Glycosphingolipids and Glyceroglucolipids of Glandular Epithelial

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Glycosphingolipids and Glyceroglucolipids of Glandular Epithelial Tissue BRONISLAW L. SLOMIANY and AMALIA SLOMIANY Department of Medicine, New York Medical College, New York, NY 10029

A major problem encountered in the analysis of glycolipids is the assurance that glycolipids are removed from the tissue in high yield. Utilization of the classical methods for the isolation of lipids have introduced some limitations with regard to the extractibility of highly polar glycosphingolipids and hence have led to many false statements and to misconceptions that the glycosphingolipid compositions are well explored. Our systematic investigations into the nature of ABH antigens of gastric mucosa have resulted in the isolation and identification of a number of fucolipids, Forssman glycosphingolipid variants and sulfated glycosphingolipids, which have differences in their internal composition, anomeric configuration, length of oligosaccharide chains and degree of complexity. The successful isolation of these glycolipids was the result of a new methodological approach that considered the effect of carbohydrate moiety on the solubility of glycosphingolipids and their tight association with other membrane components. Our extensive studies on glycosphingolipids of gastric mucosa indicate that in order to obtain complete solubilization of this class of compounds, entirely different methodological approaches must be considered. In spite of the assumption that the mucous glycolipids and glycoproteins are similar to, or possibly derived from those found on cell surfaces, glycosphingolipids have not been found to be constituents of mucus secretions. However, the presence of a new type of glycolipid (glyceroglucolipid) has been demonstrated. This implies that glycosphingolipids are confined to membranous structures of the cell in which they may vary in composition, content and expression, and that this may be essential for certain specialized functions of the cell. A protective role of glyceroglucolipids in the cell may be speculated from their localization in the gastric mucous barrier and their resistance to chemical and biological degradation in the most obnoxious of environments. 0-8412-0556-6/80/47-128-14957.00/ 0 © 1980 American Chemical Society Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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In t h i s a r t i c l e we review developments i n methodological approaches f o r i s o l a t i o n of g l y c o s p h i n g o l i p i d s i n high y i e l d s ; demonstration of g l y c o s p h i n g o l i p i d complexity as w e l l as species, i n d i v i d u a l and organ s p e c i f i c i t y ; demonstration of d i s t i n c t i v e features o f the e p i t h e l i a l t i s s u e versus i t s s e c r e t i o n ; and desc r i p t i o n o f a new group of g l y c o l i p i d s which are confined to mucous s e c r e t i o n s . The G l y c o s p h i n g o l i p i d s

of G a s t r i c Mucosa and S a l i v a r y Glands

In e a r l y attempts to i s o l a t e blood group ABH antigens the idea of " l i p i d - h a p t e n " has been c r i t i c i z e d since the antigens were not e x t r a c t a b l e by ether o r alcohol-ether mixtures (1_, 2) , but i n stead the ABH a c t i v i t y was found i n more p o l a r solvents (3). The presence o f complex g l y c o s p h i n g o l i p i d s i n animal t i s s u e s and t h e i r e x t r a c t i b i l i t y were not known, hence the s o l u b i l i t y p r o p e r t i e s were s u f f i c i e n t to support the idea that ABH blood group antigens are g l y c o p r o t e i n s . In e a r l y s t u d i e s , the immunologically a c t i v e l i p i d s were obtained by s o l u b i l i t y d i f f e r e n c e s i n organic solvents and by p r e c i p i t a t i o n as metal conrplexes (£, 5). More r e c e n t l y , the i s o l a t i o n and c h a r a c t e r i z a t i o n of g l y c o s p h i n g o l i p i d s has been g r e a t l y advanced, but the problem with complex components has not been completely solved. In contrast t o the well-known blood group a c t i v e g l y c o p r o t e i n s of g a s t r i c mucosa and g a s t r i c s e c r e t i o n , l i t t l e was known about these g l y c o s p h i n g o l i p i d s except f o r the e a r l y work of Masamune and Siojima (6). E x t r a c t i o n with Chloroform/Methano1. E x t r a c t i o n of hog g a s t r i c mucosa with the conventional mixture o f chloroform/methan o l (2/1, v/v) r e s u l t e d i n the i s o l a t i o n o f s e v e r a l glycosphingol i p i d s . The blood-group a c t i v e g l y c o s p h i n g o l i p i d s p u r i f i e d from t h i s e x t r a c t contained up to seven carbohydrate residues i n the molecule (Table 1, s t r u c t u r e s 1,2,3,4). The heptahexosylceramides 1 and 2 e x h i b i t e d strong blood group A a c t i v i t y and d i f f e r e d from each other i n linkages of the subterminal galactose to N-acetylglucosamine residues (type 1 and type 2 chains) and i n f a t t y a c i d composition (7,8). G l y c o s p h i n g o l i p i d 1 with the type 1 chain had 11.4% hydroxylated acids and 15.7% of C22-C24, whereas glycos p h i n g o l i p i d 2 with type 2 chain had 35.4% hydroxylated f a t t y acids and 44.4% of C 2-Co. f a t t y a c i d s . The d i f f e r e n c e s i n f a t t y acids apparently were s u f f i c i e n t to a f f e c t chromatographic m o b i l i t y of these compounds, and permitted t h e i r i s o l a t i o n as two d i s t i n c t bands. This enabled us to show f o r the f i r s t time the existence o f two types o f chains i n A - a c t i v e g l y c o s p h i n g o l i p i d s , s i n c e only type 2 chains were found i n A and H g l y c o s p h i n g o l i p i d s o f the erythrocytes (9,10). A l s o , the i s o l a t e d g l y c o s p h i n g o l i p i d s d i f f e r e d from those o f human erythrocytes by having an a d d i t i o n a l galactose residue adjacent to the l a c t o s y l p o r t i o n o f the carbohydrate chain. 2

Further studies of blood group a c t i v i t y of various i s o l a t e d g l y c o s p h i n g o l i p i d s l e d to i s o l a t i o n o f components 3 and 4

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

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( T a b l e I ) . The h e x a h e x o s y l e e r a m i d e 3 was A - a c t i v e b u t l a c k e d N - a c e t y l g l u c o s a m i n e ; i t s a c t i v i t y i n A - a n t i - A s y s t e m was somewhat d i m i n i s h e d as compared t o t h a t o f t h e h e p t a h e x o s y l c e r a m i d e s 1 a n d 2 (11). T h e a b s e n c e o f N - a c e t y l g l u c o s a m i n e was a l s o d e tected i n tetrahexosylceramide 4, which manifested H a c t i v i t y ( 1 2 ) b u t a g a i n e x h i b i t e d weaker a n t i g e n i c p o t e n c y t h a n t h a t shown f o r H - a c t i v e p e n t a - , o c t a - and decahexosylceramides of the erythrocytes (10).This decrease i n antigenic a c t i v i t y apparently r e s u l t s from t h e p r o x i m i t y o f t h e a n t i g e n i c determinant and hydrophobic p o r t i o n o f t h e s e g l y c o s p h i n g o l i p i d s , o t i t may b e d u e t o t h e a b s e n c e o f N - a c e t y l g l u c o s a m i n e , w h i c h i n some s u b t l e w a y i n f l u e n c e s the a c t i v i t y of the antigenic determinants. The f u c o s e - c o n t a i n i n g g l y c o s p h i n g o l i p i d s , so abundant i n hog g a s t r i c mucosa, were n o t d e t e c t e d i n r a t s u b l i n g u a l and s u b m a x i l l a r y glands (13), although both tissues are f u n c t i o n a l l y s i m i l a r . O n l y t r a c e s o f f u c o s e were found i n crude g l y c o s p h i n g o l i p i d f r a c t i o n s p r i o r to t h i n - l a y e r chromatography. C u r i o u s l y enough, only t r a c e s o f f u c o s e were a l s o found i n t h e g l y c o p r o t e i n s o f r a t s u b l i n g u a l (14) a n d s u b m a x i l l a r y g l a n d s ( 1 5 ) . The absence o f f u c o s e c o n t a i n i n g g l y c o s p h i n g o l i p i d s supports~The f i n d i n g s o f Kent and S a n d e r s ( 1 6 ) , who h a v e s t u d i e d t h e d i s t r i b u t i o n o f b l o o d g r o u p A antigen throughout the d i g e s t i v e t r a c t o f r a t and found i t s highest content i n large i n t e s t i n e . Their data, together with our r e s u l t s , suggest gradient d i s t r i b u t i o n of fucose-containing glyc o s p h i n g o l i p i d s and glycoproteins throughout the d i g e s t i v e t r a c t o f t h e r a t and p o s s i b l y o f o t h e r mammalian s p e c i e s . The n e u t r a l g l y c o s p h i n g o l i p i d s (Table I I ) contained g l u c o s e , g a l a c t o s e and N a c e t y l g a l a c t o s a m i n e . N - a c e t y l g l u c o s a m i n e was f o u n d o n l y i n v e r y s m a l l amounts i n t h e g a n g l i o s i d e f r a c t i o n s o f t h e g l a n d s . The s u b m a x i l l a r y and e s p e c i a l l y s u b l i n g u a l glands e x h i b i t e d a h i g h cont e n t o f t h e s u l f a t e d g l y c o s p h i n g o l i p i d s . These were composed o f mono- and d i - h e x o s e s u l f a t i d e , w i t h t h e former b e i n g predominant i n b o t h types o f g l a n d s . The h i g h content o f s u l f a t e d g l y c o s p h i n g o l i p i d s i s i n agreement w i t h h i s t o l o g i c a l s t u d i e s o f P r i t c h a r d a n d Rusen (17) and P r i t c h a r d (18) o n t h e d i s t r i b u t i o n o f r a d i o s u l f a t e i n r a t s a l i v a r y g l a n d s . The abundance o f s u l f a t e d g l y c o s p h i n g o l i p i d s i n s a l i v a r y g l a n d s may i n d i c a t e t h a t t h e y p a r t i c i p a t e i n the secretory processes of these glands. Buffered Tetrahydrofuran. In 1973, Tettamanti et a l . (19) d e s c r i b e d a n i m p r o v e d p r o c e d u r e f o r t h e e x t r a c t i o n , s e p a r a t i o n and p u r i f i c a t i o n of b r a i n g a n g l i o s i d e s . In t h i s method, the b r a i n t i s s u e was s u b j e c t e d t o h o m o g e n i z a t i o n a n d e x t r a c t i o n w i t h b u f f e r e d ( p o t a s s i u m phosphate b u f f e r , pH 6.8) t e t r a h y d r o f u r a n . F o l l o w i n g c e n t r i f u g a t i o n , d i e t h y l e t h e r was added a n d t h e m i x t u r e s e p a r a t e d i n t o o r g a n i c and aqueous phase. The g a n g l i o s i d e s , r e c o v e r e d e x c l u s i v e l y i n t h e aqueous p h a s e , were t h e n f r e e d o f r e s i dual phospholipids and other minor contaminants ( i . e . peptides)by c o l u m n c h r o m a t o g r a p h y on s i l i c a g e l . T h i s p r o c e d u r e , as shown b y t h e a u t h o r s , w a s s u p e r i o r t o t h e commonly u s e d chloroform/methanol


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Table I . S t r u c t u r e s o f the g l y c o s p h i n g o l i p i d s c h a r a c t e r i z e d from hog and dog g a s t r i c mucosa.

Glycolipid Structure 1. GalNAcal->3GalBl->3GlcNAc61^3Gal61->4Gal61->4Glcei^lCer 2 t laFuc 2.

GalNAcal-K5Gal Bl+4GlcNAcei+3Gal gl-»4Gal gl-*4Glc31->lCer 2 t laFuc

3.

GalNAcal-K3Gal ei+3Gal Sl+4Gal 01+4G l c W C e r 2 laFuc

4. 5.

Fucal+2Galal->3GalBl->4Glcl-*lCer G alNAcal+3G a l g 1+4G1 cNAc £1+3G a l 61+4G 1 eg 1->1 Ce r 2 laFuc

6.

7.

8.

9.

3 laFuc

GalNAcal-K5Gal 31+3/ 4G1 cNAc l+3Gal 1+4G1 c 1+lCer 2 laFuc Gall+4GlcNAcl-*3Gall+4Glcl+lCer 3 t lFuc GalNAcal+3Gall+3Gall->4Glcl+lCer 2 i laFuc GalNAcal+SGalNAcBl+SGalal^Gaiei^Glcl+lCer


Glandular

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G a INAc a l +3G a INAc 0.

\

3 Galal+4GalBl->4Glcl+lCer 4

/ GalBl->3GalNAc31 11.

GalNAcal+3GalNAcei

\ 3 Galal+4GalBl+4Glcl+lCer 4

/ Fucai->2GalBl->3GalNAc3l 12.

GalNAcal+3Gall+4GlcNAcl lGlcNAc 2 \ + f 3 6 laFuc Gal1+4G1cNAcl+4G1cNAcl+3Gal1+4 6 Glcl+lCer

/ GlcNAcl+4GlcNAcl lGlcNAc 4

13.

Gal 1 4 lGlcNAc

GalNAcal+3Gall+4GlcNAcl 2 + 1 Fuc

V + 3 6 Gall+4GlcNAcl+4GlcNAcl+3Gall+4 6 4 Glcl+lCer / f G a INAca 1+3G a 11+4G1 cNAc 1 1 2 GlcNAc f 4 laFuc t lGlcNAc 14.

GalNAcal+3Galgl+4Galgl 3

t 1 Fuc

Gal31+4Gaiei+4Glc31+lCer

/ GalNAcal+3Gal3l


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GalNAcal+3Gal 3l+4Gal 31 2 +

\ 3

laFuc

Gal 31+4Glc31+lCer

GalNAcal+3Gal3r 16.

GalNAcal+3Gal31+4Gal31 2 \ t 3 laFuc Gal31+4Glc31+lCer 6 GlcNAc31+4Gal31

17.

/

GalNAcal+3Gal31+4Gal31 2 \ laFuc

\ 3 Gal31+4Glc31+lCer 6

/ Galgl 18.

Gal31+4GlcNAc3l+4Gal31

*

\

f

3 Gal31+4Glc31+lCer 6

laFuc

GlcNAc31+4Gal3l 19.

/

GalNAcal+3Gal3l+3/4GlcNAc3l \ laF uc

3 G a 131+4G1 cNAc 3 1+3G a 131+4 6 Gal31+4Glc31+lCer

/ Fucal+2Gal3l+3/4GlcNAc31 2 0.

GalNAca1+3G a 131+3/4G1cNAc31

I f

>»

laFuc

3 G a 131+4G1 cNAc3 1+4G1 cNAcfB 1+3 6 Galgl+4GlcBl+lCer

Fucal+2Gal3l+3/4GlcNAc3l^


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BGal 1

21.

4 3G 1 cNAc 1 GalNAcal+3Gal31+3/4GlcNAc3\ + 2 3 6 t Gal31+4GlcNAc31+3Gal31+4 laFuc 6 Gal31+4Glc31->lCer Fucal+2Gal3l+3/4GlcNAc31

22.

GalNAcal+3Gal31+3/4GlcNAc31 2 \ i 3 laFuc Gal31+4GlcNAc31+4GlcNAc31+3 6 Gal3l+4Glc31+lCer Fuca 1+2G a 131+3/ 4G1 cNAc 31 * 6 1 3GlcNAc 4 1 3Gal

Table I I The Composition and Molar Ratios o f Carbohydrates gual and Submaxillary G l y c o s p h i n g o l i p i d s .

Glycosphingolipid

G

l

c

G

a

RSL RSM RSL Glucosylceramide Galactosylceramide Dihexosylceramide Trihexosylceramide Tetrahexosylceramide Pentahexosylceramide Monosialoganglioside Disialoganglioside Monohexose s u l f a t i d e Dihexose s u l f a t i d e

GlcNAc

l

RSM

RSL

o f Rat S u b l i n -

GalNAc RSM

RSL

RSM

1,.0 1,.0 1. 0 0.97 1. 95 1. 92 3. 02 1. 02 0.98 1. 0 1 .0 1,.0 0.96

1 .0 1 .0 1 .0 1 .0 1 .0 1 .0

1,,0 1,.0 1,.0 1,.0 1,.0 1,.0

1.0 0.99 1.91 1.90 2.95 0.97 0.28 0.95 t r . 1.0 0.98

1.01 0.98 0.06 0.31 0.10 t r .

RSL, r a t s u b l i n g u a l : RSM. r a t submaxillary; t r . , t r a c e s (From Ref. 13 )


0.94 0.97 0.05 0.09

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/water p a r t i t i o n systems (20,21). A p p l i c a t i o n o f b u f f e r e d tetrahydrofuran e x t r a c t i o n to g a s t r i c mucosa, followed by c a r e f u l examination o f the aqueous phase f o r various g l y c o s p h i n g o l i p i d s , i n d i c a t e d that t h i s phase contained s i a l o g l y c o s p h i n g o l i p i d s and considerable q u a n t i t i e s of n e u t r a l g l y c o s p h i n g o l i p i d s (22). These were separated from the a c i d i c g l y c o s p h i n g o l i p i d s by DEAE-Sephadex column chromatography (23). The n e u t r a l g l y c o s p h i n g o l i p i d f r a c t i o n of hog g a s t r i c mucosa was shown to c o n s i s t mostly o f f u c o l i p i d s C 4,25), whereas that o f dog g a s t r i c mucosa e x h i b i t e d a high content of N-acetylgalactosaminecontaining g l y c o s p h i n g o l i p i d s (26). Of the eight f u c o l i p i d s p u r i f i e d from the n e u t r a l g l y c o l i p i d f r a c t i o n o f the aqueous phase o f b u f f e r e d tetrahydrofuran e x t r a c t of hog g a s t r i c mucosa, four were found to be i d e n t i c a l with those (Table I, compounds 1-4) i s o l a t e d p r e v i o u s l y by the chloroform/methanol e x t r a c t i o n procedure (7^,8^,1^,12) and the e l u c i d a t e d s t r u c t u r e s o f four new compounds (5-8) are l i s t e d i n Table I (24,27). F u c o l i p i d s 5,6 and 8 e x h i b i ted blood group A - a c t i v i t y , whereas f u c o l i p i d 7 was not a c t i v e i n the A anti-A, B anti-B o r H anti-H systems. In f u c o l i p i d 6, the subterminal galactose was l i n k e d t o the next sugar i n the chain, N-acetylglucosamine by both 1+3 (40%) and 1+4 (60%) l i n k a g e s . F u c o l i p i d 8 was s t r u c t u r a l l y r e l a t e d to compound 3 and, although i t e x h i b i t e d blood group A - a c t i v i t y , i t s carbohydrate chain was devoid o f N-acetylglucosamine. F u c o l i p i d 7 had a carbohydrate chain i d e n t i c a l i n s t r u c t u r e to that i n the g l y c o l i p i d from normal and human adenocarcinoma t i s s u e (28). Thus, i t became apparent that t h i s g l y c o s p h i n g o l i p i d i s not only present i n human glandular t i s s u e but a l s o i n glandular t i s s u e o f other species and may not n e c e s s a r i l y be a s p e c i f i c antigen of malignant c e l l s . 2

The carbohydrate chain o f f u c o l i p i d 5 contained seven sugar residue and d i f f e r e d from the known A - a c t i v e g l y c o s p h i n g o l i p i d s by the presence o f a second fucose residue, l i n k e d to C-3 o f the i n t e r n a l N-acetylglucosamine. I d e n t i f i c a t i o n o f t h i s glycosphingol i p i d provided the f i r s t evidence f o r the existence o f d i f u c o s y l blood group A - a c t i v e g l y c o s p h i n g o l i p i d s with carbohydrate s t r u c tures i d e n t i c a l to d i f u c o s y l o l i g o s a c c h a r i d e s o f g l y c o p r o t e i n o r i g i n (29), suggesting that the same carbohydrate chains may be l i n k e d to a l i p i d o r p r o t e i n core. A s i m i l a r g l y c o l i p i d was i s o l a t e d l a t e r from dog i n t e s t i n e (30). Examination o f the n e u t r a l g l y c o l i p i d s i n the aqueous f r a c t i o n o f a b u f f e r e d tetrahydrofuran e x t r a c t o f dog g a s t r i c mucosa i n d i c a t e d presence o f g l y c o s p h i n g o l i p i d s containing s i g n i f i c a n t amounts of N-acetylgalactosamine, but only traces o f fucose (26). A s i m i l a r conclusion as to the content o f f u c o l i p i d s i n dog gast r i c mucosa was reached e a r l i e r by McKibbin and L y e r l y (31). Extensive p u r i f i c a t i o n o f the g l y c o s p h i n g o l i p i d s present i n the n e u t r a l f r a c t i o n r e s u l t e d i n the i s o l a t i o n o f three d i s t i n c t g l y c o l i p i d s e x h i b i t i n g Forssman a n t i g e n i c a c t i v i t y (Table I, compounds 9,10,11). T h i n - l a y e r chromatographs o f these g l y c o l i p i d s i s i l l u s t r a t e d i n F i g . 1. Chemical analyses o f the p u r i f i e d


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Thin-layer chromatography of

the glycolipids with Forssman activity (1) Glycolipid 11, Table I; (2) glycolipid 9, Table II; (3) glycolipid 10, Table I. Solvent system: chloroform/methanol/water (60/ 35/8, v/v/v). Visualization: orcinol reagent. (26)

Figure 2. Thin-layer chromatogram of purified glycolipid 11 (Table I) and its enzymatic hydrolysis products (1) Native glycolipid; (2) glycolipid obtained by treatment of the native compound with a-fucosidase; (3) glycolipid obtained by the sequential treatment of the native compound with a-fucosidase, /? galactosidase and / ? - N acetylhexosaminidase; (4) glycolipid obtained by treatment of the defucosylated compound (from line 2) with a-N-acetylgalactosaminidase and f3-N-acetylhexosaminidase; (5) glycolipid obtained by treatment of the defucosylated compound (from line 2) with j3-galactosidase, a-N-acetylgalactosaminidase and /3-N-acetylhexosaminidase; (6) glycolipid from line 5 after incubation with a- and /?galactosidase. Standards: (7a) glucosylceramide; (7b) lactosylceramide; (7c) triglycosylceramide; (7d) glycolipid 9, Table 1; (7e) glycolipid 10, Table 1. Solvent system: chloroform/methanol/water (65/35/8, v/v/v). Visualization: orcinol reagent. (33)


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compounds i n d i c a t e d t h a t the carbohydrate moiety of g l y c o l i p i d 9 c o n s i s t e d o f glucose, galactose and N-acetylgalactosamine i n a molar r a t i o o f 1:2:2. The same carbohydrates, but i n a molar r a t i o o f 1:3:3, were present i n g l y c o l i p i d 10, whereas g l y c o l i p i d 11 contained glucose, fucose, galactose and N-acetylgalactosamine i n a molar r a t i o o f 1:1:3:3 (26). Further s t r u c t u r a l s t u d i e s (32,33) revealed that the carbohydrate moiety o f g l y c o l i p i d 9 i s chemicall y i d e n t i c a l with that of Forssman hapten, c h a r a c t e r i z e d prev i o u s l y from kidney and i n t e s t i n e o f dog (34,35,36) and from spleen and kidney of horse (37,38). The r e s u l t s o f chemical and enzymatic analyses of g l y c o l i p i d 10 suggested that t h i s compound contains two terminal sugar r e s i d u e s , galactose and N - a c e t y l galactosamine, and thus has a branched s t r u c t u r e . S u s c e p t i b i l i t y of g l y c o l i p i d 10 to g l y c o s i d a s e degradation i n the sequence: 3-galactosidase, 3-N-acetylhexosaminidase, and the sequence: a-N-acetylgalactosaminidase, 3-N-acetylhexosaminidase i n d i c a t e d that the s i d e chains are composed o f 3Gal+3GalNAc and aGalNAc+3 GalNAc d i s a c c h a r i d e s . P a r a l l e l s t u d i e s on permethylated fragments of such enzymic degradation products e s t a b l i s h e d that the above d i s a c c h a r i d e chains are l i n k e d by 1+4 and 1+3 bonds, r e s p e c t i v e l y , to the galactose residue adjacent to lactosylceramide o f the gly-* c o l i p i d core. The presence o f two s i d e chains, aGalNAc(l+3)gGalNAc and aFuc(l+2)3Gal(l+3)3GalNAc, i n g l y c o l i p i d 11 was c l e a r l y demonstrated with the a i d of g l y c o s y l h y d r o l a s e s ( F i g . 2) and permethylation a n a l y s i s . Furthermore, the n a t i v e g l y c o l i p i d 11 exhib i t e d both Forssman and H a n t i g e n i c a c t i v i t i e s . D e f u c o s y l a t i o n o f t h i s g l y c o l i p i d (0.1 M t r i c h l o r o a c e t i c a c i d , 100°C f o r 2 h) r e s u l t e d i n the l o s s o f i t s H - a c t i v i t y but had no e f f e c t on i t s r e a c t i v i t y with Forssman anti-serum o r on i t s a b i l i t y to i n h i b i t hemagglutination i n the A/anti-A system. The l a t t e r a c t i v i t y , shared by a l l three compounds (9,10,11), i s thought to be due to the presence of a terminal a-N-acetylgalactosamine residue i n the Forssman antigen and blood group A determinant (39). I t has been suggested e a r l i e r (39) that Forssman antigen may not be a s i n g l e compound. This view was supported by Gahmberg and Hakomori (40) who i s o l a t e d two polymorphic v a r i a n t s of Forssman g l y c o l i p i d from hamster f i b r o b l a s t s . Both v a r i a n t s , however, shared the common terminal s t r u c t u r e composed o f three sugar r e sidues, GalNAc(al+3)GalNAc(31+3)Gal. Our data i n d i c a t e that t h i s terminal s t r u c t u r e i s not only common f o r the Forssman g l y c o l i p i d v a r i a n t s c o n t a i n i n g s t r a i g h t carbohydrate chains, but a l s o can be located on the t e r m i n i o f g l y c o l i p i d s with branched s t r u c t u r e s which c a r r y more than one a n t i g e n i c determinant. I s o l a t i o n o f the Forssman hapten v a r i a n t s from the aqueous phase o f b u f f e r e d t e t r a hydrofuran l i p i d e x t r a c t s i n d i c a t e s that g l y c o l i p i d s bearing Forssman antigen may e x h i b i t considerable water s o l u b i l i t y . This behavior may be d i r e c t l y r e l a t e d to the r e l a t i v e l y strong antigen i c p r o p e r t i e s of Forssman hapten under the p h y s i o l o g i c a l condit i o n s . In accord with these r e s u l t s the term "Forssman antigen" should r e f e r to g l y c o s p h i n g o l i p i d s bearing a terminal


10.

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Epithelial

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159

GalNAc (al+3) GalNAc s t r u c t u r e and should not be used with r e f e r e n c e to one p a r t i c u l a r chemical e n t i t y , i . e . globopentaglycosylceramide. S u l f a t e d g l y c o s p h i n g o l i p i d s (41,42) from the b u f f e r e d t e t r a hydrofuran l i p i d e x t r a c t were also i n v e s t i g a t e d i n our l a b o r a t o r y . In l i p i d e x t r a c t s o f hog g a s t r i c mucosa these g l y c o l i p i d s were found mainly i n the organic phase. A f t e r r i g o r o u s p u r i f i c a t i o n , three s u l f a t e d g l y c o s p h i n g o l i p i d s were obtained i n a homogeneous form. These were i d e n t i f i e d as galactosylceramide s u l f a t e , l a c t o sylceramide s u l f a t e and t r i g l y c o s y l c e r a m i d e s u l f a t e . The s t r u c tures of these compounds are presented i n Table I I I . The presence of g a l a c t o s y l and lactosylceramide s u l f a t e s i n g a s t r i c mucosa and the small i n t e s t i n e has been reported e a r l i e r (31), whereas the i s o l a t e d t r i g l y c o s y l c e r a m i d e s u l f a t e (compound 3, Table I I I ) , not reported h e r e t o f o r e , provided the f i r s t i n d i c a t i o n that s u l f a t e d carbohydrates a l s o occur i n more complex g l y c o s p h i n g o l i p i d s . Succ e s s f u l i s o l a t i o n o f t h i s s u l f a t e d g l y c o l i p i d represents another example o f the s u p e r i o r i t y o f b u f f e r e d t e t r a h y d r o f u r a n e x t r a c t i o n over the conventional chloroform/methanol procedure. Butanol E x t r a c t i o n . Development o f a butanol ext r a c t i o n procedure f o r the i s o l a t i o n o f complex g l y c o s p h i n g o l i p i d s from e r y t h r o c y t e membrane (43) prompted us to apply t h i s method, with m o d i f i c a t i o n , to hog g a s t r i c mucosa. The aqueous phase, a f t e r n-butanol e x t r a c t i o n , was subjected to a l k a l i n e treatment to degrade the g l y c o p r o t e i n s s u s c e p t i b l e to the 3 - e l i m i n a t i o n r e a c t i o n . The products of a l k a l i n e degradation were d i a l y z e d and the prot e i n s separated from the g l y c o l i p i d s by chromatography on C e l l e x P column (44). The g l y c o l i p i d f r a c t i o n was then a c e t y l a t e d , chromatographed on a F l o r i s i l column and p u r i f i e d to homogeneity ( i n the a c e t y l a t e d form) by t h i n - l a y e r chromatography. Although s e v e r a l g l y c o l i p i d bands were detected, only two i n d i v i d u a l compounds were s u c c e s s f u l l y p u r i f i e d to homogeneity (Table I, compounds 12,13). Both g l y c o l i p i d s e x h i b i t e d blood group A - a c t i v i t y and t h e i r carbohydrate p o r t i o n s were h i g h l y enriched i n N - a c e t y l glucosamine. Results of chemical analyses (44) i n d i c a t e d that g l y c o l i p i d 12 contained twelve sugar u n i t s , and g l y c o l i p i d 13 contained eighteen sugar u n i t s . In g l y c o l i p i d 12 one residue of f u cose, one residue o f N-acetylgalactosamine and two out o f s i x r e sidues of N-acetylglucosamine were l o c a t e d at non-reducing t e r m i n i . G l y c o l i p i d 13 contained two terminal residues o f fucose, two r e sidues o f N-acetylgalactosamine and two terminal residues o f Nacetylglucosamine. A n a l y s i s o f the g l y c o l i p i d fragments recovered a f t e r three complete steps of Smith degradation o f glycosphingol i p i d s 12 and 13 showed, i n both g l y c o l i p i d s , the presence of g l u cose, galactose and N-acetylglucosamine i n the molar r a t i o s of 1:2:2. P a r t i a l a c i d h y d r o l y s i s o f these fragments r e s u l t e d mainly i n the formation o f Gal+Glc+ceramide, GlcNAc+Gal+Glc+ceramide and GlcNAc+GlcNAc+Gal+Glc+ceramide (44) . T h i s suggested that the seq u e n t i a l arrangement of the sugar u n i t s i n the saccharide chains adjacent to the ceramide core i n both g l y c o l i p i d s was


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Table I I I S u l f a t e d G l y c o s p h i n g o l i p i d s of G a s t r i c Mucosa

Glycolipid 1. 2. 3. 4. 5.

Structure

S03H+3Gal+ceramide S0 H+3Gall+4Glc+ceramide SOH+3G a l 1+4G a 11+4G1 c+cer amide S0 H+6G1 cNAcB 1+3G a l 31+4G1 c+cer amide Gal3l+4GlcNAc(6^S0 H) 3l+3Gal3l+4Glc+ceramide 3

3

3

3


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Gal+GlcNAc+GlcNAc+Gal+Glc+ceramide. In each g l y c o l i p i d , one r e s i due o f galactose present i n the backbone pentasaccharide was i n volved i n branching. Among other f e a t u r e s , noted f o r the f i r s t time i n g l y c o s p h i n g o l i p i d s was the occurrence of d i - ( N - a c e t y l ) c h i t o b i o s e . T h i s sequence, o r i g i n a l l y reported i n the carbohydrate chains o f porcine (45,46) and horse (47) blood group a c t i v e g l y c o p r o t e i n s , was a l s o r e c e n t l y found i n tKe complex g l y c o s p h i n g o l i pids o f e r y t h r o c y t e membrane (48). The presence of g l y c o l i p i d s with carbohydrate s t r u c t u r e s i d e n t i c a l to those found i n o l i g o saccharides of g l y c o p r o t e i n o r i g i n l e n t f u r t h e r support f o r the existence o f a common pathway f o r the b i o s y n t h e s i s o f blood groupa c t i v e g l y c o p r o t e i n s and g l y c o s p h i n g o l i p i d s . Sodium Acetate E x t r a c t i o n . In our f u r t h e r s t u d i e s o f f u c o l i p i d s o f hog g a s t r i c mucosa, we have found that the residue l e f t a f t e r very thorough e x t r a c t i o n o f l i p i d s Cchloroform/methanol, 2/1, twice f o r 24 h at room temperature) s t i l l contained c o n s i derable q u a n t i t i e s of more complex g l y c o s p h i n g o l i p i d s , which were e x t r a c t a b l e with a mixture o f methanol/chloroform/water cont a i n i n g sodium a c e t a t e . A c c o r d i n g l y , we have developed a procedure which i n v o l v e s i n i t i a l p r e - e x t r a c t i o n o f mucosa scrapings with chloroform/methanol (2/1, v/v) to remove simple g l y c o l i p i d s , f o l lowed by e x t r a c t i o n o f the r e s i d u e with sodium acetate i n methanol/ chloroform/water (60/30/8, v / v / v ) . The h i g h e s t y i e l d of g l y c o l i p i d s was obtained with 0.4 M sodium acetate i n the above methan o l / chloroform/ water system (49). G l y c o s p h i n g o l i p i d s recovered i n such e x t r a c t s i n c l u d e d n e u t r a l g l y c o l i p i d s c o n t a i n i n g fucose as well as a c i d i c g l y c o l i p i d s c o n t a i n i n g both s i a l i c a c i d and s u l f a t e . Separation of these g l y c o l i p i d s i n t o n e u t r a l and a c i d i c components was accomplished by DEAE-Sephadex column chromatography (23). The n e u t r a l g l y c o l i p i d f r a c t i o n was then p e r a c e t y l a t e d and chromatographed on a F l o r i s i l column. The f u c o l i p i d s were contained mainly i n the 1,2-dichloroethane/acetone (1/1, v/v) e l u a t e . T h i s f r a c t i o n , a f t e r extensive p u r i f i c a t i o n on t h i n - l a y e r p l a t e s , y i e l d e d f i v e i n d i v i d u a l f u c o l i p i d s (49,50), four o f which e x h i b i t e d blood group A - a c t i v i t y (Table I, compounds 14-17) and one (compound 18) i n a c t i v e i n the ABH system. Common f e a t u r e s o f a l l f i v e f u c o l i p i d s were a carbohydrate chain with two branches and h i g h enrichment of g a l a c t o s e . In f u c o l i p i d s 14-17, one of the branches was t e r minated by the blood group A-determinant, while the others terminated e i t h e r with a-N-acetylgalactosamine (compounds 14 and 15), 3-N-acetylglucosamine (compound 16) or 3-galactose (compound 1 7 ) . F u c o l i p i d 18, which lacked ABH blood group determinants, a l s o contained two branches, one terminating with 3-galactose and the other with 3-N-acetylglucosamine. The f a c t that only one type of complex g l y c o s p h i n g o l i p i d (enr i c h e d i n galactose) was obtained may have r e f l e c t e d the procedure of p u r i f i c a t i o n , e s p e c i a l l y the choice o f a F l o r i s i l column and the solvents used f o r e l u t i o n of a c e t y l a t e d compounds. This p o s s i b i l i t y became obvious when the n e u t r a l g l y c o l i p i d f r a c t i o n o f the


162

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sodium acetate e x t r a c t s of hog g a s t r i c mucosa was subjected ( i n the a c e t y l a t e d form) to chromatography on a s i l i c i c a c i d column (51,52). The 1,2-dichloroethane/acetone (1/1, v/v) eluate from t h i s column contained two a d d i t i o n a l f u c o l i p i d s (each 12 sugar residues long), whereas the acetone f r a c t i o n contained f u c o l i p i d s with 14 sugar u n i t s . The subsequent f r a c t i o n , e l u t e d with acetone/ methanol (1/1, v / v ) , contained f u c o l i p i d s w i t h 18-24 sugar u n i t s ; and the l a s t f r a c t i o n , e l u t e d with methanol/chloroform/water (90/10/2), c o n s i s t e d o f f u c o l i p i d s with 28-36 sugar residues (53). The i s o l a t e d f u c o l i p i d s i n t h e i r n a t i v e form, d i d not migrate on t h i n - l a y e r p l a t e s i n the solvent systems used f o r p u r i f i c a t i o n o f the p r e v i o u s l y described blood group ABH f u c o l i p i d s (22,54). However, i n the a c e t y l a t e d form a l l o f the compounds studied e x h i b i ted good m o b i l i t i e s i n s e v e r a l solvent systems ( F i g . 3 and 4 ) . The proposed s t r u c t u r e s f o r g l y c o l i p i d s p u r i f i e d from 1,2-dichloroethane/acetone (compounds 19,20) and acetone (compounds 21,22) f r a c t i o n s are given i n Table I. The most i n t e r e s t i n g f e a t u r e s o f these four f u c o l i p i d s were the presence o f two a n t i g e n i c determinants (A and H) on the same g l y c o l i p i d molecule and the s i m i l a r i t y o f the o l i g o s a c c h a r i d e chains to those present i n the blood group (A+H) a c t i v e g l y c o p r o t e i n s (45,46). The carbohydrate and sphingosine composition o f the major f u c o l i p i d s p u r i f i e d from the acetone/methanol and methanol/chloroform/water f r a c t i o n s are given i n Table IV. F u c o l i p i d s 23-25 were present i n the acetone/methanol f r a c t i o n , whereas the methanol/ chloroform/water eluate contained f u c o l i p i d s 26-28 (53). In hemag g l u t i n a t i o n - i n h i b i t i o n assays a l l s i x compounds were potent i n h i b i t o r s o f a g g l u t i n a t i o n o f human group A - c e l l s by anti-A serum (1.5-3.1 yg/0.1 ml) and human 0 - c e l l s by a n t i - H l e c t i n (2.1-4.5 yg/0.1 ml), i n d i c a t i n g that the carbohydrate chain o f each f u c o l i p i d bears two types o f blood group determinant, A and H. Although the s t r u c t u r e s o f these f u c o l i p i d s are not yet e l u c i d a t e d , c e r t a i n features o f the saccharide chains can be suggested on the b a s i s o f carbohydrate a n a l y s i s , immunological assays and the s u s c e p t i b i l i t y o f the n a t i v e and d e f u c o s y l a t e d g l y c o s p h i n g o l i p i d s to the action o f s p e c i f i c exoglycosidases. These data i n d i c a t e that the carbohydrate chain o f f u c o l i p i d 23 contains four branches, two terminated by 3-galactose, one by the blood group A (GalNAcal+3[Fucal->2]Gal-) a n t i g e n i c determinant and one by the blood group H (Fucal->2Gal-) determinant; f u c o l i p i d 24 contains two branches terminated by the blood group A determinant, one by the H and one by 3-galactose; f u c o l i p i d 25 contains two branches terminated by the A determinant, one by H and two by 3-galactose; f u c o l i p i d 26 contains three branches terminated by the A determinant, one by H and two by 3-galactose; f u c o l i p i d 27 contains three branches terminated by the A determinant, one by H and three by 3-galactose; and fucol i p i d 28 contains three branches terminated by the A determinant, two by H, two by 3-galactose and one by 3-N-acetylglucosamine. L i p i d s e x t r a c t e d from hog g a s t r i c mucosa with 0.4 M sodium acetate i n methanol/chloroform/water were a l s o i n v e s t i g a t e d f o r


SLOMIANY AND

Figure 3.

SLOMIANY

Glandular

Epithelial

Tissue

163

Thin-layer chromatography of the acetylated blood group (A-\-H) complex fucolipids

(1) Fucolipid 19, Table I; (2) fucolipid 20, Table I; (3), fucolipid 21, Table I; (4) fucolipid 22, Table I. Solvent system: chloroform/acetone/methanol/water (52/40/00/4, by volume), plate A; 1,2-dichloroethane/methanol/ water (80/25/2, v/v/v), plate B; 1,2-dichloroethane/acetone/methanol/water (50/40/10/4, by volume), plate C. Visualization: orcinol reagent. (52)

Figure 4.

Thin-layer chromatography of

the acetylated highly complex fucolipids from hog gastric mucosa Left plate, developed in chloroform/methanol/2M NH,OH (40/15/1.5, v/v/v). (1) Fucolipid 19, Table I; (2) fucolipid 24, Table IV; (3) fucolipid 23, Table IV; (4) fucolipid 26, Table IV; (5) fucolipid 25, Table IV. Right plate, developed in chloroform/methanol/water (60/40/10, v/v/v). (1) Fucolipid 26, Table IV; (2) fucolipid 27, Table IV; (3) fucolipid 28, Table IV. Visualization: orcinol reagent (53)


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Table IV. Molar Ratios o f Sphingosine and Carbohydrates i n the Highly Complex F u c o l i p i d s from G a s t r i c Mucosa.

Fucolipid

23 24 25 26 27 28

a

Molar r a t i o s a Fuc Gal Glc

GlcNAc

2.01 7.86 2.84 7.50 2.90 9.81 3.81 9.77 3.85 11.78 4.63 12.40

5.92 6.77 7.85 9.68 11.89 13.60

1.0 1.0 1.0 1.0 1.0 1.0

GalNAc Sphingosine No. o f sugar residues 1.0 18 1.05 20-21 0.9 1.78 24 2.02 0.8 3.12 28 0.8 32 0.9 2.79 35-36 3.04 0.7

R e l a t i v e to Glc=l

(From Ref. £ 3 )


10.

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Epithelial

Tissue

165

the presence of s u l f a t e d g l y c o s p h i n g o l i p i d s . For t h i s , the a c i d i c g l y c o l i p i d s , e l u t e d from DEAE-Sephadex with sodium acetate i n methanol/chloroform/water, were a c e t y l a t e d and separated on a s i l i c i c a c i d column i n t o s e v e r a l f r a c t i o n s (55,56). The 1,2-dichloroethane and 1,2-dichloroethane/acetone eluates contained mainly s i a l o g l y c o s p h i n g o l i p i d s , together with traces of the d i and trihexose s u l f a t i d e s described p r e v i o u s l y (41). F r a c t i o n s e l u t e d with more p o l a r solvents contained s e v e r a l new s u l f a t e d g l y c o s p h i n g o l i p i d s . Some of these g l y c o l i p i d s contained s u l f a t e and s i a l i c a c i d . Whether these compounds represent homogeneous g l y c o s p h i n g o l i p i d s containing both s i a l i c a c i d and s u l f a t e on the same molecule or are a mixture of s u l f a t e d and s i a l y l a t e d glycos p h i n g o l i p i d s remains to be e s t a b l i s h e d . However, two of the c h a r a c t e r i z e d s u l f a t e d g l y c o s p h i n g o l i p i d s (55,56) were devoid of s i a l i c a c i d and contained glucose, galactose, N-acetylglucosamine and s u l f a t e i n molar r a t i o s of 1:1:1:1 and 1:2:1:1, r e s p e c t i v e l y . The proposed s t r u c t u r e s o f these g l y c o l i p i d s are shown i n Table I I I (compounds 4 and 5). These newly i d e n t i f i e d compounds d i f f e r from p r e v i o u s l y c h a r a c t e r i z e d s u l f a t e d g l y c o s p h i n g o l i p i d s (41,42) with respect to sugar composition, length of the carbohydrate chain and the s i t e of s u l f a t i o n . Results of periodate o x i d a t i o n and permethylation analyses showed that both compounds contain N-acetylglucosamine 6 - s u l f a t e . To our knowledge, s u l f a t e d g l y c o s p h i n g o l i p i d s cont a i n i n g s u l f a t e d N-acetylglucosamine have not been p r e v i o u s l y desc r i b e d i n mammalian g a s t r i c mucosa or other t i s s u e s . However, N-acetylglucosamine 6 - s u l f a t e was found i n blood group (A+H) s u l f a t e d g l y c o p r o t e i n s of hog g a s t r i c mucosa (45,46). This again i n d i c a t e s that i n glandular e p i t h e l i a l t i s s u e the same o l i g o saccharides may be l i n k e d to a l i p i d or p r o t e i n core. New Approach to I s o l a t i o n of G l y c o s p h i n g o l i p i d s . Prog r e s s i v e d i s c o v e r i e s of more complex g l y c o s p h i n g o l i p i d s , r e v e a l i n g l y s i m i l a r i n s t r u c t u r e to g l y c o p r o t e i n s , i n d i c a t e that current techniques f o r the i s o l a t i o n of g l y c o s p h i n g o l i p i d s are inadequate and do not permit complete recovery of a l l c o n s t i t u e n t s by any one procedure. S i z e and complexity of the carbohydrate p o r t i o n governs e x t r a c t i b i l i t y and lends to some of these g l y c o s p h i n g o l i p i d s the p r o p e r t i e s of g l y c o p r o t e i n s . Therefore, they are e i t h e r l e f t behind during the e x t r a c t i o n or are c l a s s i f i e d as g l y c o p r o t e i n s . To overcome the problem of g l y c o p r o t e i n - l i k e p r o p e r t i e s of complex g l y c o s p h i n g o l i p i d s and at the same time to i s o l a t e short-chain g l y c o s p h i n g o l i p i d s which may be i n strong a s s o c i a t i o n with other components of the c e l l membrane, we have r e c e n t l y introduced a new approach f o r the i s o l a t i o n of g l y c o s p h i n g o l i p i d s (unpublished). In t h i s procedure, g a s t r i c mucosa i s homogenized i n s o l u b i l i z i n g buff e r (sodium s u l f i t e ) and t r e a t e d s e q u e n t i a l l y with RNA-ase and DNA-ase to decrease the v i s c o s i t y o f the homogenate, and then subj e c t e d to a l k a l i n e degradation (3-elimination) and pronase digest i o n . The r e s u l t a n t t i s s u e d i g e s t i s extracted with chloroform/


166

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methanol (2/1, v/v) to remove s h o r t - c h a i n g l y c o s p h i n g o l i p i d s and the aqueous phase i s adjusted to 1% with a z w i t t e r i o n i c detergent. A f t e r c e n t r i f u g a t i o n , the c l e a r supernatant f r a c t i o n i s subjected to g e l f i l t r a t i o n (Bio-Gel P-60) and chromatography on DEAE-Sephadex. Following molecular s i z i n g the Bio-Gel P-4 and/or P-6 columns, the g l y c o l i p i d s are a c e t y l a t e d and p u r i f i e d to i n d i v i d u a l components by chromatography on t h i n - l a y e r p l a t e s o r on Bio-Beads SX-1 columns. Since the e n t i r e process o f i s o l a t i o n i s conducted i n a s o l u t e phase and i n the presence o f a detergent, the a r t i f a c t u a l entrapment o f g l y c o s p h i n g o l i p i d s i s e l i m i n a t e d . Glycolipids

of Mucous S e c r e t i o n

The o r a l , g a s t r o i n t e s t i n a l , b r o n c h i a l , pulmonary and reproductive t r a c t s o f higher animals secrete copious q u a n t i t i e s o f viscous mucus which f u n c t i o n s mainly as a l u b r i c a n t and p r o t e c t i v e agent. The v i s c o u s p r o p e r t i e s o f the mucous s e c r e t i o n s are the r e s u l t o f the presence of high molecular weight g l y c o p r o t e i n s c a l l e d mucins (57). These g l y c o p r o t e i n s have been s t u d i e d e x t e n s i v e l y (see f o r review r e f . £7,^58); however, u n t i l r e c e n t l y no informat i o n was a v a i l a b l e on g l y c o l i p i d s of mucous s e c r e t i o n s . Furthermore, the general assumption was that both mucous 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 are s i m i l a r to, o r p o s s i b l y d e r i v e d from, those found on the c e l l surfaces (59). To provide data on the nature o f g l y c o l i p i d s of mucous s e c r e t i o n s , we have analyzed g l y c o l i p i d cons t i t u e n t s o f the l i p i d extracts derived from g a s t r i c s e c r e t i o n , g a s t r i c mucous b a r r i e r , s a l i v a and a l v e o l a r lavage. Analyses o f the l i p i d extracts from human g a s t r i c s e c r e t i o n revealed that g l y c o l i p i d s c o n s t i t u t e about 30% o f the l i p i d f r a c t i o n (60), whereas i n g a s t r i c s e c r e t i o n s from dog Heidenhain pouch and from l i g a t e d r a t stomach, the g l y c o l i p i d f r a c t i o n comp r i s e s up to 50% o f the t o t a l l i p i d s (61). On t h i n - l a y e r chromatography, the g l y c o l i p i d f r a c t i o n from human s e c r e t i o n could be separated i n t o nine i n d i v i d u a l components, f i v e g l y c o l i p i d components were present i n the g a s t r i c s e c r e t i o n o f dog, and four i n the g a s t r i c s e c r e t i o n o f r a t (61,62). Each o f the p u r i f i e d g l y c o l i p i d s contained f a t t y a c i d s , glucose and g l y c e r y l - monoethers. In a d d i t i o n , two g l y c o l i p i d s from human g a s t r i c s e c r e t i o n were s u l f a t e d . None o f these g l y c o l i p i d s contained sphingosine, phosphorus o r alkenyl ethers (61,63). A l l o f the g l y c o l i p i d s Were susc e p t i b l e to d e a c y l a t i o n under m i l d a l k a l i n e c o n d i t i o n s , indicating the presence o f e s t e r - l i n k e d f a t t y a c i d s , and the s u l f a t e d compounds were a l s o s u s c e p t i b l e to a c i d s o l v o l y s i s ( F i g . 5). Results of s t r u c t u r a l analyses performed on the major g l y c o l i p i d components o f human g a s t r i c s e c r e t i o n i n d i c a t e d that the g l y c o l i p i d s o f g a s t r i c s e c r e t i o n are composed of one or more glucose residues l i n k e d to a monoalkylmonoacylglycerol l i p i d core (64,65). The proposed s t r u c t u r e s f o r g l y c o l i p i d s o f human g a s t r i c s e c r e t i o n are presented i n Table V.


10.

SLOMIANY

A N D SLOMIANY

Glandular

Epithelial

Tissue

167

Figure 5. Thin-layer chromatogram of the major sulfated glycolipid from human gastric secretion (1) Native glycolipid, compound 4, Table V; (2) desulfated glycolipid; (3) desulfated and deacylated glycolipid; (4) digalactosyl diglyceride standard. Solvent system: chloroform/ methanol/water (65/25/4, v/v/v). Visualization: orcinol reagent. (64)

Table V . Glyceroglucolipids

Glycolipid 1. 2. 3. 4. 5.

o f Human G a s t r i c

Secretion.

Structure

Glcal+3-1,(3)-0-alkyl-2-0-acylglycerol Glcal->6Glcal+6Glcal+6Glcal+6Glcal->6Glcal+3-l,

(3)-0-alkyl-2-0acylglycerol Glcal+6Glcal->6Glcal+6Glcal->6Glcal->6Glcal+6Glcal->6Glcal->3-l, (3)-0-alkyl-2-0-acylglycerol SO„H-6G1ca1+6G1ca1+6G1ca1+3-1,(3)-0-alkyl-2-0-acylglycerol S0 H-6Glcal+6Glcal+6Glcal+6Glcal+3-l,(3)-0-alkyl-2-0-acylglycerol 3


168

CELL SURFACE

GLYCOLIPIDS

Our s t u d i e s on the o r i g i n o f g l y c e r o g l u c o l i p i d s i n g a s t r i c s e c r e t i o n e s t a b l i s h e d that these compounds are present not only i n the s o l u b l e p o r t i o n o f g a s t r i c s e c r e t i o n ( d i s s o l v e d mucin), but a l s o i n the g a s t r i c mucous b a r r i e r and i n the preformed i n t r a c e l l u l a r mucus contained w i t h i n the s e c r e t o r y granules o f the e p i t h e l i a l c e l l s (66). Furthermore, we have demonstrated that i n s t i l l a t i o n of v a r i o u s noxious agents such as ethanol and hyperosmotic NaCl causes d e p l e t i o n o f g l y c e r o g l u c o l i p i d s from g a s t r i c mucous b a r r i e r (67). S i m i l a r d e p l e t i o n o f g l y c e r o g l u c o l i p i d s was observed i n various g a s t r o i n t e s t i n a l d i s o r d e r s ( g a s t r i t i s , g a s t r i c u l c e r s ) (68). These data c l e a r l y e s t a b l i s h the importance o f glyceroglucol i p i d s as an e s s e n t i a l component o f g a s t r i c s e c r e t i o n and suggest the p o s s i b i l i t y o f t h e i r involvement i n the defense mechanism against the i n j u r y o f the mucosal s u r f a c e s . In f u r t h e r studies on the g l y c o l i p i d s of mucous s e c r e t i o n s , we have d i r e c t e d our a t t e n t i o n to s a l i v a (69,70). Since glycoprot e i n s o f s a l i v a r y and g a s t r i c s e c r e t i o n bear considerable s t r u c t u r a l and immunological s i m i l a r i t i e s (71,72), i t was o f i n t e r e s t to determine whether the g l y c o l i p i d s o f s a l i v a resemble those o f g a s t r i c s e c r e t i o n . Accordingly, we have i s o l a t e d a g l y c o l i p i d f r a c t i o n from l i p i d e x t r a c t s of whole human s a l i v a and s t u d i e d the composition and s t r u c t u r e o f seven i n d i v i d u a l g l y c o l i p i d components ( F i g . 6). A l l seven compounds were found t o contain g l u cose, f a t t y acids and glyceryl-monoethers. One o f the g l y c o l i p i d s a l s o contained s u l f a t e (70). Results o f chemical analyses (Table V I ) , i n d i c a t e d that these g l y c o l i p i d s are s t r u c t u r a l l y r e l a t e d to those o f g a s t r i c s e c r e t i o n , i . e . they contain p o l y g l u c o s y l carbohydrate chains l i n k e d to monoalkylmonoacylglycerol. Again, glycos p h i n g o l i p i d s were not detected. Our data are c o n s i s t e n t with the r e s u l t s o f e a r l i e r studies on the b i o s y n t h e s i s o f carbohydratec o n t a i n i n g substances i n the s a l i v a r y glands of mice (73), i n which s t i m u l a t i o n with i s o p r o t e r e n o l increased the synthesis o f g l y c o l i p i d o f g l y c e r o g l y c o l i p i d type. A l s o , P r i t c h a r d ' s studies (74) on s u l f o l i p i d formation i n r a t submandibular glands have demonstrated the presence o f a s u l f o t r a n s f e r a s e c a t a l y z i n g the t r a n s f e r o f l a b e l l e d s u l f a t e from 3 -phosphoadenosine-5 -phosphos u l f a t e to an endogenous l i p i d acceptor. This r a d i o - l a b e l l e d s u l f o l i p i d produced by submandibular gland was shown to be o f the g l y c e r o g l y c o l i p i d type. Our recent s t u d i e s (75,76) on the o r i g i n o f g l y c e r o g l u c o l i p i d s i n the s a l i v a i n d i c a t e that tEese compounds are elaborated by the p a r o t i d and submandibular glands and that t h e i r l e v e l s are elevated i n the s a l i v a r y s e c r e t i o n s derived from i n d i v i d u a l s with a high r a t e o f s a l i v a r y c a l c u l u s formation. Whether there i s a d i r e c t a s s o c i a t i o n between the g l y c e r o g l u c o l i p i d content o f the s a l i v a and the development o f plaque, c a l c u l u s and p e r i o d o n t a l disease remains to be e s t a b l i s h e d . For the a n a l y s i s o f e x t r a c e l l u l a r g l y c o l i p i d s o f r e s p i r a t o r y t r a c t , we have chosen the a c e l l u l a r m a t e r i a l l i n i n g the a l v e o l i of mammalian lungs. This unique l i p i d - p r o t e i n mixture, r e s p o n s i b l e f o r the r e d u c t i o n o f a l v e o l a r surface f o r c e s during r e s p i r a t i o n , f

1


10.

SLOMIANY

A N D SLOMIANY

Glandular

Epithelial

Tissue

169

Figure 6. Thin-layer chromatogram of the glycolipids purified from human saliva (see Table VI for structures) (1) Glycolipid 1; (2) glycolipid 2; (3) glycolipid 3; (4) glycolipid 4; (5) desulfated glycolipid 5; (6) glycolipid 6; glycolipid 6; (7) glycolipid 7. Solvent system: chloroform/methanol/water (65/35/8, v/v/v). Visualization: orcinol reagent. (10)

Table VI. G l y c e r o g l u c o l i p i d s of Human S a l i v a .

Glycolipid

Structure

1. Glcal+3-1, (3) - 0 - a l k y l - 2 - 0 - a c y l g l y c e r o l 2,3.Glcal+6Glcal+3-l,(3)-0-alkyl-2-0-acylglycerol 4. Glcal+6Glcal+6Glcal+3-l, ( 3 ) - 0 - a l k y l - 2 - 0 - a c y l g l y c e r o l 5. S0 H-6Glcal+6Glcal+6Glcal+3-l, ( 3 ) - 0 - a l k y l - 2 - 0 - a c y l g l y c e r o l 6. Glcal->6Glcal->6Glcal+6Glcal->6Glcal+6Glcal->3-l, (3)-0-alkyl-2-0acylglycerol 7. Glco:.l-*6Gl c 1+6G1 ca 1+6G 1 ca 1+6G 1 ca 1+6G1 cal+6G 1 cal+6G 1ca1+3-1, C3)-0-alkyl-2-0-acylglycerol 3

a


170

CELL SURFACE

GLYCOLIPIDS

includes the s u r f a c e - a c t i v e phospholipids and other moieties such as n e u t r a l l i p i d s , p r o t e i n s and carbohydrates (77,78,79). I n v e s t i gations on the nature o f the carbohydrate component of pulmonary s u r f a c t a n t i n d i c a t e d that t h i s m a t e r i a l i s not only a s s o c i a t e d with a p r o t e i n but also i s present i n the l i p i d e x t r a c t (80). Analyses o f the l i p i d e x t r a c t s from a l v e o l a r lavage o f r a b b i t , p e r formed i n our l a b o r a t o r y (81,82), showed that the carbohydrate component a s s o c i a t e d with l i p i d s c o n s i s t s e x c l u s i v e l y o f glucose. About 60% o f t h i s carbohydrate was a s s o c i a t e d with n e u t r a l glycol i p i d s and 40% with a c i d i c g l y c o l i p i d s . Extensive p u r i f i c a t i o n o f the g l y c o l i p i d s present i n these f r a c t i o n s r e s u l t e d i n the i s o l a t i o n o f four i n d i v i d u a l components. Three o f these g l y c o l i p i d s contained glucose, f a t t y acids and glycerl-monoethers, whereas the major a c i d i c g l y c o l i p i d , i n a d d i t i o n t o the above components, cont a i n e d s u l f a t e e s t e r (82). The s t r u c t u r e s o f these g l y c o l i p i d s are shown i n Table VII. Our data (81,82) on g l y c o l i p i d s o f the a l v e o l a r l i n i n g l a y e r of r a b b i t lungs c l e a r l y show that these compounds, as those o f g a s t r i c s e c r e t i o n and s a l i v a , belong to the g l y c e r o g l u c o l i p i d c l a s s . Thus, i t appears that an a c e l l u l a r g l y c o l i p i d s i n the sec r e t i o n s o f the alimentary t r a c t and i n the a l v e o l a r l i n i n g l a y e r o f mammalian lungs are e n t i r e l y d i f f e r e n t from those found i n c e l l membranes. The p h y s i o l o g i c a l importance o f s e c r e t o r y g l y c o l i p i d s i s s t i l l unknown. G l y c e r o g l u c o l i p i d s present i n mucous s e c r e t i o n s of the alimentary t r a c t are p a r t o f the p r o t e c t i v e l i n i n g o f the surface e p i t h e l i a l c e l l s and i n s a l i v a they may be involved i n the process o f tooth p e l l i c l e formation, whereas i n the a c e l l u l a r m a t e r i a l l i n i n g the surfaces o f a l v e o l i g l y c e r o g l u c o l i p i d s may p a r t i c i p a t e i n spreading o f the s u r f a c t a n t l a y e r w i t h i n the a l veolus. The Nature o f ABH Blood Group Antigens i n Mucous S e c r e t i o n The occurrence and nature o f blood s p e c i f i c antigens i n t i s sue and i n mucous s e c r e t i o n s has been studied by a number o f i n v e s t i g a t o r s (£3,84,85^,86^,87); the e a r l y data suggested that mucous s e c r e t i o n s contain water-soluble antigens whereas red c e l l s and most o f the other t i s s u e s contain only the a l c o h o l - s o l u b l e a n t i gens. In s p i t e o f t h a t , the d i s c o v e r y o f blood group-active glycos p h i n g o l i p i d s and g l y c o p r o t e i n s from the same source (see f o r r e view r e f . 2j2,5£,58,8^,89,9£) l e d to the proposal o f t h e i r coexistence i n the t i s s u e s and to the assumption (59) that secret i o n s represent a l s o a mixture o f blood group-active glycosphingol i p i d s and g l y c o p r o t e i n s . Furthermore, evidence was presented on the g l y c o p r o t e i n nature of ABH antigens o f erythrocytes (91,92, 93), which were known t o contain antigens o f the glycosphingol i p i d character only. Our s t u d i e s on g l y c o l i p i d s o f g a s t r i c s e c r e t i o n (62,63,64,65) and s a l i v a (69,70) showed that these s e c r e t i o n s do not contain g l y c o s p h i n g o l i p i T s ; i n s t e a d g l y c e r o g l u c o l i p i d s were found. To


10.

SLOMIANY AND SLOMIANY

Glandular

Epithelial

Tissue

171

Table V I I . G l y c e r o g l u c o l i p i d s o f A l v e o l a r Lavage from Rabbit.

Glycolipid 1. 2. 3. 4.

Structure

Glcal->3-l, ( 3 ) - 0 - a l k y l - 2 - 0 - a c y l g l y c e r o l Glcal+6Glcal+6Glcal+6Glcal+6Glcal+3-l,(3)-0-alkyl-2-0-acylglycerol Glcal->6Glcal+6Glcal->6Glcal->6Glcal->6Glcal+3-l, (3)-0-alkyl-2-0acylglycerol S0 H-6Glcal+6Glcal+6Glcal->6Glcal->3-l, ( 3 ) - 0 - a l k y l - 2 - 0 - a c y l glycerol 3

Table V I I I . ABH blood group a c t i v i t y i n human s a l i v a and g a s t r i c s e c r e t i o n . Assay 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Material

Activity*

Native g a s t r i c s e c r e t i o n Native s a l i v a Delipidated gastric secretion Delipidated saliva Native and d e l i p i d a t e d g a s t r i c s e c r e t i o n t r e a t e d with 0.5 M NaOH, (60 h, room temperature) Native and d e l i p i d a t e d s a l i v a t r e a t e d with 0.5 M NaOH (60 h, room temperature) A l k a l i n e degradation o f g a s t r i c s e c r e t i o n i n the presence o f A - a c t i v e g l y c o s p h i n g o l i p i d A l k a l i n e degradation o f s a l i v a i n the presence of A - a c t i v e g l y c o s p h i n g o l i p i d L i p i d extract of g a s t r i c secretion L i p i d extract of s a l i v a Glycolipid fraction of saliva Glycolipid fraction of gastric secretion Blood group A - a c t i v e g l y c o s p h i n g o l i p i d i n the presence o f l i p i d e x t r a c t from s a l i v a or g a s t r i c secretion

* (+) s i g n i f i e s i n h i b i t i o n o f hemagglutination (-) i n d i c a t e s hemagglutination


+ + + +

+ +

+

172

CELL SURFACE

GLYCOLIPIDS

determine the nature of blood group ABH antigens i n s a l i v a and g a s t r i c s e c r e t i o n , the n a t i v e and d e l i p i d a t e d samples, t o t a l l i p i d s , and p u r i f i e d g l y c o l i p i d s were t e s t e d f o r a n t i g e n i c a c t i v i t y . The lack o f i n h i b i t i o n o f a g g l u t i n a t i o n with t o t a l l i p i d s and with p u r i f i e d g l y c o l i p i d s c l e a r l y i n d i c a t e d that the a n t i g e n i c propert i e s o f s a l i v a and g a s t r i c s e c r e t i o n were not r e l a t e d t o the l i p i d p o r t i o n o f these s e c r e t i o n s (94,95). The n a t i v e a c t i v i t i e s o f sal i v a and g a s t r i c s e c r e t i o n were abolished by treatment with a l k a l i which i s known to destroy blood group-active g l y c o p r o t e i n s , but i s completely i n e f f e c t i v e i n degradation o f g l y c o s p h i n g o l i p i d s . However, n e i t h e r a l k a l i nor the presence o f n a t i v e g l y c o l i p i d s from s a l i v a o r g a s t r i c s e c r e t i o n were capable o f d i m i n i s h i n g the a n t i g e n i c potency o f added blood group A g l y c o s p h i n g o l i p i d s (Table V I I I ) . A l s o , the removal o f l i p i d s p r i o r to the hemagglut i n a t i o n - i n h i b i t i o n assay d i d not decrease the n a t i v e a c t i v i t y o f the samples; t o the contrary, a s l i g h t increase i n potency per mg o f residue was noted. These data c l e a r l y i n d i c a t e that g l y c o p r o t e i n s (water-soluble antigens) are r e s p o n s i b l e f o r the blood group a c t i v i t y o f the sec r e t i o n s and t h e i r presence i n s e c r e t o r y t i s s u e i s only temporary, whereas g l y c o s p h i n g o l i p i d s thus f a r i s o l a t e d from a number o f t i s sues represent antigens which are an i n t e g r a l p a r t o f the c e l l membranes (94,95). T h i s d i s t i n c t i v e feature o f e p i t h e l i a l - s e c r e t o r y t i s s u e versus i t s s e c r e t i o n does not e x p l a i n the o r i g i n o f blood group antigens o f the e r y t h r o c y t e s . The coexistence o f g l y c o s p h i n g o l i p i d and g l y c o p r o t e i n ABH antigens i s s t i l l disputed. According to Koscielak e t a l . (96) the erythrocyte stroma i s only equipped with antigens o f g l y c o s p h i n g o l i p i d nature. This i s s t r o n g l y opposed by others (91,92,95) who have provided evidence that erythrocyte membrane antigens are o f dual o r i g i n . I t i s poss i b l e that r i g o r o u s s e p a r a t i o n o f blood group-active g l y c o p r o t e i n s and g l y c o s p h i n g o l i p i d s between s e c r e t i o n and s e c r e t o r y t i s s u e i s not a p p l i c a b l e to erythrocytes, which represent an unusual type o f tissue entirely.

Acknowledgement. This study has been supported by Grants AM No. 21684-02 and 25372-01 from N a t i o n a l I n s t i t u t e o f A r t h r i t i s , Metabolism and D i g e s t i v e Diseases, N a t i o n a l I n s t i t u t e s of Health, United States P u b l i c Health S e r v i c e .


10. SLOMIANY AND SLOMIANY

Glandular Epithelial Tissue

173

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