Glycosaminoglycans and Proteoglycans of Normal and … · 1000) by a modified Elson-Morgan reaction...
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[CANCER RESEARCH 39, 2802-2806, July 1979] 0008-5472/79/0039-0000$02.00 Glycosaminoglycans and Proteoglycans of Normal and Tumoral Cartilages of Humans and Rats1 Paulo A. S. Mourão,2 Yara M. Michelacci, and Olga M. S. Toledo Laboratdrio de Investigacáoem Reumatologia, Faculdade de Medicina da Universidade de SãoPaulo, Caixa Postal 8100, 01246 SãoPaulo, SP, Brazil (P. A. S. M.. 0. M. S. Ti, and Departamento de Bioquirnica e Farmacologia da Escola Paulista de Medicina, SãoPaulo, SP,BrazilfY. M. M.j ABSTRACT Differences in the glycosaminoglycans and proteoglycans synthesized by ‘ ‘young, ‘ ‘ ‘ ‘adult, ‘ ‘ and tumoral chondrocytes are reported. Young cartilage and human chondrosarcoma contain chondroitin 4- and 6-sulfates, whereas adult human cartilage contains almost exclusively chondroitin 6-sulfate. High keratan sulfate content is reported in adult cartilage, whereas it is almost absent in young and tumoral cartilages. The electrophoretic pattern and keratan sulfate content in these proteoglycans from adult cartilage are clearly distinct from those of the young and tumoral cartilages. The high molecular weight is the distinguishing property of the glycosa minoglycan synthesized by tumoral chondrocytes. INTRODUCTION Several groups of workers (12, 13, 25) have established the existence of variations in the GAG' content of articular carti lage, corresponding to distance from the articular surface. Increases in the relative proportion of the chondroitin 4-sulfate (1 9, 20) and in the average molecular weight of the chondroitin sulfates (i 0) were found to correspond with distance from the articular surface of the epiphysis of young mammals. A species specific distribution of chondroitin 4- and 6-sulfates in the epiphysial cartilages of adult mammals has been reported recently by Mourãoand Dietrich (i 9). Chondroitin 6-sulfate is present in high relative proportions in adult epiphysial carti lages in most of the mammals studied whereas, in some species of the order Rodentia, chondroitin 4-sulfate is the only chon droitin sulfate present. Choi et al. (2) reported that chondroitin 4-sulfate is the exclusive chondroitin sulfate produced by a transplantable rat chondrosarcoma, while Mourãoand Dietrich (19) observed that normal cartilages of young and adult rats also contain almost exclusively chondroitin 4-sulfate. Although the chemical composition of human chondrosarcoma has been reported by Meyer et al. (15) and by Anderson et al. (1), the GAG have not been analyzed by newly developed biochemical methods. This paper gives a comparative analysis of GAG extracted from young, adult, and tumoral cartilages of humans and rats. The objective of this study is to compare GAG synthesized by , ‘young, ‘ ‘‘ ‘old, ‘ ‘and tumoral chondrocytes of these 2 mam mals. , This research was aided by Grant 77/0748 from Fundaçao de Amparo a Pesqulsa do Estado de SãoPaulo(FAPESP). 2 Supported by FINEP (Flnanciadora de Estudos e Projetos) and CNPq (Con selho Nacional de Desenvolvimento CientIfico e Tecnológico). 3 The abbreviations used are: GAG, glycosaminoglycan(s); aDi-4S, 2-aceta mido-2-deoxy-3-O-(8-o-gluco-4-enepyranosyluronic acid)-4-0-sulfo-o-galac toss; ADi-6S, 2-acetamido-2-deoxy-3-O-(fl-o-gluco-4-enepyranosyluronic acid) 6-O-sulfo-o-galactose. Received October 11, 1978; accepted April 12, 1979. GAG. Fresh cartilages were obtained from young and adult mammals and checked for ‘ ‘normality' ‘ by macroscopic ex amination. Human chondrosarcoma from the upper femoral end was the kind gift of Dr. 0. Gianotti Filho (Departamento de Anatomia PatolOgica, Hospital A. C. Camargo, SãoPaulo, Brazil). Transplantable rat chondrosarcoma was provided by Dr. R. R. Brentani (LaboratOrio de Oncologia Experimental, Faculdade de Medicina da Universidade de SãoPaulo). GAG was extracted from cartilages as previously described (20). Standard chondroitin 4- and 6-sulfate and dermatan sulfate were purchased from Miles Laboratories (Elkhart, Ind.). Kera tan sulfate was prepared from bovine cornea, according to the method of Meyer et al. (i 6). The barium salt of heparitin sulfate from bovine lung, obtained through the courtesy of Dr. L. L. Coleman, Upjohn Co., Kalamazoo, Mich., was converted to its ammonium salt as described previously (6). Heparitin sulfates B, C, and D from bovine lung tissues were prepared as previ ously described (5). Proteoglycans. Fresh canilages were immersed in 10 ml of acetone and kept for 24 hr at 5°with 2 fresh changes of acetone. The dried tissue (50 mg) was macerated and sus pended in 1.0 ml of 4.0 M guanidine hydrochloride. In some experiments, protease inhibitors [1 m@ phenylmethylsulfonyl fluoride, 2 mM EDTA, and soy bean trypsin inhibitor (0.2 mg/ ml)] were added to the guanidine hydrochloride solution. The mixture was maintained at 5°for 48 hr under agitation and then dialyzed for 4 hr against distilled water. Enzymes. Chondroitinases AC and ABC were purchased from Miles Laboratories. Chondroitinase C was prepared as described by Michelacci and Dietrich (18). Crude extract from Flavobacterium heparinum was prepared as previously de scribed (3). Crude extract from Pseudomonas sp. , which cat alyze an extensive cleavage of keratan sulfate, was prepared according to the method of Nakazawa et al. (22). Trypsin was purchased from Sigma Chemical Co. (St. Louis, Mo.). Other Chemicals. Agarose was purchased from L'lndustrie Biologique FrançaiseS.A. (Gennevilliers, Seine, France); 1.3- diaminopropane was from Aldrich Chemical Co. (Milwaukee, Wis.); amino sugars and galactose were from British Drug House Chemical Ltd. Identification and Quantitation of GAG and Proteoglycans. The GAG and proteoglycans were identified by a combination of agarose gel electrophoresis (4) and enzymatic degradation with specific mucopolysaccharidases and trypsin. The prod ucts formed by the action of the enzymes on the GAG and proteoglycans were identified by paper chromatography (24) and agarose gel electrophoresis (4). The agarose gel slides (5.0 x 7.5 cm) were prepared with 1.2% agarose in 0.05 M 1,3-diaminopropane acetate, pH 9.0 (nondissociative condi tions). In some experiments, 6 M urea was added to the buffer 2802 CANCERRESEARCHVOL. 39 MATERIALS AND METHODS on April 6, 2021. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
Transcript of Glycosaminoglycans and Proteoglycans of Normal and … · 1000) by a modified Elson-Morgan reaction...
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[CANCER RESEARCH 39, 2802-2806, July 1979]0008-5472/79/0039-0000$02.00
Glycosaminoglycans and Proteoglycans of Normal and Tumoral Cartilagesof Humans and Rats1
Paulo A. S. Mourão,2 Yara M. Michelacci, and Olga M. S. Toledo
Laboratdrio de Investigacáoem Reumatologia, Faculdade de Medicina da Universidade de SãoPaulo, Caixa Postal 8100, 0 1246 SãoPaulo, SP, Brazil(P. A. S. M.. 0. M. S. Ti, and Departamento de Bioquirnica e Farmacologia da Escola Paulista de Medicina, SãoPaulo, SP, BrazilfY. M. M.j
ABSTRACT
Differences in the glycosaminoglycans and proteoglycanssynthesized by ‘‘young,‘â€ẫ€˜â€˜adult,‘‘and tumoral chondrocytesare reported. Young cartilage and human chondrosarcomacontain chondroitin 4- and 6-sulfates, whereas adult humancartilage contains almost exclusively chondroitin 6-sulfate.High keratan sulfate content is reported in adult cartilage,whereas it is almost absent in young and tumoral cartilages.The electrophoretic pattern and keratan sulfate content inthese proteoglycans from adult cartilage are clearly distinctfrom those of the young and tumoral cartilages. The highmolecular weight is the distinguishing property of the glycosaminoglycan synthesized by tumoral chondrocytes.
INTRODUCTION
Several groups of workers (12, 13, 25) have established theexistence of variations in the GAG' content of articular cartilage, corresponding to distance from the articular surface.Increases in the relative proportion of the chondroitin 4-sulfate(1 9, 20) and in the average molecular weight of the chondroitinsulfates (i 0) were found to correspond with distance from thearticular surface of the epiphysis of young mammals. A speciesspecific distribution of chondroitin 4- and 6-sulfates in theepiphysial cartilages of adult mammals has been reportedrecently by Mourãoand Dietrich (i 9). Chondroitin 6-sulfate ispresent in high relative proportions in adult epiphysial cartilages in most of the mammals studied whereas, in some speciesof the order Rodentia, chondroitin 4-sulfate is the only chondroitin sulfate present. Choi et al. (2) reported that chondroitin4-sulfate is the exclusive chondroitin sulfate produced by atransplantable rat chondrosarcoma, while Mourãoand Dietrich(19) observed that normal cartilages of young and adult ratsalso contain almost exclusively chondroitin 4-sulfate. Althoughthe chemical composition of human chondrosarcoma has beenreported by Meyer et al. (15) and by Anderson et al. (1), theGAG have not been analyzed by newly developed biochemicalmethods.
This paper gives a comparative analysis of GAG extractedfrom young, adult, and tumoral cartilages of humans and rats.The objective of this study is to compare GAG synthesized by, ‘young, ‘‘‘‘old, ‘‘and tumoral chondrocytes of these 2 mam
mals.
, This research was aided by Grant 77/0748 from Fundaçao de Amparo aPesqulsa do Estado de SãoPaulo (FAPESP).
2 Supported by FINEP (Flnanciadora de Estudos e Projetos) and CNPq (Con
selho Nacional de Desenvolvimento CientIfico e Tecnológico).3 The abbreviations used are: GAG, glycosaminoglycan(s); aDi-4S, 2-aceta
mido-2-deoxy-3-O-(8-o-gluco-4-enepyranosyluronic acid)-4-0-sulfo-o-galactoss; ADi-6S, 2-acetamido-2-deoxy-3-O-(fl-o-gluco-4-enepyranosyluronic acid)6-O-sulfo-o-galactose.
Received October 11, 1978; accepted April 12, 1979.
GAG. Fresh cartilageswere obtainedfrom young and adultmammals and checked for ‘‘normality'â€b̃y macroscopic examination. Human chondrosarcoma from the upper femoralend was the kind gift of Dr. 0. Gianotti Filho (Departamento deAnatomia PatolOgica, Hospital A. C. Camargo, SãoPaulo,Brazil). Transplantable rat chondrosarcoma was provided byDr. R. R. Brentani (LaboratOrio de Oncologia Experimental,Faculdade de Medicina da Universidade de SãoPaulo). GAGwas extracted from cartilages as previously described (20).Standard chondroitin 4- and 6-sulfate and dermatan sulfatewere purchased from Miles Laboratories (Elkhart, Ind.). Keratan sulfate was prepared from bovine cornea, according to themethod of Meyer et al. (i 6). The barium salt of heparitin sulfatefrom bovine lung, obtained through the courtesy of Dr. L. L.Coleman, Upjohn Co., Kalamazoo, Mich., was converted to itsammonium salt as described previously (6). Heparitin sulfatesB, C, and D from bovine lung tissues were prepared as previously described (5).
Proteoglycans. Fresh canilages were immersed in 10 ml ofacetone and kept for 24 hr at 5°with 2 fresh changes ofacetone. The dried tissue (50 mg) was macerated and suspended in 1.0 ml of 4.0 M guanidine hydrochloride. In someexperiments, protease inhibitors [1 m@ phenylmethylsulfonylfluoride, 2 mM EDTA, and soy bean trypsin inhibitor (0.2 mg/ml)] were added to the guanidine hydrochloride solution. Themixture was maintained at 5°for 48 hr under agitation andthen dialyzed for 4 hr against distilled water.
Enzymes. Chondroitinases AC and ABC were purchasedfrom Miles Laboratories. Chondroitinase C was prepared asdescribed by Michelacci and Dietrich (18). Crude extract fromFlavobacterium heparinum was prepared as previously described (3). Crude extract from Pseudomonas sp. , which catalyze an extensive cleavage of keratan sulfate, was preparedaccording to the method of Nakazawa et al. (22). Trypsin waspurchased from Sigma Chemical Co. (St. Louis, Mo.).
Other Chemicals. Agarose was purchased from L'lndustrieBiologique FrançaiseS.A. (Gennevilliers, Seine, France); 1.3-diaminopropane was from Aldrich Chemical Co. (Milwaukee,Wis.); amino sugars and galactose were from British DrugHouse Chemical Ltd.
Identification and Quantitation of GAG and Proteoglycans.The GAG and proteoglycans were identified by a combinationof agarose gel electrophoresis (4) and enzymatic degradationwith specific mucopolysaccharidases and trypsin. The products formed by the action of the enzymes on the GAG andproteoglycans were identified by paper chromatography (24)and agarose gel electrophoresis (4). The agarose gel slides(5.0 x 7.5 cm) were prepared with 1.2% agarose in 0.05 M1,3-diaminopropane acetate, pH 9.0 (nondissociative conditions). In some experiments, 6 M urea was added to the buffer
2802 CANCERRESEARCHVOL. 39
MATERIALS AND METHODS
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Concentration andchemical composition of GAG in differentcartilagesHexosaminesand galactose(%CartilageTotal
GAG (%dry wt of tie
sue)aUronic
acidcontent (%
dry wt of tissue)in
totalGAG)MW.Galac-
Galac- Glucosatoss tosaminemineYoung7.62.9
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+ Chase ABCF.H.extract
4,IJf
1
, , , I 1@ I I
3
@—I@ III10 20 30
Disaccharideproductsformedby theactionof chondroitinaseACuponGAG from normal cartllages andchondrosarcomaChondroitinase
AC products(%)aCartilage
Mammal ADI-4S@Di-6SYoung
Rat 100
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10 20
ProteoglycansfromnormalhumancartilagesandchondrosarcomaProteoglycan
fractions(%)bElectrophore
ExtractionCartilagep1p2Without
proteaseYoung
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P. A. S. Mour@oet al.
composition of heparitin sulfate. Fractionation and characterization of fouracidic mucopolysaccharides in heparitin sulfate from beef lung tissue.Biochim. Biophys. Acta, 237: 430-441 , 1971.
7. Dietrich, C. P., Nader, H. B., and Mourão,P. A. S. Differentiation of Hunter'sand Hurler's syndromes by the analysis of the excreted mucopolysaccharides. Biochem. Med., 8: 371 -379, 1973.
8. Dische, Z. A new specific color reaction of hexuronic acids. J. Biol. Chem.,167: 189—192,1947.
9. Hilborn, J. C., and Anastassiadis, P. A. Acrylamide gel electrophoresis ofacidic mucopolysaccharides. Anal. Biochem., 31: 51-55, 1969.
10. Jones, I. L, and Lemperg, R. Chondroitin sulphate of calf knee-joint cartilage. Biochim. Biophys. Acta, 392: 310—318, 1975.
11. Kaplan, D., and Meyer, K. Ageing of human cartilage. Nature (Lond.), 183:1267-1268, 1959.
12. Lemperg, R., Larsson, S. E., and Hjertquist, 5. 0. The glycosaminoglycansof bovinearticularcartilage.I. Concentrationanddistributionin differentlayers in relation to age. Calcif. Tissue Res., 15: 237—251, 1974.
13. Maroudas, A., Muir, H., and Wingharn, J. The correlation of fixed negativecharge with glycosaminoglycan content of human articular cartilage.Blochim. Biophys. Acta, 177: 492-500, 1969.
14. Mathews, M. B., and Glacov, S. Acid mucopolysaccharide patterns in aginghuman cartilage. J. Clin. Invest., 45: 1103—1111, 1966.
15. Meyer, K., Davidson, E., Linker, A., and Hoffman, P. The acid mucopolysaccharides of connective tissue. Biochim. Biophys. Acta, 2 1: 506—518, 19@6.
4 5 6 lhA 4D@O$.AC
16. Meyer, K., Linker, A., Davidson, E. A., and Weissman, B. The mucopolysaccharides of bovine cornea. J. Biol. Chem., 205: 61 1-620, 19@3.
17. Michelacci, Y. M., and Dietrich, C. P. Structure of chondroitin sulfates:analyses of the products formed from chondroitin sulfates A and C by theaction of the chondroitinases C and AC from Flavobacterium heparinum.Biochim. Biophys. Acta, 45 1: 436—443.1976.
18. Michelaccl, Y. M., and Dietrich, C. P. Chondroitinase C from Flavobacteriumheparinum. J. Biol. Chem., 251: 1154—1158, 1976.
19. Mourão,P. A. S., and Dietrich, C. P. Chondroitin sulfates of the epiphysialcartilages of different mammals. Comp. Biochim. Physiol., 62: 115—117,1979.
20. Mourão,P. A. S.. Rozenfeld, S.. Laredo, J., and Dietrich, C. P. The distribution of chondroitin sulfates in articular and growth cartilages of humanbones. Biochim. Biophys. Acta, 428: 19-26, 1976.
21 . Mourão,P. A. 5., Toledo, 5. P. A., Nader, H. B., and Diefrich, C. P. Excretionof chondroitln sulfate C with low sulfate content by patients with generalizedplatyspondyly (brachyolmia). Biochem. Med., 7: 4 15—423,1973.
22. Nakazawa, K., Suzuki, N., and Suzuki, S. Sequential degradation of keratansulfate by bacterial enzymes and purification of a sulfatase in the enzymaticsystem. J. Blot. Chem.. 250: 905-91 1, 1975.
23. ROndIe,C. J. M., and Morgan, W. I. J. The determinationof glycosamineand galactosamine. Biochem. J., 61: 586-589, 1955.
24. Saito, H., Yamagata,T., and Suzuki, S. Enzymaticmethodsfor the determinatlon of small quantities of isomeric chondroitin sulfate. J. Biol. Chem.,
243: 1536—1542, 1968.25. Stockwell, R. A., and Scott, J. E. DistributIOnof acid glycosaminoglycans in
human articular cartilage. Nature (Lond.). 215: 1376-1 378, 1969.
FRACTION
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Fig. 2. Agarose gel elecfrophoresis of purified GAGsamplesfrom adult humancartilage. To about I .0 mg of GAG and 1.5 mg of 4.0 PAguanidine hydrochlorideextractfromadultnormalcartilagewereadded1.0mlofcrudeextractfromF.heparinum (to prepare a pure sample of keratan sulfate) and 1.0 ml of crudeextract from Pseudomonas sp. (to prepare a pure sample of chondroitin sulfate).Theincubationconditionsaredescribedin “MaterialsandMethods.―AfterIncubation, the precipitate formed was removed by centrifugation, and 2 volumesof ethanol were added to the clear supematant. After 12 hr at —10@,theprecipitate formed was collected by centritugation, washed once with 3.0 ml of80% ethanol, and dried. About 100 @gof each sample were submitted to agarosegel electrophoresls in 0.05 M 1,3-diaminopropane acetate buffer, pH 9.0, andstained with toluidine blue as previously described. Column 1, GAG plus F.hepannum extract; Column 2, GAG plus Pseudomonas sp. extract: Column 3,proteoglycan (P0) pIus F. heparinum extract: Column 4, proteoglycan plusPseudomonas sp. extract; ST. mixture of standard keratan sulfate (KS) andchondroitin 4- and 6-sulfate (CS).
SAN PS
Chasi AC@ Chos. C
Fig. 1. Paper chromatogram of the chondroitinase AC and C digest of theGAG from normal cartilages and chondrosarcoma. Three x 1o@' unit of thechondroitinase AC (Chase AC) and 2.8 x 1O@ unit of the chondroitinase C(Chase C) were incubated with 100 @igof GAG in 0.05 PAethylenediamine acetatebuffer, pH 8.0, in a final volume of 20 4 After 12 hr incubation at 37°for thechondroitinase AC and at 200 for the chondroitinase C, the mixtures were spottedon Whatman No. 1 paper and subjected to descending chromatography inisobutyric acid: 1 M NH3(5:3, v/v) for 48 hr. After the run, the reducing productswere located by silver nitrate staining. GAG from rat cartilages: 1, young: 2,adult: 3. tumoral. GAG from human cartilages: 4, young: 5, adult: 6, tumoral.ChA, chondroitin 4-sulfate: ChC, chondroitin 6-sulfate.
2806 CANCERRESEARCHVOL. 39
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1979;39:2802-2806. Cancer Res Paulo A. S. Mourão, Yara M. Michelacci and Olga M. S. Toledo Cartilages of Humans and RatsGlycosaminoglycans and Proteoglycans of Normal and Tumoral
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