Glycolipids of Rat Small Intestine with Special Reference to Epithelial

6

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Glycolipids of Rat Small Intestine with Special Reference to Epithelial Cells in Relation to Differentiation M. E. BREIMER, G. C. HANSSON, K.-A. KARLSSON, and H. LEFFLER Department of Medical Biochemistry, University of Göteborg, Göteborg, Sweden Saccharides may be structurally very complex. In addition to the variation in type and sequence of monomers as for peptide, the heterocyclic carbohydrate monomer may vary in ring size, the glycosidic bond may have both different positions and configurations, and there is often branching of the saccharide chains. A great variability may also mean a rich biochemical language (provided there is specificity of expression) and this is one of the reasons why cell surface carbohydrates are being considered in biological recognition (1, 2). The membrane-bound carbohydrates exist as glycoproteins and glycolipids. Although the functional importance of these substances is far from proven they appear to be essential parts in phenomena such as cellular adhesion, control of differentiation and cell growth, and the binding by cells of enzymes, hormones and toxins. One system that we consider of great interest for the study of cell surface glycolipids is the small intestine. Firstly, the epithelial cells lining the intestine exist in a great number on the enlarged surface area and each cell has in itself a large cell surface involved in transport processes and recognition phenomena. Secondly, these cells, arranged as a single columnar layer on the basement membrane, are rapidly renewed (1-3 days) and undergo a successive maturation on their way from the crypt depth to the villus tip (3). Thirdly, these cells are possible to prepare by a gentle washing technique (4), the oldest, less strongly adhered cells (villus tip) being obtained in the first, and the youngest, cells (crypt) obtained in the final fractions. Lastly, the concentration of complex glycolipids is high in relation to protein (see 5), which may be explained by a large amount of surface membrane in relation to other membranes. Our study was divided into two different parts and applied on two separate strains of rat, which were shown to differ in blood groups. In the first stage, following improvement and adaptation of methods, glycolipids were prepared and characterized from pooled whole small intestine of the black and white strain. In the second stage, the knowledge of the general glycolipid 0-8412-0556-6/80/ 47-128-079S6.50/ 0 © 1980 American Chemical Society Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

80

C E L L SURFACE GLYCOLIPIDS

composition allowed a c h a r a c t e r i z a t i o n on a smaller s c a l e of e p i t h e l i a l c e l l s and n o n - e p i t h e l i a l r e s i d u e , and a comparison of the two s t r a i n s . The d i f f e r e n t components of t i s s u e are v i s u a l i z e d i n F i g . 1. The experience obtained has now been used f o r a s i m i l a r i n v e s t i g a t i o n on human m a t e r i a l ( i n p r e p a r a t i o n ) . Methods The animals used were from inbred s t r a i n s of white, and black and white r a t . The p r e p a r a t i o n of e p i t h e l i a l c e l l s i n separate stages of d i f f e r e n t i a t i o n was modified from the technique of Weiser (4). The completeness of removal of e p i t h e l i a l c e l l s from n o n - e p i t h e l i a l residue was checked by conventional microscopy. The p r e p a r a t i o n of t o t a l g l y c o s p h i n g o l i p i d s f r e e of contaminants has been improved to an important extent but i s based on conventional steps such as chloroform-methanol e x t r a c t i o n , m i l d a l k a l i n e degradation, d i a l y s i s , a c e t y l a t i o n and chromatography on DEAE-cellulose and s i l i c i c a c i d . T h i n - l a y e r chromatography was done on HPTLC p l a t e s with s i l i c a g e l 60 (Merck). Conditions f o r mass spectrometry (6,7) and NMR spectroscopy (8, 9_ 10) have been described. Gas chromatography a f t e r degradation of n a t i v e or permethylated g l y c o l i p i d s was done according to standardized t e c h niques (11) except that the a n a l y s i s was performed on c a p i l l a r y columns. 9

Non-Epithelial Tissue The non-acid p a t t e r n of the r e s i d u e a f t e r exhaustive washing and removal of e p i t h e l i a l c e l l s from small i n t e s t i n e i s shown i n F i g . 2, f o r the b l a c k and white ( B ) and white (W ) s t r a i n , r e s p e c t i v e l y . The two samples look i d e n t i c a l with a major compoment corresponding to four sugars. Most of the g l y c o l i p i d s have been i s o l a t e d and c h a r a c t e r i z e d . To present an overview the t o t a l g l y c o l i p i d s of white r a t were subjected to a novel a p p l i c a t i o n of mass spectrometry (7) a f t e r permethylation and r e d u c t i o n with L i A l H ^ . F i g s . 3 and 4 show some of the r e s u l t s . The mixture of g l y c o l i p i d d e r i v a t i v e s i s introduced i n t o the i o n source and s u c c e s s i v e l y heated (5°C/min) as shown on the s c a l e below the curves. Scans (each scan producing a mass spectrum such as that of F i g . 7) were taken each 38 sec, and the change i n r e l a t i v e i n t e n s i t y of s e l e c t e d ions f o r separate g l y c o l i p i d s was r e produced as curves along the temperature and scan s c a l e s . In t h i s case the ions s e l e c t e d contained the complete saccharide and the f a t t y a c i d as shown i n the e x p l a i n i n g formulas ( u s u a l l y r e l a t i v e l y abundant i o n s , which i s demonstrated f o r the A a c t i v e g l y c o l i p i d s i n F i g s . 7 and 8). Curves corresponding to s p e c i f i c ions f o r nine separate g l y c o l i p i d s are reproduced. Two s e r i e s of compounds are shown, one without ( F i g . 3) and the other with hexosamine ( F i g . 4 ) . The curve i n F i g . 3 f o r m/e 516 (monohexosylg

s

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

6.

BREIMER ET A L .

Epithelial

Cells

Figure 1. Acid and non-acid glycosphingolipids were prepared and characterized from different compartments of rat small intestine: non-epithelial residue, total epithelial cells, and epithelial cells of different maturity (crypt, intermediate, and villus fractions).

Figure 2.

Thin-layer chromatogram of non-acid glycolipids of small intestine of black and white (B) and white (W)rat

The following samples were applied: 40 fig of total glycolipids (t); glycolipids corresponding to 4 mg protein of non-epithelial residue (s); glycolipids corresponding to 2 mg protein for epithelial cells of villus (v), intermediate (i), and crypt (c) fractions. Figures in the margins indicate number of sugars. Anisaldehyde was used for the detection, and the solvent was chloroform-methanol-water 60:35:8 (by volume).


81

82

C E L L SURFACE

-CH-CH-CH-C

-CH-^H-CH-C.-H,,

Hex-

Hex—o—Hex-

1 3

-Hex-o-Hex-

Hex-o-Hex-

-Hex-

GLYCOLIPIDS

-Hex-o—CH CH2

-Hex-o—Hex—o—CH CH2

-Hex-o-cH C H -

-CH-CH-CH-C

1 3

H-

7

-CH-CH=CH-C

1 3

H_

7

1536 (CH ) 2

Hex-

-Hex-o-Hex-o—Hex—o—Hex-

-Hex-o—CH.CH-

1 4

Figure 3. Selected ion monitoring from mass spectrometry of a permethylatedreduced mixture of non-acid glycolipids from non-epithelial residue of the white rat The curves reproduced correspond to relative abundance of saccharide plus fatty acid ions (see formulas) of glycolipids lacking hexosamine as a function of evaporation temperature. A total of 200 fig was evaporated by a temperature rise of 5°C/min, and spectra were recorded each 38 sec. The electron energy was 34 eV, acceleration voltage 4 kV, trap current 500 fiA, and ion source temperature 280°C.


BREIMER ET A L .

Epithelial

Cells

HexN-o—Hex—o—Hex—o—Hex-

-Hex—o—Hex—o—Hex—o—CH C H -

Hex N - o - H e x -

(CH ) 2

HexN-o-Hex-

-Hex—o—Hex—o—Hex-

1 4

-Hex—o—CH_CH-

-1359(x3)5 150°

Figure 4.

' ' I " 21 200°

37 250°

Selected ion monitoring of saccharide plus fatty acid ions of hexosaminecontaining glycolipids from the same experiment as for Figure 3


84


ceramide) appears at lower temperature while those f o r higher members ( F i g s . 3 and 4) come up l a t e r , i n some cases i n d i c a t i n g a complete s e p a r a t i o n of g l y c o l i p i d s p e c i e s . The r e l a t i v e i n t e n s i t i e s of the separate bands on the chromatogram ( F i g . 2) are not d i r e c t l y comparable with the i o n curves as the r e l a t i v e abundance of ions decreases r a p i d l y with i o n mass. The space a v a i l a b l e does not allow a more d e t a i l e d presenta t i o n . Mass spectra and s e l e c t e d i o n monitoring of the permethylated (non-reduced) mixture supplement the information with sequence data (6, 7) that allow the formulas w r i t t e n i n F i g s . 3 and 4. The nature of the o l i g o m e r i c hexosylceramides was f u r t h e r s u b s t a n t i a t e d by NMR spectroscopy and degradation of some pure or p a r t i a l l y p u r i f i e d f r a c t i o n s . The hexosamine-lacking compounds were separated from those c o n t a i n i n g hexosamine by use of a c e t y l ated d e r i v a t i v e s and s i l i c i c a c i d column chromatography. F i g . 5 shows NMR s p e c t r a of d e r i v a t i z e d tetrahexosylceramide ( f r a c t i o n A) and a mixture ( f r a c t i o n B) of pentahexosylceramide (major p a r t ) and f u c o s y l t e t r a h e x o s y l c e r a m i d e . The two ^"resonances and the exresonance at about 5.0 ppm ( f r a c t i o n A) are comparable with those of trihexosylceramide of human e r y t h r o c y t e membrane (8). Theref o r e , the second a-resonance at about 5.1 ppm (the sharp s i g n a l c l o s e to that i s due to ethanol) may o r i g i n a t e i n a terminal Gal (the r a t i o of G a l r G l c as shown by degradation was 3:1). One Gal i s bond 1->3 (5.1 ppm) and the other 1->4 (5.0 ppm). The spectrum of the mixture (B) shows the same s i g n a l s but the second Gala has now about doubled i n i n t e n s i t y compared with the f i r s t Gala, suggesting that the major pentahexosylceramide i s f o r m a l l y der i v e d from the tetrahexosylceramide by a d d i t i o n of another Gala1-K3. Therefore, the o l i g o m e r i c hexosylceramides (we have det e c t e d by mass spectrometry and t h i n - l a y e r chromatography up to eight hexoses) may be formed by a s e q u e n t i a l a d d i t i o n of Gala1->3 to g l o b o t r i a o s y l c e r a m i d e ( F i g . 6). The minor f u c o l i p i d i s probabl y a l s o derived from the tetrahexosylceramide, i n t h i s case the fucose having caused an u p f i e l d l o c a t i o n of the two Gala resonances ( i n d i c a t e d by d o t s ) . The t e t r a g l y c o s y l c e r a m i d e with terminal hexosamine ( F i g . 4) was shown to c o n s i s t of about one t h i r d of c y t o l i p i n K and two t h i r d s of c y t o l i p i n R ( g l o b o t e t r a o s y l - and i s o g l o b o t e t r a o s y l ceramide, r e s p e c t i v e l y , see F i g . 6). The higher members detected i n t h i s s e r i e s ( F i g . 4) are probably formed by an e l o n g a t i o n of g l o b o t r i a o s y l c e r a m i d e as f o r the f i r s t s e r i e s and a termination by GalNAcg1-*3 ( F i g . 6). Of p a r t i c u l a r i n t e r e s t was the i d e n t i f i c a t i o n of a blood group B a c t i v e hexaglycosylceramide based on galactosamine ( F i g . 6). The g l y c o l i p i d s detected i n n o n - e p i t h e l i a l t i s s u e are summarized i n F i g . 6. The g a n g l i o s i d e composition w i l l be commented on below. Epithelial

Cells

The g l y c o l i p i d p a t t e r n of e p i t h e l i a l c e l l s i s d i s t i n c t l y


6.

BREIMER ET A L .

Epithelial

Cells

85

5.0

4,0 6 (ppm)

•

5^0

4.0 5(ppm)

Figure 5. NMR spectra of two permethylated-reduced glycolipid samples (A and B) lacking hexosamine and isolated from whole intestine of black and white rat; 2 mg in 0.5 mL chloroform and 2300 pulses at 40°C (sample A), and 1 mg in 0.5 mL chloroform and 5300 pulses at 40°C (sample B).


86

CELL SURFACE GLYCOLIPIDS

Figure 6.

Thin-layer pattern with deduced chemical formulas of non-acid glycolipids of white rat non-epithelial residue (cf. Figure 2).


6.

BREIMER ET A L .

Epithelial

Cells

87

d i f f e r e n t from that of n o n — e p i t h e l i a l t i s s u e ( F i g , 2), Bands corresponding to one and three sugars are dominating. In a d d i t i o n , there are a number of compounds that have been prepared from pooled whole i n t e s t i n e s of the black and white s t r a i n . Two s e r i e s of f u c o l i p i d s were i d e n t i f i e d , one with blood group H and one with blood group A determinants. Mass spectra of permethylatedreduced d e r i v a t i v e s of two of the A - g l y c o l i p i d s are shown i n F i g s . 7 and 8, r e s p e c t i v e l y . In a d d i t i o n , a 6-sugar A a c t i v e compound was c h a r a c t e r i z e d , thus completing a s e r i e s with 4, 6 and 12 sugars. Concerning the 12 sugar compound the mass spectra of the permethylated d e r i v a t i v e (not shown) and of the permethylatedreduced d e r i v a t i v e (Fig.8) are remarkable i n that they together a f f o r d a c o n c l u s i o n on the type, number and sequence of sugars i n c l u d i n g branching of the chain, i n a d d i t i o n to ceramide s t r u c t u r e (to be p u b l i s h e d ) . The saccharide plus f a t t y a c i d peaks i n the i n t e r v a l m/e 2835-2977 ( F i g . 8) are evidence f o r f i v e hexoses, f i v e hexosamines, two fucoses and a v a r y i n g f a t t y a c i d , mainly from 16:0 (m/e 2835) to 24:0 (m/e 2947) nonhydroxy f a t t y a c i d , but a l s o 24:0 hydroxy a c i d (m/e 2977). In the spectrum of the non-reduced d e r i v a t i v e (not shown) m/e 396 showed that the dominating base i s phytosphingosine. According to the r e l a t i v e i n t e n s i t y of the s e r i e s of peaks at m/e 2835-2977 the major molecular species contained phytosphingosine and 20:0 nonhydroxy f a t t y a c i d . Evidence f o r the sequence and branching point was obtained by the absence or presence of several i o n s . Some primary and secondary ( l o s s of methanol, mass 32) ions with a successive increase i n the number of sugars from the non-reducing end are shown up to nine sugars (m/e 1915). The absence of sequence ions between m/e 871 and 1915 speaks against a l i n e a r sequence between these two fragmentation p o i n t s . (There were analogous ions obtained from the non-reduced d e r i v a t i v e ) . The absence of ions f o r smaller saccharides with two fucoses ( i n spectra of both d e r i v a t i v e s ) i s evidence f o r fucose l o c a t i o n i n separate c h a i n s . F i n a l l y , there i s a number of rearrangement ions c o n t a i n i n g the f a t t y a c i d and an i n c r e a s i n g part of the saccharide from the ceramide end (some of them i n d i c a t e d below the formula). These ions have taken up one or two hydrogens depending on the l o c a t i o n of the branch (see peaks at m/e 614, 818, 1049, 1239, 1470, 1848, 2093, 2324). Therefore, the evidence obtained from the two d e r i v a t i v e s i s c o n c l u s i v e concerning the sequence of the 12-sugar g l y c o l i p i d . This substance represents the l a r g e s t biomolecule s t r u c t u r a l l y determined by mass spectrometry thus f a r . Compared with the B - a c t i v e g l y c o l i p i d found i n n o n - e p i t h e l i a l t i s s u e , the f u c o l i p i d s i n e p i t h e l i a l c e l l s were based on glucosamine i n s t e a d of galactosamine (see F i g . 9). The H a c t i v e f u c o l i p i d s of b l a c k and white r a t had three and f i v e sugars, r e s p e c t i v e l y . The g l y c o l i p i d s found i n e p i t h e l i a l c e l l s of the two s t r a i n s are summarized i n F i g . 10.



60

40

t

d

80

100

300

246

400

296 409- 1 502

613^ 1 614 /

500

I , i 1.1,., 600

501*1

700

800

828 \

844

0—Hexose•

1000

T

991 992 980 924

900

900< 901

I

NMe

1036

1

0

I

1 I

I

0

V

V

1200

1081

1209

1300

1267

1400

n-1

1409

1500

AIaL

n-1

1521

Figure 7. Mass spectrum of permethylated-reduced derivative (60 fig) of a blood-group A active tetraglycosylceramide. Electron energy was 44 eV, acceleration voltage 4 kV, trap current 100 fiA, ion source temperature 290°C, and probe temperature 215°C.

200

187

Hexos ami ne-

246

1600

oo

m O r *< o o r

n

>

c

r r

00


1700

1900

2000

2100

M2300 i*.

2891-624*1 2268

2200

\

2400

V

(2947-885-885)

C H

900

2700

2800

2900

|

2891

1300

3000

2947

1200

1500

1600

2947-855-624^2 M 7 0

.H„

Journal of the American Chemical Society

2835

lMl,ii.,i,4. 1000 1 100

2600

m/e

4lll

1400

Me Me

I I I ICH — C, _

976-58 1237*2 918 1032-58 048* 1 1 049 1 239 \ 974 855 \ | 992*1 1181*2 1915-624* 1 993 1 183 ,871 * 292 \

2500

JJi

2947-624* 1 2324

700

640

81 7* 818 761*1

1237

I

CH,

2947 j

Figure 8. Mass spectrum of permethylated-reduced derivative (90 fxg) of a blood-group A active dodecaglycos^lceramide. Electron energy was 29 eV, acceleration voltage 2.9 kV, trap current 500 fiA, and evaporation temperature 305°C. In-beam technique was used (23).

1800

1915

2947-855* 1 2093

2891-855*1 2037 ( /

600

\

\

IliniiLl

500

He

i

o!

X

0

61 3* 1 61 4 557*1

V

Hexose-}-0 — Hexosami

llllUuililltii 11 200 300 400

2947-855-246*2 1848

100

J

101 X0.6

Hexosamine-

M - 3232

1700

90


A

(min)

Figure 9. Open tubular gas chromatogram of partially methylated alditol acetates obtained from blood-group A active tetraglycosylceramide (A) and hexaglycosylceramide (B), respectively. Stationary phase was OV-1, and carrier gas was N . Column temperature was kept at 175°C for 14 min, then raised l°C/min. The designation above the peaks indicate actual binding positions. 2



Figure 10.

Thin-layer pattern with deduced chemical formulas of non-acid glycolipids of epihtelial cells of the two rat strains (cf. Figure 2)

> r

92


D i f f e r e n c e s between the two compartments and between s t r a i n s As shown, the g l y c o l i p i d patterns of e p i t h e l i a l c e l l s and n o n - e p i t h e l i a l residue are d i s t i n c t l y d i f f e r e n t . G l y c o l i p i d s with one, two, three and four hexoses e x i s t i n both compartments. Concerning the two globosides these are present only i n the none p i t h e l i a l f r a c t i o n , which i s demonstrated both by chromatography and by the absence of s p e c i f i c ions at mass spectrometry and s e l e c t e d i o n monitoring of e p i t h e l i a l g l y c o l i p i d s . The g l y c o l i p i d s with f i v e to e i g h t hexoses are a l s o present only i n n o n - e p i t h e l i a l t i s s u e , as are the g l y c o l i p i d s with one hexosamine and a v a r y i n g number of hexoses. F u c o l i p i d s are present i n both compartments. However, the blood group B a c t i v e compound of n o n - e p i t h e l i a l c e l l s (absent i n e p i t h e l i a l c e l l s ) i s based on GalNAc while the H and A a c t i v e substances s p e c i f i c f o r e p i t h e l i a l c e l l s have GlcNAc i n t h e i r core saccharide. In f a c t , GlcNAc seems to be absent from a l l none p i t h e l i a l g l y c o l i p i d s . The minor f u c o l i p i d based on t e t r a h e x o s y l ceramide (as i n d i c a t e d i n f r a c t i o n B of F i g . 5) was obtained from pooled t i s s u e . This g l y c o l i p i d has been shown to be l o c a t e d i n the e p i t h e l i a l c e l l s . In both compartments there are minor slow-moving substances on t h i n - l a y e r chromatography. For example, when p u r i f y i n g and e n r i c h i n g the 12-sugar A a c t i v e g l y c o l i p i d from black and white r a t there appeared more p o l a r m a t e r i a l i n very low amounts, probably being g l y c o l i p i d s having more than 12 sugars. The d i f f e r e n c e between the two s t r a i n s of r a t , the black and white and the white s t r a i n , seems r a t h e r c l e a r . The n o n - e p i t h e l i a l t i s s u e i s i d e n t i c a l f o r the two, i n c l u d i n g the blood group B a c t i v e substance. The d i f f e r e n c e i s found i n the e p i t h e l i a l c e l l s and only concerning f u c o l i p i d s . This i s i l l u s t r a t e d i n F i g . 11 by s e l e c t e d i o n curves a f t e r mass spectrometry and summarized i n F i g . 10. There i s a q u a l i t a t i v e d i f f e r e n c e i n the blood group A type g l y c o l i p i d s with 4, 6 and 12 sugars, these being absent i n e p i t h e l i a l c e l l s of the white r a t . In F i g . 11 there are curves f o r the 4- and 6-sugar compounds (m/e 1125 and 1560, r e s p e c t i v e l y ) i n the black and white but not i n the white r a t . However, the 3and 5-sugar H-type g l y c o l i p i d s (m/e 894 and 1329) e x i s t i n both samples. The 10-sugar H-type g l y c o l i p i d , present i n the white r a t does not show up i n the black and white r a t , probably due to a complete GalNAcd g l y c o s y l a t i o n of the 10-sugar but not of the 3and 5-sugar g l y c o l i p i d s . For some reason the H-type 3-sugar g l y c o l i p i d i s r e l a t i v e l y more abundant i n black and white than i n white r a t ( F i g s . 2, 10 and 11). These r e s u l t s obtained by chemical means were confirmed by immunology, which showed the black and white r a t g l y c o l i p i d s to be blood group A a c t i v e , while those of the white r a t were non-active (Table I ) .



y

Figure 11. Selected ion monitoring from mass spectrometry of glycolipids of epithelial cells of the two rat strains. A total of 200 fig each of the permethylated-reduced mixture was evaporated by a temperature rise of 5°C/min, and mass spectra were recorded each 38 sec. Electron energy was 34 eV acceleration voltage 4 kV, trap current 500 jiA, and ion source temperature 290°C.


:c

Antisera d i l u t i o n

Antisera d i l u t i o n

1:0

1:1

ND means: not determined

"

"

Non-acid g l y c o l i p i d s (mg)

Blood group B a c t i v i t y

Blood group H a c t i v i t y

Blood group A a c t i v i t y

8.2

2+

1.14

Epithelial Cells

10.9

+

ND

1.19

Non-Epithelial Residue

White Rat (8 r a t s )

The Blood Group A c t i v i t i e s Concern G l y c o l i p i d F r a c t i o n s .

14.6

8.2

+

ND

-

4+ ND

1.26

Non-Epithelial Residue

1.32

Epithelial Cells

Black and White Rat (7 r a t s )

Some C h a r a c t e r i s t i c s of D i f f e r e n t Compartments of Rat Small I n t e s t i n e .

T o t a l p r o t e i n (g)

Table I.

6.

BREIMER ET A L .

Epithelial

95

Cells

Gangliosides The s i t u a t i o n f o r g a n g l i o s i d e s i s a l s o complex, with a number of separate s p e c i e s . However, two of these are q u i t e dominating, and are hematoside with N-acetyl and N - g l y c o l o y l s u b s t i t u t i o n , r e s p e c t i v e l y . F i g . 12 shows that the N - a c e t y l type e x i s t s i n none p i t h e l i a l while the N - g l y c o l o y l type i s mostly present i n e p i t h e l ial cells. E p i t h e l i a l C e l l s of D i f f e r e n t L o c a t i o n and M a t u r i t y E p i t h e l i a l c e l l s of small i n t e s t i n e were prepared i n a f r a c t i o n a l way (4), the o l d e r , l e s s adherent v i l l u s t i p c e l l s being washed out by EDTA-containing phosphate b u f f e r f i r s t , while mitot i c crypt c e l l s appeared i n the f i n a l f r a c t i o n s . The enzyme c h a r a c t e r i s t i c s of the s e r i e s of f r a c t i o n s obtained ( F i g . 13) followed conventional c r i t e r i a f o r d i f f e r e n t i a t e d ( v i l l u s ) and l e s s d i f f e r e n t i a t e d (crypt) c e l l s (3, h). The thymidine kinase a c t i v i t y decreased from crypt to v i l l u s while the a c t i v i t y of a l k a l i n e phosphatase increased ( F i g . 13). The c e l l s obtained were pooled i n three f r a c t i o n s , a v i l l u s ( v ) , an intermediate ( i ) , and a crypt (c) f r a c t i o n . The patterns of g l y c o l i p i d s of these are shown i n F i g . 2 (non-acid) and F i g . 12 ( a c i d ) . The only s i g n i f i c a n t d i f f e r e n c e s between the three l o c a l s concern the three major g l y c o l i p i d s and are a succ e s s i v e i n c r e a s e o f monoglycosylceramide ( F i g . 2) and hematoside ( F i g . 12) from crypt to v i l l u s , but a successive decrease of t r i hexosylceramide ( F i g . 2). These f a c t s have been n o t i c e d before (12, 13). Other d i f f e r e n c e s e x i s t but we have not y e t r e s o l v e d and q u a n t i t a t e d a l l minor bands to allow comments on t h i s . There i s a l s o a change i n r e l a t i v e i n t e n s i t y o f the two bands of each of mono- and trihexosylceramide ( F i g . 2 ) . The slower-moving band i s i n c r e a s i n g towards the v i l l u s . Analogous changes are a l s o apparent f o r minor g l y c o l i p i d s . The reason f o r the two bands i s a heterogeneity i n the ceramide p o r t i o n , mainly concerning 2-hydr o x y l a t i o n of the f a t t y a c i d . As the base i s almost e x c l u s i v e l y phytosphingosine a change i n the mass s p e c t r a l fragments f o r ceramide i n d i c a t e d by the formula of F i g . 14 should r e f l e c t the f a t t y a c i d change. Monitoring of these ions through the temperature i n t e r v a l shown should give the composition of a l l g l y c o l i p i d s present. However, as mono- and trihexosylceramides dominate the two major peaks i n d i c a t e d a t about 190°C and 225°C mainly r e f l e c t these two g l y c o l i p i d s , r e s p e c t i v e l y . One should a l s o bear i n mind that the r e l a t i v e p r o p o r t i o n of these two substances changes between the two f r a c t i o n s (see F i g . 2, f r a c t i o n s B and B ) . With t h i s knowledge one may i n t e r p r e t e from the curves of F i g . 14 a r e l a t i v e lengthening o f the f a t t y a c i d and an increased hydr o x y l a t i o n from crypt to v i l l u s . The r e l a t i v e i n c r e a s e i n c h a i n length i s shown by m/e 722 (24:0 hydroxy) compared with m/e 666 (20:0 hydroxy) and m/e 610 (16:0 hydroxy) i n the two f r a c t i o n s , y


c

C E L L SURFACE

Figure 12.

GLYCOLIPIDS

Thin-layer chromatogram of gangliosides of small intestine of black and white (B) and white (W) rat

The fractions and amounts were analogous to those of Figure 2, except for the total fractions (t), where 20 fig glycolipid were used. Bands for N-acetyl (a) and 'N-glycoloyl (b) type of hematoside are indicated. Resorcinol was used for the detection, and the solvent was methyl acetate-2-propanol-CaCl (8 mg/mL)-NH (5M) 45:35:15:10 (by volume). 2

3


1PD WPHVdS

S

IV 13 H3WI3HH


98


but a l s o by m/e 636 (20:0) which i s q u i t e dominating i n the t r i hexosylceramide peak of the crypt f r a c t i o n (225°C) while m/e 692 (24:0) i s the most abundant ion of the v i l l u s f r a c t i o n . The change i n h y d r o x y l a t i o n i s not c l e a r from the curves of Fig.14 without an i n t e g r a t i o n . However, from e a r l i e r experience of the behaviour of molecular species of g l y c o l i p i d s on t h i n - l a y e r chromatography (14) and knowledge of major f a t t y acids present ( F i g . 14) one may conclude that the two bands ( F i g . 2) are mainl y composed of 20, 22, 23 and 24 carbon nonhydroxy acids (upper band) and 20, 22, 23 and 24 carbon hydroxy f a t t y acids (lower band). The change i n f a t t y a c i d composition may be shown f o r separ a t e major or minor g l y c o l i p i d s i n the mixture by s e l e c t i n g fragments s p e c i f i c f o r the species i n question, namely saccharide plus f a t t y a c i d ions which are r e l a t i v e l y abundant (see spectra of F i g s . 7 and 8). One example of t h i s i s shown f o r t e t r a h e x o s y l ceramide i n F i g . 15. The change i s s i m i l a r to that of the t o t a l g l y c o l i p i d s ( F i g . 14). However, an analogous r e t r i e v a l f o r the 4-sugar A-type g l y c o l i p i d (compare F i g . 7) d i d not demonstrate that c l e a r d i f f e r e n c e i n chain length between v i l l u s and crypt cells. Discussion Small i n t e s t i n e i s r e l a t i v e l y r i c h i n g l y c o s p h i n g o l i p i d s (Table I ) . Compared to myelin, a m e t a b o l i c a l l y s t a b l e p o l y membrane s t r u c t u r e (15), a l s o with a high content of g l y c o l i p i d (one sugar), small i n t e s t i n e has a p a t t e r n o f t e n dominated by complex f u c o l i p i d s (5, 16). Of p a r t i c u l a r i n t e r e s t i s the f i n d i n g i n t h i s and recent works (5, 16) of the l o c a l i z a t i o n of these more complex substances to e p i t h e l i a l c e l l s which are s t r u c t u r a l l y complex and asymmetrical. These c e l l s are involved i n important transport and r e c o g n i t i o n processes and have a r a p i d turnover ( 3 ) . This s i t u a t i o n has provided us with an i n t e r e s t i n g object f o r the study of s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s of g l y c o s p h i n g o l i p i d s . Although there i s strong evidence f o r one p a r t i c u l a r g a n g l i o s i d e being the s p e c i f i c receptor f o r c h o l e r a t o x i n (1), there i s at present no good idea about a p h y s i o l o g i c a l f u n c t i o n of a g l y c o l i p i d . A p o s s i b l e exception i s s u l f a t i d e , the only substance with a rather consequent s t o i c h i o m e t r i c r e l a t i o n to a surface membrane f u n c t i o n , i n t h i s case Na and K transport (17, 18). Although the postulated r o l e ( s e l e c t i o n of K ions, 17) i s due to the s u l f a t e group, the sugar part c a r r y i n g t h i s group may be s p e c i f i c a l l y r e quired c l o s e to the membrane matrix. +

+

+

In our i n i t i a l studies reported here of g l y c o l i p i d s of r a t small i n t e s t i n e , p r e p a r a t i v e and s t r u c t u r a l methods were adapted to c h a r a c t e r i z e e p i t h e l i a l and n o n - e p i t h e l i a l t i s s u e and e p i t h e l i a l c e l l s of d i f f e r e n t l o c a t i o n and l e v e l of d i f f e r e n t i a t i o n . The two compartments were d i s t i n c t l y d i f f e r e n t with core saccharides with GalNAc being r e s t r i c t e d to n o n - e p i t h e l i a l c e l l s while those



Villus

ia

0i

-CH-CH— C H

CryPt

Non-hydroxy f a t t y acids

Hydroxy f a t t y acids 580

610 636

666

20:0

Figure 14. Selected ion monitoring from mass spectrometry of villus and crypt epithelial glycolipids of the black and white rat. Relative abundance of ceramide ions was reproduced. A total of 100 fig each of the permethylated mixture was evaporated by a temperature rise of 5°C/min, and spectra were recorded each 38 sec. Electron energy was 49 eV, acceleration voltage 4 kV, trap current 500 fiA, and ion source temperature 290°C.

2

Carbohydrate chain-j-CH CH

16:0

692

722

24:0


2

1

I' 21 200°

1 1

''I'' 37 250°

o

o

scan

5 150°

— 1158

— 1128

Crypt

Hydroxy f a t t y acids 1158

1128

• • i• • 21 200°

Non-hydroxy f a t t y acids

16:0

37 250°

1214

1184

20:0

Figure 15. Monitoring from mass spectrometry of saccharide plus fatty acid ions of tetrahexosylceramide of villus and crypt epithelial cells of black and white rat. Data'were retrieved from 200 /xg of the permethylated-reduced mixtures of total glycolipids, were evaporated at 5°C/min. Spectra recorded each 38 sec. Electron energy was 34 eV, acceleration voltage 4 kV, trap current 500 yiA, and ion source temperature 280°C.

5 150°

•'l' '

— 1158

Villus

Hex—o—Hex—o—Hex-o—Hex—o—CH.CH-

NMe

CH

1270

1240

24:0

6.

BREIMER ET A L .

Epithelial

Cells

101

with GlcNAc were confined to e p i t h e l i a l c e l l s . An unusual blood group B a c t i v e hexaglycosylceramide based on GalNAc and r e s t r i c t ed to n o n - e p i t h e l i a l c e l l s may be i d e n t i c a l with a g l y c o l i p i d detected i n r a t macrophages and granuloma (19). A l l other fucol i p i d s were found i n e p i t h e l i a l c e l l s and based on GlcNAc or l a c k ing hexosamine. Two s e r i e s of f u c o l i p i d s were found i n the black and white s t r a i n , one H a c t i v e with 3, 5 and 10 sugars, and one A a c t i v e with 4, 6 and 12 sugars. The f u c o l i p i d s with 3 and 4 sugars are novel species and based simply on l a c t o s y l c e r a m i d e , demonstrating that the simple d e r i v a t i v e s of reducing l a c t o s e found i n milk (20) have counterparts i n membrane g l y c o l i p i d s . In l a r g e i n t e s t i n e of r a t we have detected d i f u c o s y l substances which are absent from small i n t e s t i n e (unpublished). Further work may show i f these a l s o are analogous to the simple l a c t o s e saccha r i d e s i n milk (20). I t w i l l be i n t e r e s t i n g to see i f the novel s e r i e s of g l y c o l i p i d s i n n o n - e p i t h e l i a l t i s s u e , probably formed by a s e q u e n t i a l a d d i t i o n of Gala, have s p e c i f i c immunological p r o p e r t i e s or can bind c e r t a i n l e c t i n s . Apparently, the two s t r a i n s of r a t both have these g l y c o l i p i d s but d i f f e r i n e p i t h e l i a l c e l l s being blood group A p o s i t i v e or negative. The d i f f e r e n c e between the two s t r a i n s may be explained by the absence of an a-N-acetylgalactosaminyltransferase i n the white s t r a i n . Of i n t e r e s t i s the lack of A a c t i v i t y i n red c e l l s and red c e l l g l y c o l i p i d s of the black and white r a t , which i s s t r o n g l y A p o s i t i v e i n i n t e s t i n a l g l y c o l i p i d s . Both s t r a i n s had, however, B a c t i v i t y both i n t h e i r i n t a c t red c e l l s and i n red c e l l g l y c o l i p i d s . Whether t h i s B a c t i v i t y i s based on the same g l y c o l i p i d as found i n n o n - e p i t h e l i a l t i s s u e remains to be shown. We have p r e l i m i n a r y evidence that t h i s g l y c o l i p i d i s a major component of the complex g l y c o l i p i d s of r a t l i v e r . According to Table I, the e p i t h e l i a l c e l l s of the black and white s t r a i n were r i c h e r i n g l y c o l i p i d s , and according to F i g s . 10 and 11 the same s t r a i n contained more f u c o l i p i d expressed as the Htype 3-sugar g l y c o l i p i d . In view of current d i s c u s s i o n s on a p o s s i b l e r o l e of c e l l surface saccharides i n c o n t r o l of growth and d i f f e r e n t i a t i o n (1, 2), the changes found i n e p i t h e l i a l c e l l s undergoing a successive maturation from crypt to v i l l u s t i p are, as a f i r s t impression, s u r p r i s i n g l y small. An increase i n monoglycosylceramide and hematoside and a decrease i n trihexosylceramide, the three major g l y c o l i p i d components, was found. A l s o , the ceramide of these g l y c o l i p i d s undergoes a successive change from crypt to v i l l u s with a chain lengthening and a 2-hydroxylation of the f a t t y a c i d . Concerning the more complex f u c o l i p i d s , these are present already i n the crypt c e l l s (see F i g . 2 f o r 10- and 12-sugar compounds) i n d i c a t i n g "a need" f o r these surface saccharides already i n crypt c e l l s . An extension of the saccharide chains p a r a l l e l to the process of maturation (1, 2) was t h e r e f o r e not found. One should, however, bear i n mind the extreme complexity of the e p i t h e l i a l c e l l being h i g h l y asymmetric with a surface


102


membrane (where g l y c o l i p i d s are supposed to be located) d i v i d e d mainly i n t o a brush border, f a c i n g the i n t e s t i n a l lumen, and a b a s o l a t e r a l membrane, being i n contact with other e p i t h e l i a l c e l l s and the b a s a l membrane. So f a r we have only studied whole c e l l s and not yet r e s o l v e d minor components f o r a p r e c i s e q u a n t i t a t i o n . A s u b c e l l u l a r f r a c t i o n a t i o n i n t o separate type of surface membrane and g l y c o l i p i d a n a l y s i s may r e v e a l i n t e r e s t i n g both q u a l i t a t i v e and q u a n t i t a t i v e d i f f e r e n c e s . In f a c t , Lewis and coworkers (21) have shown that preparations of brush border and b a s o l a t e r a l membranes of guinea-pig small i n t e s t i n e had d i f f e r e n t g l y c o l i p i d p a t t e r n s . The g l y c e r o l i p i d s of the two regions were f a i r l y s i m i l a r but t r i - and t e t r a g l y c o s y l c e r a m i d e s were more concentrated i n the b a s o l a t e r a l membranes, whereas mono- and diglycosylceramides and s u l f a t i d e were enriched i n the brush border membranes. For human (16) and dog small i n t e s t i n e 05, 22) i t has been shown that globoside and the Forssman hapten, r e s p e c t i v e l y , are l o c a t e d i n n o n - e p i t h e l i a l c e l l s , while f u c o l i p i d s are present i n e p i t h e l i a l c e l l s . This i s s i m i l a r to the f i n d i n g s of t h i s paper. A l s o , g l y c o l i p i d s of e p i t h e l i a l c e l l s (5, 16^, 22) had a more hydroxylated ceramide (phytosphingosine and 2-hydroxy f a t t y acid) than n o n - e p i t h e l i a l c e l l s (sphingosine and nonhydroxy f a t t y a c i d ) . An analogous s i t u a t i o n was found f o r r a t small i n t e s t i n e , a l though the d i f f e r e n c e s were not that c l e a r c u t , as nonhydroxy a c i d s were a l s o present i n e p i t h e l i a l c e l l s and phytosphingosine was a l s o present to some extent i n n o n - e p i t h e l i a l c e l l s . The extent of 2-hydroxylation increased from crypt to v i l l u s t i p ( F i g s . 2 and 14). The meaning of these d i f f e r e n c e s i n ceramide h y d r o x y l a t i o n (from one to three hydroxy groups) i s not known. A model has, however, been proposed, with a system of l a t e r a l l y o r i e n t e d hydrogen bonds along the membrane at t h i s l e v e l of ceramide i n the membrane matrix (17). The e p i t h e l i a l c e l l s of i n t e s t i n e , e s p e c i a l l y those of the v i l l u s , are exposed to an i n t e s t i n a l content of h i g h l y v a r y i n g composition (both h y d r o p h i l i c and hydrophobic) and may need a more t i g h t and s t a b l e surface membrane produced by an increased h y d r o x y l a t i o n of ceramide. As already mentioned the e p i t h e l i a l c e l l s of small i n t e s t i n e are involved i n a number of enlarged transport processes and a l s o i n b i o l o g i c a l r e c o g n i t i o n . S u r p r i s i n g l y , the a c i d g l y c o l i p i d f r a c t i o n of r a t small i n t e s t i n e lacked the animal s u l f a t i d e (ceramide g a l a c t o s e - 3 - s u l f a t e ) , which i s a major component of human i n t e s t i n e (16) and a l s o of small i n t e s t i n e of s e v e r a l animals (cat, guinea-pig, hen and r a b b i t , unpublished). As f o r the r a t , t h i s l i p i d was absent i n small i n t e s t i n e of mouse and cod f i s h (unpublished). The lack of s u l f a t i d e i s unexpected i n view of the postulated r o l e of t h i s l i p i d as a K receptor i n Na and K transport (17, 18) and the dominance of N a t r a n s p o r t i n small i n t e s t i n e as a primary d r i v e f o r the transport of a number of other molecules. However, i n these cases the molecule may be r e placed by the g l y c e r o l - b a s e d s u l f a t i d e , which i s removed i n the +

+

+


+

6.

BREIMER ET AL.

Epithelial Cells

103

standard procedure of mild alkaline degradation. The recognition processes of interest in relation to cell surface saccharides and intestinal epithelial cells are of at least two kinds. One is the exposure of primarily the brush border membrane for a number of foreign molecules and microorganisms (or products of these) in the intestinal contents. A role for carbohydrate in the binding of bacteria in the mechanism of infection in epithelia has been postulated (1). The second kind of recognition is the association of autologous cells with each other, which should take place in the alteral membranes, and the attachment of the cells to the basal membrane during movement from crypt to villus tip. As a first step in the study of small intestine the present work has defined to some extent the difference concerning cell surface glycolipids between epithelial and non-epithelial cells and between whole epithelial cells of different maturity. As a next step it would be relevant to investigate the composition of separate types of surface membranes. Also, the large intestine of the same strains of rat, with a somewhat separate profiel of functions, may profile supplementary information. Acknowledgement The work was supported by a grant from the Swedish Medical Research Council (No. 3967). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Hughes, C.L.; Sharon, N. Trends Biochem. Sci. 1978, 3, N 275. Marchesi, V.T.; Ginsburg, V.; Robbins, P.W.; Fox, C.F.; Eds. "Cell Surface Carbohydrates and Biological Recognition"; Alan R. Liss, Inc.; New York, 1978. Lipkin, M. Physiol. Rev. 1973, 53, 891. Weiser, M.M. J.Biol. Chem. 1973, 248, 2536. McKibbin, J.M. J. Lipid Res. 1978, 19, 131. Karlsson, K.-A. InWitting,L.A., Ed. "Glycolipid Methodology"; American Oil Chemists' Society: Champaign, Illinois, 1976; p. 97. Breimer, M.E.; Hansson, G.C.; Karlsson, K.-A.; Leffler, H.: Pimlott, W.; Samuelsson, B.E. Biomed. Mass Spectrom. 1979, 6, 231. Falk, K.-E.; Karlsson, K.-A.; Samuelsson, B.E. Arch. Biochem. Biophys. 1979, 192, 164. Falk, K.-E.; Karlsson, K.-A.; Samuelsson, B.E. Arch. Biochem. Biophys. 1979, 192, 177. Falk, K.-E.; Karlsson, K.-A.; Samuelsson, B.E. Arch. Biochem. Biophys. 1979, 192, 191.


104


11.

Laine, R.A.; Stellner, K.; Hakomori, S.-i. In Korn, E.D., Ed. "Methods in Membrane Biology"; Plenum Press: New York, 1974; Vol. 2, p. 205. Bouhours, J.-F.; Glickman, R.M. Biochim. Biophys. Acta 1976, 441, 123. Glickman, R.M.; Bouhours, J.-F. Biochim. Biophys. Acta 1976, 424, 17. Karlsson, K.-A.; Samuelsson, B.E.; Steen, G.O. Biochim. Biophys. Acta 1973, 306, 317. Morgan, I.G.; Gombos, G.; Tettamanti, G. In Horowitz, M.I.; Pigman, W.; Eds. "The Glycoconjugates"; Academic Press: New York, 1977, Vol. I, p. 351. Falk, K.-E.; Karlsson, K.-A.; Leffler, H.; Samuelsson, B.E. FEBS Lett. 1979, 101, 273. Karlsson, K.-A. In Abrahamsson, S.; Pascher, I.; Eds. "Structure of Biological Membranes"; Plenum Press: New York, 1977, p. 245. Hansson, G.C.; Heilbronn, E.; Karlsson, K.-A.; Samuelsson, B.E. J. Lipid Res. 1979, 20, 509. Hanada, E.; Handa, S.; Konno, K.; Yamakawa, T. J. Biochem. 1978, 83, 85. Kobata, A. In Horowitz, M.I.; Pigman, W.; Eds. "The Glycoconjugates"; Academic Press: New York, 1977, Vol. I, p. 423. Michell, R.H.; Coleman, R.; Lewis, B.A. Biochem. Soc. Trans. 1976, 4, 1017. Smith, E.L.; McKibbin, J.M.; Karlsson, K.-A.; Pascher, I.; Samuelsson, B.E. Biochim. Biophys. Acta 1975, 388, 171. Dell, A.; Williams, D.H.; Morris, H.R.; Smith, G.A.; Feeney, J.; Roberts, G.C.K. J. Am. Chem. Soc. 1975, 97, 2497.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

RECEIVED

December 10, 1979.


Glycolipids of Rat Small Intestine with Special Reference to Epithelial

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