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Comp. Biochem. Physiol. Vol. 73B, No. 4, pp. 857 to 863, 1982 0305-0491 82 120857-07503.00~0 Printed in Great Britain. © 1982 Pergamon Press Ltd
DISTRIBUTION OF H E P A R I N A N D OTHER S U L F A T E D G L Y C O S A M I N O G L Y C A N S IN
VERTEBRATES*
PURIFICACION B. GOMF, S and CARt P. DIETRICH
Departmento de Bioquimica, Escola Paulista de Medicina, CP 20372, 01000 Silo Paulo. SP, Brazil
(Receiced 7 May 1982)
Abstract--1. A comparative study on the distribution of sulfated glycosaminoglycans in several tissues of six specimens of different vertebrate classes is reported.
2. Each tissue has a characteristic composition differing from each other regarding the relative amount, type and molecular size of chondroitin 4, 6-sulfate, dermatan sulfate and heparan sulfitte.
3. The same tissue from different vertebrates has, in general, the same types of sulfated glycosamino- glycans, but with different molecular weights.
4. Exception to this rule wits observed for the distribution of heparin which was present only in a few tissues of the 6 vertebrates studied.
5. The possible involvement of the sulfated glycosaminoglycans in cell recognition and/or adhesive- ness is discussed in view of this characteristic distribution.
INTRODUCTION
We have previously repor ted that each tissue of several m a m m a l i a n species has a character is t ic sul- fated g lycosaminoglycan compos i t ion , differing from each o ther regarding the relative amount , type and molecular size of chondro i t in 4,6-sulfates, de rma tan sulfate, heparan sulfate and hepar in (Dietrich et al., 1976; Toledo & Dietrich, 1977). It was also shown that these c o m p o u n d s are present in variable amoun t s and types in 22 species of inver tebrates be longing to representa t ive classes of the animal k ingdom (Cassaro & Dietrich, 1977). Conversely no sulfated glycos- aminoglycans could be found in different species of bacteria, p r o toz oa and fungi analysed. These results led to the suggestion that the emergence of sulfated g lycosaminoglycans co r r e sponds to the emergence of t issue-organized life forms (Cassaro & Dietrich, 1977).
These and o ther data are in agreement with the hypothes is that these c o m p o u n d s might be involved in the process of cell differentiation, confer ing to the cells some of their par t icular proper t ies such as recog- ni t ion and /o r adhesiveness (Dietrich et al., 1977).
In order to ga ther more in format ion concern ing this hypothesis , a compara t ive study on the distr ibu- t ion of sulfated g lycosaminoglycans in several tissues of 6 different classes of ver tebrates was under taken.
* Aided by grants from FAPESP (Fund,'l~ztio de Amparo Pesquisa do Estado de Silo Puulo), FINEP [Financia-
dora de Estudos e Projetos) and CNPq (Conselho Natio- nal do Desenvolvimento Cientifico e Tecnol6gico).
Abbreviations: ADi-OS, 2-acetamido 2-deoxy-3-O-(fl-i)- glucoenepyranosylu ronic acid)-D-galactose : ADi-4S, 2 - acetamido - 2 - deoxy - 3 - O- (glyco - 4 - enepyranosyl uronic acid}-4-O-sulfo-l>galactose; ADi-6S, 2-acetamido-2-deox- y-3-O-(fl-D-ggluco-4-enepyranosyluronic acid)-6-O-sulfo- D-galactose; GlcN 2, 6S, glucosamine, 2,6-dissulfate.
MATERIALS AND METHODS
Materials Chondroitin 4 and 6-sulfutes, dermatan sulfate, chon-
droitinases AC and ABC were purchased from Miles Laboratories {Elkhart, IN). Heparin was a kind gift from Dr. M. B. Mathews (University of Chicago). Heparan sul- fate, heparinase and heparitinases were prepared by methods previously described (Dietrich & Nader, 1974: Silva & Dietrich. 1975). Agarose was purchased from Aldrich Chemical Co. (Milwaukee, WI}.
E.\'tractioal ~1 su!ii~ted !llycosamim)cllycaJts The tissues from the following adult vertebrates were
obtained immediately after death: chicken {Gallus ,qullus), snake (XeplodoJl merre mii), lizard (Tupimmlhis te,quixin), frog (Bt
858 PURIF1CACION B. GOMES and CARL P. DIETRICH
+ , , + s ,
Fig. I. Agarose gel electrophoresis of sulfated glycosaminoglycans of different vertebrate tissues. About 10pg of sulfated glycosaminoglycans were applied to agarose gel slabs (5 × 7.5cm, 0.2 mm thickt prepared in 0.05 M diaminopropane acetate buffer pH 9.0 and subjected to electrophoresis (100V, 90 min). The compounds were then fixed with CETAVLON and stained with toluidine blue. (a) Chicken : I, ileum; 2, lung; 3, kidney; 4, liver; 5, brain. (b} Snake: 1, skin; 2, liver; 3, kidney: 4, lung; 5. muscle. (c) Lizard: 1, muscle: 2, liver; 3, ileum; 4, lung. (d) Frog: 1, skin; 2, liver; 3. kidney; 4, lung; 5, muscle. (e) Fish: 1. ileum. 2, branchia; 3, muscle; 4, skin; 5, brain. (f) Shark: 1, branchia; 2, ileum: 3, skin, Or.
origin: CHS, chondroit in sulfate; DS, dermatan sulfate: HTS, heparan sulfate, St, standard.
The precipitate was washed twice with 80~,o ethanol, dried, resuspended in 100 pl of water and analysed. The recovery of sulfated glycosaminoglycans by this procedure was com- parable to the previous method used (Dietrich et al., 1976}.
Identification and quantitation qf su(fated wlycosumino~tly- CallS
The sulfated glycosaminoglycans were identified and quantitated by a combination of agarose gel electro- phoresis and enzymatic degradation with specific enzymes as previously described (Dietrich et al., 1976). United States Pharmacopea anticoagulant-activity was used as well to identify heparin. Glycosaminoglycan quanti tat ion was per- formed by densitometry of the agarose slides after electro- phoresis and toluidine blue staining. The error of the method was of the order of +4.5~,;. The extinction coef- ficients of the glycosaminoglycans were calculated using standards of chondroitin 4-sulfate, dermatan sulfate, heparan sulfate and heparin. Six successive extractions from four different tissues have shown a 13,'~,~ variation of the total sulfated glycosaminoglycans extracted although no significant changes in the proportions of the com- pounds were observed. Paper chromatography of the products formed after enzymatic degradation was per- formed in isobutyric acid, 1 M NH 3 (5:3, v/v). The relative amounts of the disaccharide products were measured after silver nitrate and/or toluidine blue staining by methods previously described (Dietrich et al., 1973). Molecular weight determinations were performed by polyacrylamide gel electrophoresis (Dietrich & Nader, 1974) after fraction- ation of the individual glycosaminoglycans by large scale agarose gel electrophoresis in propanediamine/acetate buffer (Silva & Dietrich, 1975).
RESULTS
Sulfated glycosaminoglycan composition of selected tissues from several vertebrate species
Figure 1 shows the agarose ge e lec t rophores i s of sulfated g lycosaminog lycans ob ta ined f rom different
~>
== c J
Y
Rabbi-t
Chicken
c
> ,
. E
E
o
> , J
2 CO
Snake
LJzord
Frog
Fish
Shark
,itin sul fate
~n su l fa te
* 2 0 % Kerato suLt~ate Like * '40°/o Kerato sut~Fa'te Like
Fig. 2. Distr ibut ion of sulfated glycosaminoglycans in several tissues of different vertebrates. These are the quan- titative data obtained from the experiments shown in Fig, 1. The sulfated glycosaminoglycan composition of rabbit
tissues were obtained from Cassaro & Dietrich [1977).
Distribution of heparin and other sulfated glycosaminoglycans in vertebrates
Table 1. Sulfated glycosaminoglycans of vertebrate tissues
Tissue Species
Total Sulfated Glycosaminogly- Sulfated glycosaminoglycans (');t
cans Chondroitin Dermatan Heparan ,ug/g dry tissue sulfate sulfate sulfate Heparin
Chicken 59 25.0 58.3 16.6 <0.5 Snake 116 10.0 68.0 22.0 <0.5
Muscle Lizard 35 29.3 45.0 25.6 <0.5 Frog 105 4.0 63.0 21.0 12.0 Fish 19 13.0 57.0 30.0 <0.5 Shark 40 42.7 38.5 18.8 <0,5 Chicken 1219 15.8 67.4 11.7 5.0 Snake 565 19.0 71.0 10.0 <0.5
Skin Lizard 1383 <2.0 91.5 8.5 <0.5 Frog 277 <2.0 98.0 2.0 <0.5 Fish 87 16.0 76.0 8.0 <0.5 Shark 63 31.6 58.3 10.0 <0.5 Chicken 322 18.0 53.0 27.4 1.5 Snake 366 16.5 76.0 4.0 3.4 Lizard 515 7.4 63.3 28.1 1.2
Ileum Frog 753 5.0 70.0 25.0 <0.5 Fish 120 6.5 81,0 12,5 <0,5 Shark 156 22.7 54.5 22.7 <0.5 Chicken* 840 58.0 <2.0 21.0 <0.5 Snake 250 63.0 <2.0 37.0 <0.5
Brain Lizard 49 100.0 Frog 65 81.0 <2.0 19.0 <0.5 Fish+ 660 40.0 <2.0 18.0 <0.5 Shark 41 74.5 -,,<2.0 25.5 <0.5 Chicken 135 29.7 38.3 31.9 <0.5 Snake 756 <2.0 12.0 88.0 <0.5
Liver Lizard 1723 41.0 44.5 14.0 0.5 Frog 194 2.5 37.0 59.0 1.5 Fish 53 <2,0 68.0 31.3 0.7 Shark 209 <2.0 39.8 59.6 0.6 Chicken 622 26.3 28.9 44.0 0.7 Snake 335 <2.0 94.0 5.0 1.0
Kidney Lizard 105 11.3 60.5 28.2 <0.5 Frog 437 4.0 60.0 34.0 2,0 Shark 170 32.1 39.3 28.5 <0.5 Chicken 853 22.0 30.3 44.1 3.5
Lung Snake 325 11.0 67.0 21.0 1.0 Lizard 153 50.5 32.1 17.3 < 0.5 Frog 310 15.0 62.0 21.0 2.0
Spleen Chicken 607 39.9 46.0 14.0 <0.5 Shark 88 23.9 47.7 17.9 10.5
Branchia Fish 1676 85.4 14.0 <2.0 0.6 Shark 328 86.6 9.2 4.2 <0.5
* Contains 21% of kerato sulfate-like compound. + Contains 42°41 of kerato sulfate-like compound.
859
vertebrate tissues. Each tissue has a characteristic composition differing from each other in the relative amount and type of sulfated glycosaminoglycans. The relative and absolute amounts of sulfated glycos- aminoglycans from several tissues of different verte- brates is also shown in Fig. 2 and Table 1. Most of the tissues possess the same types of sulfated glycos- aminoglycans. For instance all the brain tissues ana- lysed contain mainly chondroitin sulfate whereas muscle and skin from the different species contain mainly dermatan sulfate. With some exceptions (kid- ney and lung) the relative proportions of sulfated gly- cosaminoglycans of the same tissue of the different species also do not vary significantly. As examples brain tissues contain about 48 80°~i chondroitin sul-
fate and 2(~40','~; heparan sulfate; muscle tissues con- tain about 40-60°/,i dermatan sulfate, 12 30"/~i of heparan sulfate. Much more significant variations are observed when the different tissues of the same species are compared (Table 1). Of particular significance was the conspicuous presence of heparan sulfate in all tissues examined as it also has been observed for mammalian tissues. Chicken and fish brain contain also an unknown glycosaminoglycan which when hy- drolysed produces glucosamine, glactose and sulfate as the main components, resembling kerato sulfate. A wide variation of the absolute amounts of the sulfated glycosaminoglycans was observed either between the same tissue of different species or between the differ- ent tissues of the same species.
860 PURIFI('A('ION B. GOMES and CARL P. DIIiTRI(H
Table 2. Molecular weight of dermatan sulfate and heparan sulfate from differ- ent tissues and species
Dermatan sulfate Heparan sulfate Tissue Vertebrate Mode value Range Mode value Range
Rabbit* 6.7 i5.6 13.5) 19.5 Chicken 33.0 18,0 70.0) 15.5
Liver Snake 35.0 (4.2 80.0! _, .0 Fro~, 30 {4.6 60.0) "~
Lizard 11 [4.0 70.01 6.2 Chicken 19.5 I5.0 70.0) Snake 21 13,7 100.0)
Skin Frog 23 Lizard 32 [7.5 85.01 Shark 22 (6.0 I00.0) ('hieken 15 14.3 52) Snake 29.0
Muscle Frog 22.0 Fish 15 14.7 42) Chicken 14.0
Kidncy Snake 3.3 Frog 7.6
Ileum Frog 16.0 Lung Chicken 25./)
(4,7 33) 17.6 331
( 8 . 5 661 (3.7 131
* Data from Toledo & Dietrich (1977).
6 0 -
0., 5 0 - -
>, LI.O - ~ ¢,
3 0 - -
0)
= '° -_1[1 /
Fig. 3. Distribution of heparin in tissues of different vertebrates. The heparin precipitated from the extracts by' the potassium acetate procedure {sec Methods) were subjectcd to elcetrophoresis as described
in Fig. I. The bands migrating as standard heparin were quantitated by densitometry.
,4l'era,qe tool. wt (g the stt!jitted .qlycosamim)ylycans /i'om tissues ~?l di[lbrent species
Table 2 shows the average molecular weight of the sulfated g lycosaminoglycans of some tissues of differ-
Table 3. Anticoagulant activity of heparins obtained from some vertebrate tissues
Species Tissue
Anticoagulant activity (IU rag)
Frog Lung > 130 Frog Muscle > 130 Chicken Ileum 140 ('hickcn Skin 180
ent species. The average mol. wt of heparan sulfate from liver varied from 6 to 20 x 10 3 whereas the mol. wt of de rma tan sulfate, on the o ther hand, is somewha t cons tan t for the same tissue of the different species. The mol. vvt of de rma tan sulfate and heparan sulfate also wMed between the different tissues of the same species. For instance the heparan sulfate ob ta ined from liver, muscle, kidney', and ileum of frog have an average mol. wt of 25, 22, 7, 6 and 16 x 10 3 respectively.
Distribution o[heparin in tissues ol d!l[lerent vertebrate species
Cont ra ry to what was observed for heparan sulfate, de rma tan sulfate and chondro i t in sulfate which are
Distribution of heparin and other sulfated glycosaminoglycans in vertebrates 861
iiiiiii iili ilii i i ii ii!i i iii i~i ~ii~i !i i iii~i i ~ iliii Ii ~~'~i7 i Tiiiiii?i~ill ~'~'~ i!i!~~ii~i~i,,iiii~ii~i iil ! i ~
i!i ¸̧ i'~i!ii~ ileal i ̧ i~ ̧ iiii!i! i~i i! iii ~i,!!ii!iiiii~i~i iiii!i i ii!iiii~ii'i~i!: 'ii~i !i~iiiiiiii!!iiiii!i!iiiiii iii iii ~
i i ~ i,~,~ ~,~,~ ~~,~~,~,~ ~ ~ ~
Fig. 4. Enzymatic degradation of sulfated glycosaminoglycans of chicken tissues. About 100/~g of sul- fated glycosaminoglycans were incubated for 12 hr with 0.01 U of chondroitinase AC, chondroitinase ABC, Heparitinases I + II and 100#g of crude extracts of F. heparinum in 0.05 M diaminopropane acetate buffer, pH 7.0 in a final volume of 20/~1. Five/A aliquots were subjected to agarose gel electro- phoresis as described in Fig. 1. After fixation and staining the remaining sulfated glycosaminoglycans were quantitated by densitometry. (a) ileum; (b) skin; (c) muscle sulfated glycosaminoglycans. 1, control; 2, after chondroitinase AC; 3, after chondroitinase ABC; 4, after heparitinases I + II; 5, after crude F.
heparinum extracts; st, standard. Other abbreviations as in Fig. 1.
widely distributed in all tissues of the different verte- brates, heparin was found only in a few tissues (Fig. 3). The ones richest in heparin were chicken lung and skin. Liver and lung of 4 of the 6 species analysed contain heparin in different concentrations. These results are similar to the ones found for different mammalian species. The anticoagulant activity of some vertebrate heparins is shown in Table 3. They had an anticoagulant activity in the range of 130-180 IU/mg.
Identification of the sulfated glycosaminoglycans The identification of the sulfated glycosaminogly-
cans from the tissues were based on the following criteria: Chondroitin sulfate: same electrophoretic migration in two buffer systems as the standard chon- droitin sulfate; degradation by chondroitinases AC and ABC but not by the heparinase and hepariti- nases; formation of 4- and 6-sulfated disaccharides after degradation with chondroitinase AC. Dermatan sulfate: same electrophoretic migration in two buffer
862 PURIFICACION B. GO,'aES and CARL P. DIETRICH
O
i I" ~"
. w ,
" Z ~ : 4 1 5
Fig. 5. Products formed from sulfated glycosaminoglycans of different skins of vertebrates after degrada- tion with specific enzymes. The incubations were performed as described in Fig. 4 except that different sulfated glycosaminoglycans were used as indicated. The mixtures were applied in Whatman No. I paper and chromatographed in isobutyric acid--I M N H 3, 5:3 v/v, for 48 hr, The products were visualized by silver nitrate staining. 1, chondroitinase AC; 2, chondroitinase ABC; 3, heparitinases 1 + II; 4, control.
(A) chicken skin: (B) shark skin: (C) snake skin,
systems as the standard dermatan sulfate; degrada- tion by chondroitinase ABC but not by chondroiti- nase AC, heparitinase and heparinase: formation of 4-sulfated disaccharide by the action of chondroiti- nase ABC. Heparan sulfate: same electrophoretic migration in two buffer systems as the standard heparan sulfate: degradation by heparitinases but not by the chondroitinases AC and ABC: formtttion of glucosamine 2,6-disulfate and N-acetylglucosamine by the action of crude induced F. heparinum extracts. Heparin: same eleetrophoretic migration in two buffer systems as the standard heparin; degradation by heparinase but not by the heparitinases, chondroi- tinases AC and ABC; anticoagulant activity similar to heparin standard.
Besides these properties the four types of sulfated glycosaminoglycans isolated from the tissues were precipitated by cetyltrimethylammonium in the agar- ose gels and exhibited the characteristic metachroma- tic colour after toluidine blue staining. Some examples of these analyses are shown in Figs 4 and 5.
D l S f t SSION
The presence of sulfated glycosaminoglycans in ver- tebrate tissues individually examined have been exten-
sively demonstrated e,g. see Mathews (1975), Seno & Sakizuka (1978), Akiyasawa & Seno (1981 ). Neverthe- less, to our knowledge, studies on comparative distri- bution of sulfated glycosaminoglycans either between different tissues of the same species of same tissues of different species have not appeared except for mam- mals (Toledo & Dietrich, 1977: Nader et al., 1980).
The results presented in this paper show that there is a tissue specific distribution of sulfated glycos- aminoglycans. The relative proportions of heparan sulfate, dermatan sulfate and chondroitin sulfate are somewhat constant for a specific tissue regardless of the vertebrate species analysed. Conservely a wide wlriation of the relative and absolute amounts of these compounds is observed when the different tissues of a given species is compared, as it has been shown previously for mammalian tissues. Also, signifi- cant variations in molecular w'eight were observed in most cases when the sulfated glycosaminoglycans obtained from the same tissue of the different verte- brate species were compared, These w~riations were also observed from different tissues of the same spe- cies. These last results suggest that in general, each tissue from each species has its particular glycos- aminoglycans. If this assumption were extended to other vertebrate tissues, we could conclude that there
Distribution of heparin and other sulfated glycosaminoglycans in vertebrates 863
is an incredible variety of heparan sulfates and derma- tan sulfates.
All these results are in agreement with the earlier proposition that the sulfated glycosaminoglycans might phty a role in cell differentiation probably act- ing as recognition markers of the tissues. On the other hand, the distribution of heparin in mammalian and vertebrate tissues do not conform to the above hy- pothesis. The absence of hcparin from all rabbit tissues, its presence in most dog and bovine tissues and a scattered distribution in other mammals (Nader et al., 1980} and vertebrate tissues suggest a different role for this compound as compared to the other sul- fated glycosaminoglycans.
Acknowledgements-- We would like to express our grati- tude do Dr. H. B. Nader for the criticisms and suggestions during the elaboration of this work as well as to Ms Marl Nobuko (University of Silo Paulo) for the help in some experiments.
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