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Acid glycosaminoglycan of eggshell membranes
BIOCHIMICAET BIOPHYSICAACTA
481
BBA 26627
ACID GLYCOSAMINOGLYCAN OF E G G S H E L L MEMBRANES
C. I. OSUOJI*
Department of Biochemistry, Trinity College, University of Dublin, Dublin (Ireland) (Received March i5th, 1971)
SUMMARY
The acid glycosaminoglycan content of eggshell membranes was characterised and compared with that of the isthmus region of hen oviduct which secretes this tissue. Results showed that whereas the eggshell membranes contain only hyaluronic acid which represents less than o.1% of the dry weight of the tissue, those of the isthrims include hyaluronic acid, dermatan sulphate, chondroitin-4-sulphate and heparan sulphate. It is suggested that hyaluronic acid m a y have a role in water retention and resistance to bacterial attack by the membranes.
Studies of acid glycosaminoglycans of mature hen oviduct (C.I. O s u o J I , u n p u b l i s h e d results) showed the presence of chondroitin-4-sulphate, dermatan sulphate, hyaluronic acid and heparan sulphate in the isthmus and uterus regions of the organ. SCHILLER1 also reported the presence of these substances in whole oviduct of oestrogen-stimulated chicks. Since eggshell membranes are secreted by the isthmusL the investigation was extended in an attempt to characterise the acid glycosaminoglycans of this tissue and to compare it with those of the organ. The membranes were collected from shell-free eggs which had broken in the iothmus and uterus during sacrifice. They were washed briefly in 0.02 M NaC1 to remove residual globulins, rinsed in distilled water and then dehydrated in acetone. The residue was digested with activated papain 3 followed by extraction with o. 5 M N a O H for 48 h at 4 °. The liberated crude acid glycosaminoglycans were precipitated with ethanol ~and redigested as previously described. Nitrogenous contaminants were removed by treatment with chloroform-amyl alcohol (3 :I, v/vj and Dowex 5o (H form), and the polysaccharide recovered by precipitation with ethanol. The final material was analysed for hexosamine 5 after hydrolysis in 4 M HC1 for 16 h at IiO °. Their identity was checked b y oxidation with ninhydrin ~ followed by paper chromatography (16 h) of the pentoses formed in pyridine-ethyl a c e t a t e - w a t e r acetic acid (5 : 5:3 : I, by vol.). Uronic acid was estimated by the carbazole reaction of * Present address: D e p a r t m e n t of Oral Pathology, The Dental School, St. Mary's Row, Birmingh a m B 4 6NN (Great Britain).
Biochim. Biophys. Acta, 244 (1971) 481-483
482
c.i. osuoji
DISCHE~. Paper chromatographic identification of the acids was carried out in the above solvent (II h) after hydrolysis of the sample in I M HC1 for I h at IOO°. Neutral sugars were liberated by hydrolysis in 0.4 M HC1 at IOO° for 4 h and separated by paper chromatography (24-36 h) in ethyl acetate-pyridine-water (8:2 :I, by vol.). In all cases, sugars were located by staining in o-aminobiphenyl reagent 8. Sulphate was estimated as described by DODGSONAND PRICE9. The polyanion-cetylpyridinium complex was chromatographed on Whatman No. 5 ° paper as described by CASTORAND DORSTEWlTZ1°. Electrophoresis was performed on cellulose acetate strips in 25 mM sodium phosphate buffer (pH 7.o, 4 V/cm.) for I½ h at room temperature. Acid glycosaminoglycans were located on chromatogram and electrophoretogram by staining in o.IYo aqueous toluidine blue. Infrared studies were made on the sodium salt of the acid glycosaminoglycans sample in Nujol using Infrascan (Hilger and Watts), and the spectrum interpreted as described by LLOYD et al. 11. Susceptibility of the substance to testicular hyaluronidase degradation was tested by incubation with the enzyme (5ooo I.U./mg) in o.I M sodium acetate buffer, pH 4.7 o at 37 ° for 24 h. Digestion products were examined by thin-layer chromatography 12. Dermatan sulphate which is resistant to the enzyme was similarly treated as a control. Owing to the very high content of keratin, most of the membranes remained undigested after prolonged proteolytic digestion and alkaline extraction. 32 mg of acid glycosaminoglycans (approx. o.o8yo of the dry weight of the starting material) were isolated. Glucuronic acid (28.90) and glucosamine (37.8%) were the only hexuronic acid and hexosamine detected (glucuronic acid/glucosamine molar ratio, o.97 ). Infrared studies showed a strong extinction peak in the 16oo-165o cm ~ regions of the spectrum (due to C = O stretching). There was also the absence of bands in the 82o, 85o and 124o cm -~ regions due to C O-S and S = O vibrations respectively. Quantitative estimation of sulphate also showed that this substance was unsulphated. The material had an electrophoretic mobility similar to that of an authentic sample of hyaluronic acid and its cetylpyridinium complex was completely dissolved when chromatographed in o.Io M MgC12. Digestion with hyaluronidase resulted in the loss of about c)5% of the substance, whereas dermatan sulphate was unaffected. It was concluded from these results that the acid glycosaminoglycans was solely hvaluronic acid. Mannose and galactose were also detected in the hydrolysate. The latter might have been derived from the acid glycosaminoglycans-protein linkage region. The above finding is intere,c.ting from the point of view that the isthmus region which secretes this tissue contains four types of acid glycosaminoglycans. There is no reason to believe that the polyanion could be a contaminant from eggshell matrix as the latter had not been deposited at the time of collection. Furthermore, BAKER AND BALCHis have shown that the acid glycosaminoglycans of eggshell are chondroitin- 4sulphate and dermatan sulphate. The isolation of hyaluronic acid from shell membranes is also at variance with the report of the above workers who found no uronic acid in the tissue. However, they noted the presence of hexosamine and added that the concentration of sugars were too low for estimation. It may well be that the low level of hyaluronic acid (estimated in this work to be less than o.1% of the dry weight of the tissue) did not allow for its detection. There is the question concerning its role in this tissue. It is possible that the resistance of the shell membranes to bacterial attack is due to their hyaluronic acid-protein matrix. A similar function in the skin has been attributed to such a matrix. However, LIFSHITZ AND B A K E R 14 attributed this resistance to the Biochim. Biophys. Acta, 244 (1971) 481-483
ACID GLYCOSAMINOGLYCAN OF EGGSHELL MEMBRANES
483
d e n s e n e t w o r k of k e r a t i n i n t h e m e m b r a n e s . I t is also p o s s i b l e t h a t h y a l u r o n i c a c i d m a y b e i n v o l v e d i n w a t e r r e t e n t i o n a n d h e n c e i n t h e m a i n t e n a n c e of m e m b r a n e t u r g i d i t y . W a t e r r e t e n t i o n is p a r t i c u l a r l y e s s e n t i a l for t h e d e v e l o p i n g e m b r y o a n d t h e h i g h m o l e c u l a r w e i g h t a n d specific v o l u m e of t h e p o l y m e r m a k e i t well s u i t e d t o t h i s f u n c t i o n . RIENITS 15 also s u g g e s t e d a role for h y a l u r o n i c a c i d i n w a t e r r e t e n t i o n . H o w e v e r , m o r e d i r e c t e v i d e n c e is r e q u i r e d t o s u p p o r t t h a t t h i s s u b s t a n c e h a s a p h y s i o l o g i c cal f u n c t i o n i n w a t e r r e t e n t i o n . T h e a u t h o r is g r a t e f u l t o D r . J . R . B a k e r for his a s s i s t a n c e a n d t o P r o f e s s o r B . S p e n c e r for t h e u s e of f a c i l i t i e s i n h i s l a b o r a t o r y . REFERENCES I S. SCHILLER, Biochim. Biophys. Acta, 32 (1959) 215. 2 E. MCNALLY, Proc. Soc. Exptl. Biol. Med., 31 (1934) 946. 3 J- E. SCOTT, in D. GLICK, iViethodsBiochem. Anal., Vol. 8, Interscience, New York, 196o, p. 145. 4 K. !~IEYER, E. A. DAVlDSON, A. LINKER AND P. HOFFMAN, Biochim. Biophys. Acta, 21 (1959)
506. G. A. LEvvY AND A. MCALLAN, Biochem. J., 73 (1959) 127. P. STOFFYN AND R. JEANLOZ, Arch. Biochem. Biophys., 52 (1954) 373. Z. DISCHE, ,[. Biol. Chem., 167 (1947) 189. H. T. GORDON, W. THORBURG AND L. N. WERtJM, Anal. Chem., 28 (1956) 849. K. S. DODGSON AND R. G. PRICE, Biochem. J., 84 (1962) lO6. C. W. CASTOR AND E. L. DORSTEWlTZ, J. Chromatogr., 13 (1964) 157. A. G. LLOYD, K. S. DODGSON, R. G. PRICE AND F. A. ROSE, Biochim. Biophys. Acta, 46 (1961) lO8. F. S. WUSTEMAN, A. G. LLOYD AND K. S. DODGSON, J. Chromatogr., 21 (1966) 32. 13 J. R. BAKER AND D. A. BALCH, Biochem. J., 82 (i962) 352. 14 A. LIFSHITZ AND R. C. BAKER, Poult. Sci., 43 (1964) 527I5 K. G. RIEXlTS, Biochem. J., 74 (196o) 27. 5 6 7 8 9 IO Ii 12
Biochim. Biophys. Acta, 244 (1971) 481-483
481
BBA 26627
ACID GLYCOSAMINOGLYCAN OF E G G S H E L L MEMBRANES
C. I. OSUOJI*
Department of Biochemistry, Trinity College, University of Dublin, Dublin (Ireland) (Received March i5th, 1971)
SUMMARY
The acid glycosaminoglycan content of eggshell membranes was characterised and compared with that of the isthmus region of hen oviduct which secretes this tissue. Results showed that whereas the eggshell membranes contain only hyaluronic acid which represents less than o.1% of the dry weight of the tissue, those of the isthrims include hyaluronic acid, dermatan sulphate, chondroitin-4-sulphate and heparan sulphate. It is suggested that hyaluronic acid m a y have a role in water retention and resistance to bacterial attack by the membranes.
Studies of acid glycosaminoglycans of mature hen oviduct (C.I. O s u o J I , u n p u b l i s h e d results) showed the presence of chondroitin-4-sulphate, dermatan sulphate, hyaluronic acid and heparan sulphate in the isthmus and uterus regions of the organ. SCHILLER1 also reported the presence of these substances in whole oviduct of oestrogen-stimulated chicks. Since eggshell membranes are secreted by the isthmusL the investigation was extended in an attempt to characterise the acid glycosaminoglycans of this tissue and to compare it with those of the organ. The membranes were collected from shell-free eggs which had broken in the iothmus and uterus during sacrifice. They were washed briefly in 0.02 M NaC1 to remove residual globulins, rinsed in distilled water and then dehydrated in acetone. The residue was digested with activated papain 3 followed by extraction with o. 5 M N a O H for 48 h at 4 °. The liberated crude acid glycosaminoglycans were precipitated with ethanol ~and redigested as previously described. Nitrogenous contaminants were removed by treatment with chloroform-amyl alcohol (3 :I, v/vj and Dowex 5o (H form), and the polysaccharide recovered by precipitation with ethanol. The final material was analysed for hexosamine 5 after hydrolysis in 4 M HC1 for 16 h at IiO °. Their identity was checked b y oxidation with ninhydrin ~ followed by paper chromatography (16 h) of the pentoses formed in pyridine-ethyl a c e t a t e - w a t e r acetic acid (5 : 5:3 : I, by vol.). Uronic acid was estimated by the carbazole reaction of * Present address: D e p a r t m e n t of Oral Pathology, The Dental School, St. Mary's Row, Birmingh a m B 4 6NN (Great Britain).
Biochim. Biophys. Acta, 244 (1971) 481-483
482
c.i. osuoji
DISCHE~. Paper chromatographic identification of the acids was carried out in the above solvent (II h) after hydrolysis of the sample in I M HC1 for I h at IOO°. Neutral sugars were liberated by hydrolysis in 0.4 M HC1 at IOO° for 4 h and separated by paper chromatography (24-36 h) in ethyl acetate-pyridine-water (8:2 :I, by vol.). In all cases, sugars were located by staining in o-aminobiphenyl reagent 8. Sulphate was estimated as described by DODGSONAND PRICE9. The polyanion-cetylpyridinium complex was chromatographed on Whatman No. 5 ° paper as described by CASTORAND DORSTEWlTZ1°. Electrophoresis was performed on cellulose acetate strips in 25 mM sodium phosphate buffer (pH 7.o, 4 V/cm.) for I½ h at room temperature. Acid glycosaminoglycans were located on chromatogram and electrophoretogram by staining in o.IYo aqueous toluidine blue. Infrared studies were made on the sodium salt of the acid glycosaminoglycans sample in Nujol using Infrascan (Hilger and Watts), and the spectrum interpreted as described by LLOYD et al. 11. Susceptibility of the substance to testicular hyaluronidase degradation was tested by incubation with the enzyme (5ooo I.U./mg) in o.I M sodium acetate buffer, pH 4.7 o at 37 ° for 24 h. Digestion products were examined by thin-layer chromatography 12. Dermatan sulphate which is resistant to the enzyme was similarly treated as a control. Owing to the very high content of keratin, most of the membranes remained undigested after prolonged proteolytic digestion and alkaline extraction. 32 mg of acid glycosaminoglycans (approx. o.o8yo of the dry weight of the starting material) were isolated. Glucuronic acid (28.90) and glucosamine (37.8%) were the only hexuronic acid and hexosamine detected (glucuronic acid/glucosamine molar ratio, o.97 ). Infrared studies showed a strong extinction peak in the 16oo-165o cm ~ regions of the spectrum (due to C = O stretching). There was also the absence of bands in the 82o, 85o and 124o cm -~ regions due to C O-S and S = O vibrations respectively. Quantitative estimation of sulphate also showed that this substance was unsulphated. The material had an electrophoretic mobility similar to that of an authentic sample of hyaluronic acid and its cetylpyridinium complex was completely dissolved when chromatographed in o.Io M MgC12. Digestion with hyaluronidase resulted in the loss of about c)5% of the substance, whereas dermatan sulphate was unaffected. It was concluded from these results that the acid glycosaminoglycans was solely hvaluronic acid. Mannose and galactose were also detected in the hydrolysate. The latter might have been derived from the acid glycosaminoglycans-protein linkage region. The above finding is intere,c.ting from the point of view that the isthmus region which secretes this tissue contains four types of acid glycosaminoglycans. There is no reason to believe that the polyanion could be a contaminant from eggshell matrix as the latter had not been deposited at the time of collection. Furthermore, BAKER AND BALCHis have shown that the acid glycosaminoglycans of eggshell are chondroitin- 4sulphate and dermatan sulphate. The isolation of hyaluronic acid from shell membranes is also at variance with the report of the above workers who found no uronic acid in the tissue. However, they noted the presence of hexosamine and added that the concentration of sugars were too low for estimation. It may well be that the low level of hyaluronic acid (estimated in this work to be less than o.1% of the dry weight of the tissue) did not allow for its detection. There is the question concerning its role in this tissue. It is possible that the resistance of the shell membranes to bacterial attack is due to their hyaluronic acid-protein matrix. A similar function in the skin has been attributed to such a matrix. However, LIFSHITZ AND B A K E R 14 attributed this resistance to the Biochim. Biophys. Acta, 244 (1971) 481-483
ACID GLYCOSAMINOGLYCAN OF EGGSHELL MEMBRANES
483
d e n s e n e t w o r k of k e r a t i n i n t h e m e m b r a n e s . I t is also p o s s i b l e t h a t h y a l u r o n i c a c i d m a y b e i n v o l v e d i n w a t e r r e t e n t i o n a n d h e n c e i n t h e m a i n t e n a n c e of m e m b r a n e t u r g i d i t y . W a t e r r e t e n t i o n is p a r t i c u l a r l y e s s e n t i a l for t h e d e v e l o p i n g e m b r y o a n d t h e h i g h m o l e c u l a r w e i g h t a n d specific v o l u m e of t h e p o l y m e r m a k e i t well s u i t e d t o t h i s f u n c t i o n . RIENITS 15 also s u g g e s t e d a role for h y a l u r o n i c a c i d i n w a t e r r e t e n t i o n . H o w e v e r , m o r e d i r e c t e v i d e n c e is r e q u i r e d t o s u p p o r t t h a t t h i s s u b s t a n c e h a s a p h y s i o l o g i c cal f u n c t i o n i n w a t e r r e t e n t i o n . T h e a u t h o r is g r a t e f u l t o D r . J . R . B a k e r for his a s s i s t a n c e a n d t o P r o f e s s o r B . S p e n c e r for t h e u s e of f a c i l i t i e s i n h i s l a b o r a t o r y . REFERENCES I S. SCHILLER, Biochim. Biophys. Acta, 32 (1959) 215. 2 E. MCNALLY, Proc. Soc. Exptl. Biol. Med., 31 (1934) 946. 3 J- E. SCOTT, in D. GLICK, iViethodsBiochem. Anal., Vol. 8, Interscience, New York, 196o, p. 145. 4 K. !~IEYER, E. A. DAVlDSON, A. LINKER AND P. HOFFMAN, Biochim. Biophys. Acta, 21 (1959)
506. G. A. LEvvY AND A. MCALLAN, Biochem. J., 73 (1959) 127. P. STOFFYN AND R. JEANLOZ, Arch. Biochem. Biophys., 52 (1954) 373. Z. DISCHE, ,[. Biol. Chem., 167 (1947) 189. H. T. GORDON, W. THORBURG AND L. N. WERtJM, Anal. Chem., 28 (1956) 849. K. S. DODGSON AND R. G. PRICE, Biochem. J., 84 (1962) lO6. C. W. CASTOR AND E. L. DORSTEWlTZ, J. Chromatogr., 13 (1964) 157. A. G. LLOYD, K. S. DODGSON, R. G. PRICE AND F. A. ROSE, Biochim. Biophys. Acta, 46 (1961) lO8. F. S. WUSTEMAN, A. G. LLOYD AND K. S. DODGSON, J. Chromatogr., 21 (1966) 32. 13 J. R. BAKER AND D. A. BALCH, Biochem. J., 82 (i962) 352. 14 A. LIFSHITZ AND R. C. BAKER, Poult. Sci., 43 (1964) 527I5 K. G. RIEXlTS, Biochem. J., 74 (196o) 27. 5 6 7 8 9 IO Ii 12
Biochim. Biophys. Acta, 244 (1971) 481-483