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Biochimica et Biophysica Acta, 498 (1977) 114--122 © Elsevier/North-Holland Biomedical Press

BBA 28256

TISSUE SPECIFIC DISTRIBUTION OF SULFATED MUCOPOLYSACCHARIDES IN MAMMALS

OLGA M.S. TOLEDO and CARL P. DIETRICH

Departamento de Bioqm'mica e Farmacologia, Escola Paulista de Medicina, C.P. 20372, 01000 S~o Paulo, SP (Brazil)

(Received December 14th, 1976)

Summary

A comparative study on the distribution of sulfated mucopolysaccharides in several tissues of five mammalian species is reported. It is shown that each tis- sue has a characteristic composition differing from each other regarding the relative amount, type and molecular size of chondroitin sulfate A/C, chondroi- tin sulfate B and heparan sulfate. It is also shown that the same tissue from dif- ferent mammals has the same types and proportions of sulfated mucopolysac- charides, but with different molecular weights. Exception to this rule was observed for the distribution of heparin which was present only in a few tissues of the five mammals studied.

The possible involvement of the sulfated mucopolysaccharides in cell recog- nition and/or adhesiveness is discussed in view of this characteristic distribu- tion.

Introduction

We have recently shown that each rat tissue and its respective mitochondria have a characteristic sulfated mucopolysaccharide composition, differing from each other regarding the relative amount, type and molecular size of chondroi- tin sulfates A/C, chondroitin sulfate B and heparitin sulfate [1]. It was also recently shown that these compounds are present in variable amounts and types in 22 species of invertebrates belonging to representative phyla of the animal kingdom [2,3]. Conversely no sulfated mucopolysaccharides could be found in different species of bacteria, protozoa and fungi analysed {Dietrich,

Abbreviat ions: c h on d r o i t in sulfate A, chondroi t in 4-sulfate; cho ndro i t in sulfate B, dermatan sul- fate; chondro i t i n sul fate C, c h on d r o i t in 6-sulfate ;A Di-4S, 2-acetamido-2-deoxy-3-O-(glyco-4-ene- pyranosyluronic acid)-4-O-sulfo-D-galactose; A Di-6S, 2-acetamido-2-deoxy-3-O-(~-D-gluco-4-ene- pyranosyluronic acid)-6-O-sulfo-D-galact ose.

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unpublished). These results led to the suggestion that the emergence of sulfated mucopolysaccharides corresponds to the emergence of tissue-organized life forms [3].

All these data are in agreement with the hypothesis that sulfated mucopoly- saccharides might be involved in the process of cell differentiation, confering to the cells some of their particular properties such as recognition and/or adhesive- ness [1 ].

In order to gather more information concerning this hypothesis, a compara- tive study on the distribution of sulfated mucopolysaccharides in mammalian tissues was undertaken.

The present paper reports the sulfated mucopolysaccharide composition of several tissues from different mammals. A preliminary communication of part of these findings has appeared [4].

Materials and Methods

Materials. Chondroitin sulfates A, B and C, chondroitinases AC and ABC were purchased from Miles Laboratories (Elkhart, Ind.). Heparin was a kind gift from Dr. M.B. Mathews {University of Chicago). Heparan sulfate B, heparinase and heparitinase were prepared by methods previously described [ 5,6]. Agarose was purchased from L'Industrie Biologique Francaise (Gennevilliers, France). Diamino propane was purchased from Aldrich Chemical Co. {Milwaukee, Wisc.).

Extraction of sulfated mucopolysaccharides. The tissues from the following adult animals were obtained immediately after death: guinea pig, rabbit, dog, hog and beef. Human tissues were obtained from autopsies of individuals that died within 12 h as a result of accidents. The tissues were ground with 10 volumes of acetone and after standing overnight at 4°C the mixture was cen- trifuged, washed once with acetone and dried under vacuum. The sulfated mucopolysaccharides were then extracted from the acetone powders essentially as previously described [1]. In those tissues which were later found to contain heparin an alternative procedure modified from Scott et al. [ 7 ] was used as fol- lows: I g of dry tissue was suspended in 10 ml of 0.05 M Tris • HC1 buffer, pH 8.0. The suspension was incubated at 30°C for 24 h with 10 mg of trypsin and a few drops of toluene. After the incubation 1.98 g of anhydrous potassium acetate were added to the incubate and the mixture shaken to insure complete solution of the salt. The pH was adjusted to 5.7 and the suspension maintained in an oven for 2 h at 60°C. About 100 mg of Celite was added to the suspen- sion which was then filtered at 60 ° C. The filtrate was collected in a centrifuge tube and kept at 3°C overnight. After removal of the supernatant by centrifu- gation the precipitate was dissolved in 0.5 ml of water. Two volumes of alcohol were added to this solution. After standing for at least 4 h in the cold the pre- cipitate formed was collected by centrifugation and dried. It was then dissolved in 10--100 pl of water and analysed for heparin. To the supernatant remaining after precipitation of heparin in the cold, two volumes of alcohol were added. The precipitate formed was collected by centrifugation, dried and resuspended in 100 pl and also analysed. The recovery of sulfated mucopolysaccharides by this last procedure was comparable to the previous method used [1]. Further-

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more this method had the advantage of separating heparin from the other sul- fated mucopolysaccharides. Recovery experiments with heparin and the other sulfated mucopolysaccharides have shown that only heparin precipitates with potassium acetate and all the other sulfated mucopolysaccharides remain in the supernatant completely free of heparin.

Identification and quantitation of sulfated mucopolysaccharides. The sul- fated mucopolysaccharides were identified and quanti tated by a combination of agarose gel electrophoresis and enzymatic degradation with specific muco- polysaccharides as previously described [1,8,9]. United States Pharmacopea antiagulant-activity assay was used as well to identify heparin. Mucopoly- saccharide quantitation was performed by densi tometry of the agarose slides after electrophoresis and toluidine blue staining. The error of the method was of the order of -+ 4.5%. The extinction coefficients of the mucopolysaccharides were calculated using standards of chondroitin sulfate A, chondroitin sulfate B, heparan sulfate and heparin. Six successsive extractions from four different tis- sues have shown a 13% variation of the total sulfated mucopolysaccharides extracted although no significant changes in the proport ions of the compounds were observed. Paper chromatography of the products formed after enzymatic degradation was performed in isobutyric acid, 1 M NH3 (5 : 3, v/v). The rela- tive amounts of the disaccharide products were measured after silver nitrate and/or toluidine blue staining by methods previously described [ 10]. Molecular weight determinations were performed by polyacrylamide gel electrophoresis [5] after fractionation of the individual mucopolysaccharides by large scale agarose gel electrophoresis in propanediamine/acetate buffer [6].

Results

Sulfated mucopolysaccharide composition of selected tissues from several mammalian species

Fig. 1 shows the agarose gel electrophoresis of sulfated mucopolysaccharides obtained from different human tissues. Each tissue has a characteristic sulfated mucopolysaccharide composit ion differing from each other in the relative amount and type of sulfated mucopolysaccharides. This characteristic distribu- tion is similar to the one reported previously for rat tissues [1]. The compara- tive sulfated mucopolysaccharide composit ion of selected tissues of different mammalians is shown in Table I and Fig. 2. Most of the tissues possess the same types of sulfated mucopolysaccharides. For instance all the brain tissues anal- ysed contain mainly chondroitin sulfate A whereas muscle from the different species contain chondroitin sulfate B and kidney, heparan sulfate. With some exceptions the relative proport ions of sulfated mucopolysaccharides of the same tissue of the different species also do not vary significantly. As examples brain tissues contain about 80% chondroitin sulfate A and 20% heparan sulfate; kidney tissues contain about 10% of chondroitin sulfate A/C, 25% of chondroi- tin sulfate B and 65% of heparan sulfate. Much more significant variations are observed when the different tissues of the same species are compared (Table I).

Average molecular weight of the sulfated mucopolysaccharides from tissues of different species

Fig. 3 shows a polyacrylamide gel electrophoresis of the sulfated mucopoly-

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t

I I I 1 1 i I t t I , S 1 23 4 S 5678

Fig. 1. S u l f a t e d m u c o p o l y s a c c h a r i d e s f r o m d i f f e r e n t h u m a n t issues. A b o u t 1 0 0 p g o f s u l f a t e d m u c o p o l y - s a c c h a r i d e s o b t a i n e d f r o m d i f f e r e n t t i ssues we re a p p l i e d in 5 X 7 .5 cm, 2 m m t h i c k aga rose gel s lab (0 .9% aga rose in 0 . 0 5 M p r o p a n e d i a m i n e / a c e t a t e b u f f e r , p H 9 .0 ) a n d s u b j e c t e d t o e l e c t r o p h o r e s i s ( 1 0 0 V f o r 1 h) a t 5°C . T h e s u l f a t e d m u c o p o l y s a c c h a r i d e s we re v i sua l ized in the gel a f t e r f i x a t i o n w i t h c e t y l t r i m e t h y l - a m m o n i u m b r o m i d e (Ce tav lon ) , d r y i n g a n d s t a in ing w i t h t o l u i d i n e b lue . S, s u l f a t e d m u c o p o l y s a c c h a r i d e s s t a n d a r d m i x t u r e ;1 , s k i n ; 2, i l eum; 3, m u s c l e ; 4, b r a i n ; 5, l ung ; 6 , a o r t a ; 7, k i d n e y ; 8, Hver. CHS, c h o n - d r o i t i n su l f a t e ; HTS , h e p a r a n su l fa te ; H E P , h e p a r i n .

saccharides extracted from liver and kidney of the different species. Some of the heparan sulfates possess different electrophoretic mobilities and different degrees of polydispersity. The calculated average molecular weight of these compounds is shown in Table II. The average molecular weight of heparan sul- fate from liver varied from 11 000 to 30 000. The same variation of molecular weight and dispersity were also found for chondroitin sulfate B which varied

! I~lSI1E ILEIJ KIDNEY

QIS~

t F [ S 1 2

Fig. 2. A g a r o s e gel e l e c t r o p h o r e s i s o f s u l f a t e d m u c o p o l y s a c c h a r i d e s f r o m se lec t ed t i s sues o f d i f f e r e n t m a m m a l i a n species . T h e e x p e r i m e n t w a s p e r f o r m e d as d e s c r i b e d in Fig . 1 e x c e p t t h a t s u l f a t e d m u c o p o l y - s a c c h a r i d e s f r o m d i f f e r e n t t i ssues w e r e u s e d as i n d i c a t e d . S, s u l f a t e d m u c o p o l y s a c c h a r i d e s s t a n d a r d m i x - t u r e ; 1, g u i n e a pig; 2, r a b b i t ; 3, d o g ; 4, h o g ; 5, h u m a n . CHS, c h o n d r o i t i n su l fa te ; H T S , h e p a r a n su l fa te ; H E P , h e p a r i n .

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T A B L E I

S U L F A T E D M U C O P O L Y S A C C H A R I D E C O M P O S I T I O N OF M A M M A L I A N T I S S U E S

Tissue M a m m a l s To ta l su l fa ted Su l fa t ed m u c o p o l y s a c c h a r i d e s (%) a m u c o p o l y -

sacchar ides C h o n d r o i t i n Ch o n d r o i t i n H e p a r a n

(pg /g d ry sul fa te AC su l fa te B sul fa te t i ssue)

He pa r in

Brain Gru inea -p ig 192 83 b < 2 17 - -

R a b b i t 143 79 < 2 21 - -

Dog 392 76 < 2 24 - -

Hog 148 79 < 2 21 - -

H u m a n 385 86 < 2 14 < 0 . 5

K i d n e y Guinea -p ig 406 < 2 22 78 - -

R a b b i t 121 12 24 62 - -

D o g 534 13 26 61 < 0 . 5

Hog 300 5 22 73 < 0 . 5 H u m a n 150 15 26 59 < 0 . 5

Liver Guinea -p ig 204 < 2 39 61 < 0 . 5

R a b b i t 106 < 2 53 47 - -

Dog 292 < 2 17 69 14.0 Hog 156 < 2 34 66 < 0 . 5

H u m a n 120 < 2 44 56 < 0 . 5

I l eum Guinea-p ig 680 16 29 51 4 .0

R a b b i t 481 10 37 52 < 0 . 5

Dog 892 2 38 57 2.0

Hog 705 6 33 51 16.0

H u m a n 299 5 50 45 < 0 . 5

A o r t a Guinea -p ig 5043 64 c 30 5 < 0 . 5

R a b b i t 5298 62 31 6 - -

H o g 4581 76 9 15 < 0 . 5

H u m a n 8291 73 10 17 < 0 . 5

L u n g Guinea -p ig 1312 44 d 25 29 3.0

R a b b i t 1754 51 19 30 < 0 . 5

Dog 1195 < 2 27 44 29.0

H o g 765 43 20 33 5.0 Beef 1516 36 27 34 3.0

H u m a n 754 < 2 43 57 < 0 . 5

Muscle Gu inea -p ig 572 3 78 20 < 0.5 R a b b i t 650 6 78 15 - -

H u m a n 316 14 61 24 ~ 0 . 5

Sk in R a b b i t 701 < 2 92 8 - -

H u m a n 1300 < 2 94 3 3 .0

Pancreas D o g 57 ~ 2 42 44 14 .0

a T h e l im i t o f d e t e c t i o n o f c h o n d r o i t i n su l fa te AC, c h o n d r o i t i n su l fa te B and h e p a r a n su l fa te by the p r e s e n t m e t h o d is a r o u n d 2 #g/g of dry t issue. F o r h e p a r i n the l imi t o f d e t e c t i o n is a r o u n d 0.5 pg/g of dry t issue.

b On ly A Di-4S was d e t e c t e d a f t e r d e g r a d a t i o n w i t h e h o n d r o i t i n a s e AC.

c A b o u t 70% o f A Di-6S and 30% o f ~ Di-4S were f o u n d a f t e r d e g r a d a t i o n w i t h c h o n d r o i t i n a s e AC. d

A b o u t 85% o f A Di-4S and 15% o f A Di-6S were f o u n d a f t e r d e g r a d a t i o n w i t h c h o n d r o i t i n a s e AC.

f rom 5000 to 63 000 in the different species. Some of the livers contained up to three types of chondroi t in sulfate B (Table II). In contrast chondroi t in sul- fate B obtained from different kidneys did not show apreciable differences. All

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ORIGIN

Fig. 3. P o l y a c r y l a m i d e gel e lec t rophores i s of su l fa ted m u c o p o l y s a c c h a r i d e s f rom liver and k idney of dif- f e ren t m a m m a l i a n species. A b o u t 50 pg of su l fa ted m u c o p o l y s a c c h a r i d e s f rom liver and k idney of differ- en t species were appl ied in 5 × 7.5 cm p o l y a c r y l a m i d e gel slabs, 2 m m th ick (6% p o l y a c r y l a m i d e in 0 .06 M barb i ta l b u f f e r pH 8.5) a nd sub jec ted to e lec t rophores l s (100 V) for 40 min . Th e su l fa ted m u c o p o l y - sacchar ides were s ta ined wi th to luidine blue (0.1% to lu id ine b lue in 1% acet ic acid). S, hepa r i t i n sulfate C; 1, gu inea pig; 2, rabbi t ; 3, dog; 4, hog; 5, h u m a n . CHS, c hond ro i t i n sulfate; HTS, h ep a ran sulfate.

T A B L E II

M O L E C U L A R W E I G H T OF S U L F A T E D M U C O P O L Y S A C C H A R I D E S FROM L I V E R A N D K I D N E Y OF D I F F E R E N T MA MMALS

Tissue M a m m a l Molecular we igh t

Chond ro i t i n sulfate B H e p a r a n sulfa te

Range Mode Value Range Mode Value

Liver Gu inea pig 4 4 0 0 - - 8 800 6 400 8 8 0 0 - - 4 0 000

R a b b i t 5 6 0 0 - - 1 3 500 6 700 13 5 0 0 - - 4 0 000

Dog 2 6 0 0 - - 7 800 5 200 10 2 0 0 - - 1 3 500 11 850 13 500----68 000

H og 4 500---61 500 -~ 61 500 - -68 000 63 500

H u m a n 11 500- -56 000 Kidney Rabb i t - - 7 000

- - 24 500 Dog - - 6 400

- - 20 000 Hog -- 8 600

- - 22 000 Human -- 7 000

-- 24 500 Lung H u m a n -- --

I l eum H u m a n - - - -

A o r t a H u m a n -- --

4 700--50 000 16 500

4 700--33 000 19 500

5 200--33 000 29 500

8 600--16 000

m

m

m

w

11 000

5 500

2 5 0 0 10 500 13 000 2 I00

18 000

3 600 47 0 0 0

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of them have shown two components with average molecular weights of about 7000 and 22 000.

The distribution of heparin in tissues of different species The amount of heparin extracted from different tissues is shown in Table I.

The relative amount of heparin varied from 3% in human skin to 29% in dog liver of the total sulfated mucopolysaccharides. An erratical distribution of heparin is observed when the different tissues of the five species are compared. For instance, in humans, heparin is present in detectable amounts only in the skin, whereas dog contains heparin in most of the tissues analysed. Only slight variations of electrophoretic migrations in agarose gel was observed for all the heparins analysed (Fig. 4). On the other hand the average molecular weight of the heparins varied from about 7500 up to 30 000 (Table III). The anticoagu- lant activity of the different heparins varied from 60 I.U./mg (human skin) up to 170 I.U./mg (dog ileum and lung).

Identification of the sulfated mucopolysaccharides The identification of the sulfated mucopolysaccharides from the tissues were

based on the following results. Chondroitin sulfate A/C: same electrophoretic migration in two buffer systems as the standard chondroitin sulfate A/C; degra- dation by chondroitinases AC and ABC but not by the heparinase and hepari- tinases, formation of 4- and 6-sulfated disaccharides after degradation with chondroitinase AC. Chondroitin sulfate B: same electrophoretic migration in two buffer systems as the standard chondroitin sulfate B; degradation by chon-

12 3 4 5 6 7 1 2 34567

[G IN

Fig. 4. Agarose and p o l y a c r y l a m i d e gel e l ec trophores i s o f heparin f r o m d i f ferent t issues o f s o m e mamma- lian species . The e x p e r i m e n t s w e r e p e r f o r m e d as descr ibed in Figs. 1 and 3 e x c e p t that beparin f r o m dif- f erent t issues w as used. 1, pig ileum; 2, dog ileum; 3, dog liver ;4, dog lung; 5, hog lung ;6, beef lung; 7, heparin standard.

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T A B L E I I I

A N T I C O A G U L A N T A C T I V I T Y A N D A V E R A G E M O L E C U L A R W E I G H T OF H E P A R I N S O B T A I N E D F R O M D I F F E R E N T TISSUES OF SOME M A M M A L S

M a m m a l s Tissue A n t i c o a g u l a n t A v e r a g e m o l e c u l a r we igh t ac t iv i ty ( I . U . / m g )

Dog I l eum 174 8 200 L u n g 171 9 300 Liver - - 9 9 0 0

30 000 P a n c r e a s 99

Hog I l eum 75 18 400 L u n g 69 7 600

Beef L u n g 150 9 300

H u m a n Skin 60 - -

droitinase ABC but not by chondroitinase AC, heparitinase and heparinase; for- mation of 4-sulfated disaccharide by the action of chondroitinase ABC. Hepari- tin sulfate: same electrophoretic migration in two buffer systems as the stan- dard heparitin sulfate; degradation by heparitinases but not by the chondroiti- nases AC and ABC; formation of glucosamine 2,6-disulfate and N-acetylglucos- amine by the action of crude induced F-heparinum extracts. Heparin: same electrophoretic migration in two buffer systems as the standard heparin; degra- dation by heparinase but not by the heparitinases, chondroitinases AC and ABC; formation of trisulfated disaccharide and sulfated tetrasaccharide by the action of the heparinase; anticoagulant activity similar to heparin standard.

Besides these properties the four types of sulfated mucopolysaccharides iso- lated from the tissues were precipitated by cetyl t r imethylammonium in the agarose gels and exhibited the characteristic metachromatic colour after tolui- dine blue staining.

Discussion

The presence of sulfated mucopolysaccharides in tissues individually exa- mined have been extensively demonstrated e.g. see refs. 11--15. Nevertheless, to our knowledge, studies on comparative distribution of sulfated mucopoly- saccharides either between different tissues of the same species or same tissues of different species have not appeared.

The results presented in this paper show that there is some tissue specific dis- tr ibution of sulfated mucopolysaccharides. The relative proport ions of heparan sulfate, chondroitin sulfate B and chondroitin sulfate A/C are somewhat con- stant for a specific tissue regardless of the mammalian species analysed.

It is also clear that each tissue obtained from five different mammals has a characteristic composit ion of sulfated mucopolysaccharides as has been shown previously for rat tissues [1]. Nevertheless, variations in microheterogeneity (e.g. molecular weight) was observed in most cases when the sulfated muco- polysaccharides obtained from the same tissue of the different mammalian spe- cies were compared. These variations were also observed when the sulfated

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mucopolysaccharides obtained from different tissues of the same species were compared. These last results suggest that in general, each tissue from each spe- cies has its particular sulfated mucopolysaccharide composition which differs from other tissues or from the same tissue of other species.

If this assumption were extended to other mammalian tissues, we could con- clude that there is an incredible variety of heparan sulfates and chondroitin sul- fates.

All these results are in agreement with the earlier proposition that the sul- fated mucopolysaccharides might play a role in cell differentiation [1]. On the other hand, the results on the distribution of heparin in mammalian tissues do not conform to the above hypothesis. The absence of heparin from all human tissues but skin, together with its presence in most dog tissues, suggests a dif- ferent role for this compound as compared to the other sulfated mucopolysac- charides.

Acknowledgements

We wish to thank Dr. Sonia M.C. Dietrich for help in the preparation of this manuscript. This work was aided by grants from F u n d a ~ o de Amparo ~ Pes- quisa do Estado de S~o Paulo (FAPESP), Conselho Nacional de Desenvolvimen- to Cientffico e Technolbgico (CNPq) and Financiadora de Estudos a Projetos (FINEP).

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