e that the l i p i d could have a very r e s t r i c t e d l o c a t i o n i n human b r a i n . R e i t e r et_ a l . (43) have shown that a second enzyme can a l s o act to degrade SGG. They found that secondary lysosomes from r a t l i v e r contained not only a r y l s u l f a t a s e A, but a l s o a l i p a s e a c t i v i t y that could act to de-acylate SGG. Under the c o n d i t i o n s used, more product was formed by the a c t i o n of the l i p a s e on SGG than by the a c t i o n of a r y l s u l f a t a s e A. These workers a l s o found that the l a t t e r enzyme could use the lyso-SGG as a substrate. I t would be


114


of i n t e r e s t to study the a c t i v i t y of the l i p a s e on GG and a l s o on the d i a c y l - c o n t a i n i n g g a l a c t o g l y c e r o l i p i d s . At the present time, the r e l a t i v e p h y s i o l o g i c a l s i g n i f i c a n c e of the two pathways of degradation of SGG has not been e s t a b l i s h e d . However, the l i p a s e a c t i v i t y has not so f a r been reported to be present i n t e s t i s . Further steps i n the catabolism of SGG i n t e s t i s - e.g. removal of the g a l a c t o s y l residue and degradation of the g l y c e r y l ether moieties - have apparently not yet been examined. I t has been shown that a B-galactosidase (E.C. 3.2.1.23) from Charonia lampas i s capable of removing the g a l a c t o s y l residue from both the GG and g a l a c t o s y l d i a c y l g l y c e r o l species d e r i v e d from r a t b r a i n (23).


Appearance of S u l f a t i d e s During T e s t i c u l a r Development One approach towards determining the p a r t i c u l a r c e l l stage at which phenotypic products ( i n the present case, c e r t a i n s p e c i f i c g l y c o l i p i d s ) of d i f f e r e n t i a t i o n appear i n t e s t i s i s to remove that organ from animals of known age and to c o r r e l a t e the appearance of the compound(s) under study with the appearance of a p a r t i c u l a r c e l l type as determined by h i s t o l o g i c examination. The time of appearance i n the t e s t i s of the v a r i o u s c e l l types i n v o l ved i n spermatogenesis has been p a r t i c u l a r l y w e l l e s t a b l i s h e d i n the case of the r a t by Clermont and Perey (44). Using t h i s approach , K o r n b l a t t et_ a l . (14) found that primary spermatocytes appeared to be the e a r l i e s t spermatogenic c e l l s to contain high l e v e l s of the SGG. Examination of the l e v e l s of SGG i n the t e s t e s of immature r a t s , hypophysectomized r a t s and normal and s t e r i l e mice i n d i c a t e d that the m a j o r i t y of the SGG was l o c a t e d i n the germinal (spermatogenic) c e l l s (as opposed to non-germinal c e l l s , such as S e r t o l i and Leydig c e l l s ) of the t e s t i s . Another f i n d i n g that r e i n f o r c e s the probable germ c e l l l o c a t i o n of the SGG was made by Suzuki et^ a l . (30). These workers fed a d u l t r a t s a d i e t d e f i c i e n t i n v i t a m i n A f o r 46 days. This r e s u l t e d i n a d e c l i n e of SGG to 13% of i t s l e v e l i n the t e s t e s of appropriate c o n t r o l animals. T o t a l l i p i d , p h o s p h o l i p i d and DNA (expressed a p p r o p r i a t e l y ) were only s l i g h t l y reduced. H i s t o l o g i c examination showed that the t e s t e s were aspermatogenic. Vitamin A i s o b v i o u s l y necessary f o r the maintenance of germ c e l l maturat i o n ; i t would be of great i n t e r e s t to determine i f i t p l a y s any s p e c i f i c r o l e i n the b i o s y n t h e s i s of SGG. A dramatic i n c r e a s e (approx. 50-fold) of the a c t i v i t y of the s u l f o t r a n s f e r a s e i n v o l v e d i n the b i o s y n t h e s i s of the SGG a l s o occurred when spermatocytes f i r s t began to appear i n r a t t e s t i s (19); the r i s e i n the a c t i v i t y of t h i s enzyme preceded by s e v e r a l days a marked r i s e i n the amount of the SGG. Studies on p r e p u b e r t a l human t e s t i s (which i s temporarily blocked i n spermatogenesis at a stage p r i o r to the appearance of primary spermatocytes) have shown that n e i t h e r SGG nor GG i s present (15,26). S i m i l a r l y , the t e s t i s of the p r e - p u b e r t a l fowl a l s o lacks sulfogalactosylceramide, the s u l f a t i d e found i n mature fowl t e s t i s (26). A l l of these f i n d -


7.

MURRAY ET A L .


ings are c o n s i s t e n t with the hypothesis that s u l f a t i d e s are synthesized i n the t e s t i s of a v a r i e t y of species when e a r l y spermatocytes appear i n that organ. L e t t s et^ a l . (45) have attempted to answer the question of which c e l l population i n r a t t e s t i s synthesizes the SGG by using methods that allowed f r a c t i o n a t i o n of d i f f e r e n t t e s t i c u l a r c e l l types. T h e i r r e s u l t s i n d i c a t e d that s u l f a t i o n of SGG occurred a t a c e l l stage p r i o r to the l a t e (pachytene and diplotene) spermatocyte stage. L e t t s et a l . (45) a l s o assayed the amount of r a d i o a c t i v e SGG i n e x t r a c t s of t e s t i s and epididymis at i n c r e a s i n g times a f t e r the i n j e c t i o n of [ S ] s u l f a t e i n t o the t e s t e s of adult r a t s . The epididymis showed no r a d i o a c t i v e SGG f o r 4 weeks f o l l o w i n g i n j e c t i o n , but e x h i b i t e d a dramatic appearance of the [35s]-labelled compound at 5 weeks. From previous studies on the k i n e t i c s of spermatogenes i s i n r a t s , i t was p o s s i b l e f o r these workers to conclude that s u l f a t e i n c o r p o r a t i o n i n t o SGG must occur p r i o r to the spermatid stage. These workers a l s o noted that the l e v e l of l a b e l l e d SGG i n t e s t i s decreased s t e a d i l y with time a f t e r i n j e c t i o n of l a b e l . Kornblatt (46) has made s i m i l a r observations to the above. With respect to the l a s t p o i n t , she found that the rate of the decrease of [35s]-labelled SGG i n t e s t i s coincided e x a c t l y with the rate of decrease of [3H]thymidine-labelled DNA l e v e l s i n t e s t i s . This i n d i c a t e s that the l o s s of l i p i d was due to c e l l death and that there was minimal turnover of SGG i n s u r v i v i n g c e l l s . To summarize, the r e s u l t s from both of these studies suggest that the SGG i s s u l fated at the e a r l y primary spermatocyte stage. The s u l f o l i p i d then appears to undergo l i t t l e or no turnover i n the germinal c e l l s during spermatogenesis and e v e n t u a l l y appears i n the spermatozoa. This i s an i n t r i g u i n g f i n d i n g which implies that the l i p i d appears i n t e s t i s at a c e l l stage w e l l before the spermatozoon and p e r s i s t s i n a m e t a b o l i c a l l y s t a b l e form throughout a l l the complex c e l l mode l l i n g processes that precede and accompany the appearance of the h i g h l y s p e c i a l i z e d sperm c e l l . I t should be noted, however, that the above studies with [35s] s u l f a t e do not exclude the p o s s i b i l i t y that other moieties of the SGG - e.g. the a c y l group - could exh i b i t turnover. Suzuki et a l . (13) showed that boar spermatozoa possessed l i t t l e or no c a p a c i t y to incorporate [35s] s u l f a t e i n t o SGG. Narasimhan et^ al.(39) have confirmed the very l i m i t e d , i f not negl i g i b l e , capacity of sperm to synthesize SGG by incubating bovine spermatozoa with l a b e l l e d g l y c e r o l and galactose. No r a d i o a c t i v i t y was detected i n the SGG f o l l o w i n g incubation with these compounds. R a d i o a c t i v i t y from these compounds was, however, found to be i n corporated i n t o SGG when they were i n j e c t e d i n t o the t e s t e s of mature r a t s . A l s o relevant to the appearance of SGG during t e s t i c u l a r d i f f e r e n t i a t i o n were the r e s u l t s of a study performed by Ishizuka and Yamakawa (47). These workers analysed the g l y c o l i p i d composition of three human seminoma ( t e s t i c u l a r ) tumors. Unlike the c o n t r o l human t e s t i c u l a r t i s s u e , no SGG or GG was detected i n the tumors. 3 5


115


116


As many malignant tumors resemble f e t a l t i s s u e i n t h e i r biochemic a l composition, t h i s r e s u l t i s c o n s i s t e n t with the observed absence of SGG from immature human t e s t i s (15,26). Another t e n t a t i v e i n t e r p r e t a t i o n of t h i s f i n d i n g i s that seminoma c e l l s d e r i v e from a c e l l stage p r i o r to that of the primary spermatocyte, thus accounting f o r t h e i r i n a b i l i t y to synthesize SGG.


Subcellular

L o c a t i o n of SGG

i n T e s t i s and

Spermatozoa

Both L e t t s et a l . (45) and Kornblatt (46), using s u b c e l l u l a r f r a c t i o n a t i o n techniques, have obtained evidence i n d i c a t i n g that at l e a s t some of the SGG i n t e s t i s i s present i n the plasma membrane f r a c t i o n of germinal c e l l s . Further s t u d i e s of t h i s subject are i n progress i n the l a b o r a t o r i e s of these workers. Levine et_ a l . (38) have i s o l a t e d head and t a i l f r a c t i o n s of bovine spermatozoa f o l l o w i n g mild treatment of these c e l l s with pronase; the SGG was found to be d i s t r i b u t e d i n both f r a c t i o n s . This l a t t e r result i s c o n s i s t e n t with a l o c a t i o n of the SGG i n the plasma membrane, as t h i s s t r u c t u r e i s continuous around the spermatozoon. I t i s apparent that treatment with a r y l s u l f a t a s e A might y i e l d information on the exposure of the s u l f a t e group of the SGG on the surface of these c e l l s and could a l s o provide a u s e f u l t o o l f o r studying the e f f e c t s on spermatozoal f u n c t i o n of modifying the s t r u c t u r e of the l i p i d . However, p r e l i m i n a r y attempts to use the a r y l s u l f a t a s e A of p i g t e s t i s (31) to d e s u l f a t e the SGG of i n t a c t bovine spermatozoa have not been s u c c e s s f u l (M. Levine and R.K. Murray, unpublished o b s e r v a t i o n s ) , d e s p i t e the f a c t that the enzyme p r e p a r a t i o n was very a c t i v e when i s o l a t e d SGG was used as a s u b s t r a t e . The production of an antiserum to SGG (48,49) may permit the a p p l i c a t i o n of immunocytochemical methods to determine both i t s c e l l u l a r and s u b c e l l u l a r l o c a t i o n s . Attempted L a b e l l i n g of G a l a c t o g l y c e r o l i p i d s Using Galactose Oxidase The s t u d i e s of Gahmberg and Hakomori (50) and Steck and Dawson (51) demonstrated the a b i l i t y of galactose o x i d a s e , i n conj u n c t i o n with NaB3H4,to l a b e l at l e a s t c e r t a i n galactose and Nacetylgalactosamine residues of c e l l surface g l y c o p r o t e i n s and g l y c o l i p i d s . In a n t i c i p a t i o n of employing t h i s method to determine whether the galactose moieties of the SGG and GG of t e s t i c u l a r c e l l s and spermatozoa are exposed on the surface of these c e l l s , Lingwood (52) has used t h i s method to attempt to l a b e l seve r a l p u r i f i e d g a l a c t o g l y c e r o l i p i d s i n v i t r o . Using c o n d i t i o n s that r e s u l t e d i n extensive l a b e l l i n g of galactosylceramide, GG was found to l a b e l to a maximum of 10% of the r a d i o a c t i v i t y i n c o r p o r ated i n t o the s p h i n g o l i p i d . In a d d i t i o n , very low l a b e l l i n g of SGG, g a l a c t o s y l d i a c y l g l y c e r o l and s u l f o g a l a c t o s y l c e r a m i d e was a l so observed, i n comparison with galactosylceramide. The l a b e l l i n g of the l a t t e r compound was not i n h i b i t e d i n the presence of GG, SGG or s u l f o g a l a c t o s y l c e r a m i d e .



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117

The chemical explanation f o r the poor l a b e l l i n g of the g a l a c t o g l y c e r o l i p i d s has not been e l u c i d a t e d . However, i t does not appear to be due to decomposition of the borohydride or to degrada t i o n of these l i p i d s during the l a b e l l i n g procedure. P o s s i b l y , some s u b t l e d i f f e r e n c e i n the p h y s i c a l s t a t e s of the g a l a c t o g l y c e r o l i p i d s as compared with the g a l a c t o s p h i n g o l i p i d s i s i n v o l v e d . I t i s a l s o of i n t e r e s t that s u l f a t i o n of the galactose residue i n h i b i t s the a c t i o n of galactose oxidase. In view of these f i n d i n g s , Lingwood (52) p o i n t s out than an i n a b i l i t y to be l a b e l l e d by the galactose oxidase procedure does not n e c e s s a r i l y mean that a g a l a c t o l i p i d i s absent from the c e l l s u r f a c e . These r e s u l t s suggest that the galactose oxidase technique - at l e a s t as p r e s e n t l y employed - i s u n l i k e l y to be u s e f u l i n determining the p o s s i b l e surface l o c a t i o n of g a l a c t o g l y c e r o l i p i d s i n t e s t i c u l a r c e l l s and spermatozoa. Antiserum

to T e s t i c u l a r

SGG

The p i o n e e r i n g s t u d i e s of Rapport and h i s colleagues (53) c l e a r l y demonstrated the a n t i g e n i c i t y of v a r i o u s g l y c o l i p i d s . Subsequent work has shown that a n t i s e r a to g l y c o l i p i d s may be used to determine t h e i r c e l l u l a r and s u b c e l l u l a r l o c a t i o n s (54). A n t i bodies to SGG and GG could thus prove u s e f u l i n i n v e s t i g a t i n g the c e l l u l a r and/or s u b c e l l u l a r l o c a t i o n of these l i p i d s i n t e s t e s and spermatozoa. The production of a n t i b o d i e s (complement-fixing) to s u l f o g a l a c t o s y l c e r a m i d e has been reported p r e v i o u s l y (55,56). Lingwood et_ a l . (48,49) have thus attempted to produce a n t i b o d i e s to SGG i n r a b b i t s . The animals were i n j e c t e d by the intravenous route with liposomes c o n t a i n i n g SGG. Antibodies to SGG were detected by a complement f i x a t i o n assay. C o n t r o l sera showed no anti-SGG a c t i v i t y , but d i d show low antibody a c t i v i t y to GG, s u l f o galactosylceramide and galactosylceramide. A l l of the anti-SGG a c t i v i t y was l o c a t e d i n the IgG f r a c t i o n . Anti-SGG was p u r i f i e d by adsorption to and e l u t i o n from c h o l e s t e r o l p a r t i c l e s coated with SGG. The e l u t e d anti-SGG reacted with SGG but not with s u l f o galactosylceramide or galactosylceramide; a low t i t e r towards GG remained. These s t u d i e s demonstrate the f e a s i b i l i t y of preparing a n t i b o d i e s to SGG. I t remains to be seen i f these a n t i b o d i e s w i l l prove u s e f u l f o r immunohistochemical approaches towards determining the l o c a t i o n of SGG i n t e s t i s and spermatozoa. S u l f o g a l a c t o l i p i d s of the T e s t i s of Various

Species

The g l y c o l i p i d s of the t e s t i s of a number of animals have been analysed to determine whether SGG i s a u n i v e r s a l c o n s t i t u e n t of t e s t i c u l a r t i s s u e of a l l chordates. The r e s u l t s of these studi e s are summarized i n Table I I I . At l e a s t four p o i n t s concerning these r e s u l t s merit comment: (1) SGG has been detected i n the t e s t e s of a l l of the l i m i t e d number of mammals so f a r examined


118



Table I I I . D i s t r i b u t i o n of S u l f o g a l a c t o l i p i d s i n the T e s t i s (or Sperm) of Various Chordata Animal

Class

Human Rat Mouse Guinea P i g Rabbit Boar Duck Fowl Salmon (milt) Trout Puffer f i s h Skate f i s h Green Monkey

Mammalia

Dog B u l l (sperm) Opossum Turtle B u l l frog

SGG

SGC

+ + + + + +

Aves

+ + + + +

Chondrichthyes Mammalia

+ + +

References (15,26) (11,14) (11,14) (11,13)

Osteichthyes

Reptilia Amphibia

SGGC

+ +

(11) (12) (26) (26) (26) (26) (57) (26) M. Levine (unpublished observations)

+ + +

The presence or absence of each of the three s u l f o g a l a c t o l i p i d s studied i s i n d i c a t e d by + or - r e s p e c t i v e l y . I t i s p o s s i b l e that trace amounts of one or other of the three g l y c o l i p i d s l i s t e d may be present i n c e r t a i n of the t e s t i c u l a r t i s s u e s marked as as i n most cases the estimates were based on v i s u a l examinations of a p p r o p r i a t e l y stained t h i n l a y e r chromatograms. A b b r e v i a t i o n s : SGC, sulfogalactosylceramide; SGGC, s u l f o g a l a c t osylglucosylceramide.



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(2) SGG has not been detected i n the t e s t e s of the l i m i t e d numbers of b i r d s , f i s h , r e p t i l e s and amphibians analysed (3) In the t e s t e s of these l a t t e r c l a s s e s of animals that l a c k SGG, two other s u l f o g a l a c t o l i p i d s were found to be the major g l y c o l i p i d s - i . e . s u l f o g a l a c t o s y l - and s u l f o g a l a c t o s y l g l u c o s y l ceramides (4) The two sphingosine-containing s u l f a t i d e s are a l s o found i n the t e s t e s of c e r t a i n mammals - f o r instance, human t e s t i s cont a i n s both of them, i n a d d i t i o n to SGG. These observations i n d i c a t e that i t should be r e v e a l i n g - i n terms of i n c r e a s i n g understanding of the mechanisms that operate to regulate the s u l f a t i d e p r o f i l e of a t i s s u e - to compare the c a p a c i t i e s of t e s t i c u l a r e x t r a c t s from one or more animals of each of the c l a s s e s l i s t e d i n Table I I I to synthesize the v a r i o u s c o n s t i t u e n t parts of the above three l i p i d s . As p a r t i a l l y d i s c u s sed e a r l i e r , the b i o s y n t h e s i s of these s u l f a t i d e s can be c o n s i dered to occur i n 3 stages: (1) assembly of the l i p i d moieties i . e . ceramide and p o s s i b l y l - 0 - a l k y l - 2 - 0 - a c y l - s n - g l y c e r o l (2) g l y c o s y l a t i o n and (3) s u l f a t i o n . The s p e c i f i c i t y of the s u l f a t i o n r e a c t i o n appears to be r e l a t i v e l y low, as the s u l f o t r a n s ferase involved i n the b i o s y n t h e s i s of SGG w i l l s u l f a t e a number of l i p i d s with a terminal (3-galactosyl residue, i n c l u d i n g GG, g a l a c t o s y l - and g a l a c t o s y l g l u c o s y l - ceramides (19,32). As human t e s t i s contains each of these three l i p i d s (15,26), t h i s can exp l a i n , at l e a s t i n p a r t , why i t e x h i b i t s a l l three s u l f a t i d e s . I t thus seems more l i k e l y that the v a r i e d s u l f a t i d e p r o f i l e s d i s played by the t e s t i s of the animals l i s t e d i n Table I I I w i l l be explained by d i f f e r i n g p o t e n t i a l s , among s p e c i e s , of that organ to synthesize the l i p i d moieties, and by the s p e c i f i c i t i e s f o r both the l i p i d acceptors and the sugar donors of the g l y c o s y l t r a n s f e r a s e s i n v o l v e d i n the second stage of s u l f a t i d e biosynthesis (cf.32). Two other p o i n t s a r i s i n g from t h i s l i n e of work a l s o deserve b r i e f d i s c u s s i o n . F i r s t l y , i t i s r e l e v a n t to mention that s u l f o quinovosyl d i g l y c e r i d e has been reported to be the major g l y c o l i p i d of the spermatozoa of sea urchins (58). I t w i l l thus be of i n t e r e s t to extend s t u d i e s of the comparative biochemistry of t e s t i c u l a r g l y c o l i p i d s to lower c l a s s e s of animals as w e l l as to f u r t h e r members of the c l a s s e s l i s t e d i n Table I I I . Secondly, i t i s apparent that, whatever the p r e c i s e phylogenetic d i s t r i b u t i o n of g l y c o l i p i d s i n t e s t i s may turn out to be, the r e s u l t s to date s t r o n g l y support the hypothesis that s u l f a t i d e s p l a y an important r o l e i n t e s t i c u l a r and/or spermatozoal f u n c t i o n i n chordates. G a l a c t o g l y c e r o l i p i d s of the Nervous System As t h i s i s a r e l a t i v e l y l a r g e subject area, i t w i l l only be touched upon i n s o f a r as i t r e l a t e s to work performed by the authors. G a l a c t o s y l d i a c y l g l y c e r o l was reported to be a c o n s t i t u ent of b r a i n i n 1963 (6); subsequently, the same l i p i d derived



120


from bovine s p i n a l cord was thoroughly c h a r a c t e r i z e d (59). Also i n 1963, GG was detected i n r a t b r a i n (10); a l a t e r study confirmed the presence of GG i n the b r a i n s of s e v e r a l other species (60). Both of these g l y c o l i p i d s are a l s o present i n p e r i p h e r a l nerves ( c f . 61). A compound corresponding i n i t s p r o p e r t i e s to d i g a l a c t o s y l d i a c y l g l y c e r o l was a l s o found to be a c o n s t i t u e n t of human b r a i n (24). Pathways f o r the b i o s y n t h e s i s of both g a l a c t o s y l - and d i g a l a c t o s y l - d i a c y l g l y c e r o l s by e x t r a c t s of r a t b r a i n have been r e f e r r e d to e a r l i e r . The presence of GG i n r a t b r a i n suggested to Levine et a l . (20) that SGG might a l s o be l o c a t e d i n that organ. These workers d i d indeed f i n d small amounts of SGG (approx. o n e - f i f t e e n t h the amount of sulfogalactosylceramide) i n adult r a t b r a i n . They a l s o detected small amounts of the same l i p i d i n r a b b i t b r a i n , but not i n a p o r t i o n of the f r o n t a l lobes of human b r a i n . In a d d i t i o n , evidence was obtained i n t h e i r study suggesting that a l e s s e r amount of s u l f o g a l a c t o s y l d i a c y l g l y c e r o l might a l s o be present i n r a t b r a i n . However, t h i s point was c l e a r l y e s t a b l i s h e d by Flynn et_ a l . (21) and P i e r i n g e r et a l . (22), who provided unequivocal evidence, i n c l u d i n g the i s o l a t i o n of s u l f o g a l a c t o s y l g l y c e r o l , f o r the presence of that compound i n r a t b r a i n . These workers found l a r g e r amounts of the d i a c y l - than of the a l k y l a c y l - c o n t a i n i n g g a l a c t o g l y c e r o l i p i d ; however, i n cont r a s t to Levine et a l . (20), they used immature (approx. 22 day old) animals. Ishizuka et a l . (23) confirmed that both l i p i d s were present i n r a t b r a i n and they developed appropriate methodology, i n c l u d i n g analyses by g a s - l i q u i d chromatography-mass spectrometry, f o r thoroughly c h a r a c t e r i z i n g them. They a l s o showed that the d i a c y l - c o n t a i n i n g l i p i d was the predominant compound i n the b r a i n s of r a t s of age up to 19 days, but t h e r e a f t e r the a l k y l a c y l type predominated, c o n s i t u t i n g 85% of the sum t o t a l of these two l i p i d s by 68 days of age. SGG was detected i n cod b r a i n , but n e i t h e r s u l f o l i p i d was detected i n normal human b r a i n nor i n the b r a i n of a case with metachromatic leukodystrophy. The s t u d i e s of Levine £t a l . (20) revealed that the turnover of the SGG i n r a t b r a i n was s i m i l a r to that of s u l f o g a l a c t o s y l ceramide. This suggested that the SGG l i k e the c l a s s i c a l s u l f a t i d e (62), might be l o c a t e d predominantly i n myelin. P i e r i n g e r et a l . (22) demonstrated that the d i a c y l form of the s u l f o g a l a c t o g l y c e r o l i p i d s present i n r a t b r a i n had a f a s t e r turnover than that of the a l k y l a c y l form. Because previous s t u d i e s (63,64) had i n d i c a t e d that the g a l a c t o s y l d i a c y l g l y c e r o l of r a t b r a i n was an e x c e l l e n t marker metabolite f o r myelination, these workers a l s o studied the p o s s i b l e a s s o c i a t i o n of the two s u l f o g a l a c t o g l y c e r o l i p i d s ( i . e . the d i a c y l and the a l k y l a c y l species) with myelinat i o n . Support f o r the a s s o c i a t i o n of these two compounds with myelination was found by showing that they were absent from r a t b r a i n before 10 days of age and that they accumulated i n that organ between 10 and 25 days of age (the period of maximum myelinat i o n ) . Further support was derived from the f i n d i n g that the s u l f o t r a n s f e r a s e involved i n the b i o s y n t h e s i s of the d i a c y l - c o n -


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t a i n i n g g a l a c t o l i p i d (and presumably, but not c o n c l u s i v e l y e s t a b l ished, a l s o of the a l k y l a c y l species) increased maximally i n act i v i t y during the same time p e r i o d . In a d d i t i o n , the amounts of the s u l f o g a l a c t o g l y c e r o l i p i d s and the a c t i v i t y of the s u l f o t r a n s ferase were g r e a t l y decreased i n the b r a i n s of non-myelinating jimpy mice. Ishizuka et a l . (23) a l s o found that synthesis of r a t b r a i n SGG was most a c t i v e around 18 days of age.


Conclusion It i s evident that knowledge of the g a l a c t o g l y c e r o l i p i d s has grown i n recent years. Instead of being recognized s o l e l y as quant i t a t i v e l y r e l a t i v e l y minor g l y c o l i p i d s of nervous t i s s u e , members of the a l k y l a c y l sub-class are now a l s o seen to c o n s t i t u t e major g l y c o l i p i d s of mammalian t e s t i s and spermatozoa. Nevertheless, t h e i r t i s s u e d i s t r i b u t i o n i s extremely r e s t r i c t e d i n comparison with that of the g l y c o s p h i n g o l i p i d s . I t w i l l be of i n t e r e s t to determine whether a d d i t i o n a l g a l a c t o g l y c e r o l i p i d s occur i n mammali a n c e l l s and a l s o i f other types of g l y c o g l y c e r o l i p i d s e x i s t . In t h i s respect, as mentioned e a r l i e r , the Slomianys (2_ ^ 9) have provided evidence that a novel s e r i e s of g l y c e r y l e t h e r - c o n t a i n i n g g l u c o g l y c e r o l i p i d s may e x i s t i n g a s t r i c j u i c e , s a l i v a and perhaps other s e c r e t i o n s . However, t h e i r r e s u l t s have not as yet r e c e i v e d independent confirmation ( c f . 65,66). With respect to f u n c t i o n , one~wonders i f the common l o c a t i o n of GG and SGG i n the b r a i n and t e s t i s of c e r t a i n species r e f l e c t s some p h y s i o l o g i c a l e n t i t y that both of these organs share - f o r instance, a blood b a r r i e r . However, the apparent absence of SGG from human b r a i n (20,23) does not support t h i s conjecture. Simil a r l y , the sharing of GG and SGG by these two "sequestered" o r gans r a i s e s thoughts as to whether t h i s could be of immunological s i g n i f i c a n c e i n some s i t u a t i o n s . Yet another l i n e of s p e c u l a t i o n i s whether the presence of r e l a t i v e l y l a r g e amounts of g l y c e r y l e t h e r - c o n t a i n i n g g a l a c t o g l y c e r o l i p i d s i n t e s t i s may somehow be r e l a t e d to the f a c t that the t e s t e s of most mammals are confined i n a scrotum maintained at a temperature lower than the r e s t of the body. These surmises r e f l e c t the humbling f a c t that there i s as yet very l i t t l e understanding of the functions of the v a r i o u s non-sulfated and s u l f a t e d g a l a c t o l i p i d s present i n mammalian c e l l s . A ray of hope f o r t h i s area i s provided by the hypothesis of Karlsson and h i s colleagues (67,68) that sulfogalactosylceramide may act as a c o f a c t o r s i t e f o r the a c t i v i t y of Na K ATPase. The p o s s i b i l i t y that SGG could be involved i n such a s i t e i n spermatozoa has been r a i s e d (16,69). 9

+

9

+

The most u s e f u l f u n c t i o n of t h i s review w i l l be i f i t stimul a t e s f u r t h e r research i n t h i s area. For t h i s reason, i t seems appropriate to conclude by posing a number of f a i r l y obvious but nevertheless b a s i c - questions, that w i l l h o p e f u l l y be answered i n future i n v e s t i g a t i o n s . What p h y s i c a l d i f f e r e n c e s e x i s t between a l k y l a c y l and d i a c y l g a l a c t o g l y c e r o l i p i d s , between g a l a c t o -



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glycerolipids and galactosphingolipids and between sulfated and non-sulfated galactolipids (assuming in all cases that the pairs of lipids mentioned differ only with respect to the specified moieties)? Assuming that physical differences do exist, what are their functional implications? What are the details of the pathway of biosynthesis of the testicular galactoglycerolipids and what factors (e.g. genetic, hormonal, enzyme specificity etc.) control the expression of this pathway during testicular differentiation? Can this pathway be interfered with by pharmacological agents (e.g. analogs of glyceryl ethers), and if so, what effects could that have on testicular and possibly nervous system function? What are the precise cellular and/or subcellular locations of the galactoglycerolipids in testicular cells and spermatozoa? Finally, what is the function of the SGG in mature spermatozoa is it involved in ion transport, in motility, in sperm-ovum interactions or is it merely a passenger molecule, having fulfilled its function at some earlier stage of the life history of these beautifully specialized cells? Acknowledgements We thank Ms. B. Palmer for her excellent technical assistance in a number of the studies whose results are summarized above. The authors are grateful for support from the Ford Foundation, from N.I.H. (Grant No. RO-1HD07889 from the National Institute of Child Health and Human Development) and from the Medical Research Council of Canada. The patience and care displayed by Ms. Stephanie Amos during the typing of this manuscript is warmly acknowledged. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9.

Sweeley, C.C.; Siddiqui, B. in "The Glycoconjugates" (Horowitz, M.; Pigman, W., eds.), Academic Press, New York, N.Y., 1976, vol. 1, pp.459-540. Carter, H.E.; Johnson, P.; Weber, E.J. Annu. Rev. Biochem., 1965, 34, 109-142. Kates, M. Advan. Lipid Res., 1970, 8, 225-265. Sastry, P.S. Advan. Lipid Res., 1974, 12, 251-340. Kates, M.; Palameta, B.; Perry, M.P.; Adams, G.A. Biochim. Biophys. Acta, 1967, 137, 213-216. Steim, J.M.; Benson, A.A. Fed. Proc., 1963, 22, 299 (abstr. no. 830). Slomiany, B.L.; Slomiany, A.; Glass, G.B.J. Eur. J. Biochem., 1977, 78, 33-39. Slomiany, B.L.; Slomiany, A.; Glass, G.B.J. Biochemistry, 1977, 16, 3954-3958. Slomiany, B.L.; Slomiany, A. Biochem. Biophys. Res. Commun. 1977, 79, 61-66.



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December 10, 1979.


Cell Surface Glycolipids - American Chemical Society

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