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Characterization of sulphated monosaccharides from stumptail monkey (Macaca arctoides) salivary mucin
Arch.7owl Biol. Vol. 26, pp. 315 to 317, 1981 Printed m Great Britain
OC03-9969/X1/040315-03102.00/O Pergamoo Press Ltd
CHARACTERIZATION OF SULPHATED MONOSACCHARIDES FROM STUMPTAIL MONKEY (MACACA ARCTOIDES) SALIVARY MUCIN L. A. Department
TABAK
and M. J.
LEVINE
of Oral Biology, School of Dentistry New York at Buffalo 14226
State University
of
Summary+_Jsing a combination of mild acid hydrolysis, ion-exchange chromatography on Dowex resins, and Smith periodate oxidation with sodium borotritide, the sulphated monosaccharide of monkey salivary mucin was identified as glucosamine-4-sulphate.
INTRODUCTION
Identification of sulphated monosaccharides and characterization of the linkage from glycoproteins can be laborious and is often impossible because material cannot be obtained. The latter problem can be partially avoided in animal species (Embery and Ward, 1976) or tissue culture (Heifetz, Kinsey and Lennarz, 1980) by utilizing 35S-labelled glycoproteins. However, when dealing with highly purified salivary mucins, the small amount of material available requires simple yet sensitive methods for identifying and characterizing the sulphated monosaccharide. Mild-acid hydrolysis had been utilized to cleave sulphated monosaccharides from the parent molecule (Pamer, Glass and Horowitz, 1968); Slomiany and Meyer, 1972; Embery and Ward, 1976) and the monosaccharide identified following complete hydrolysis. The nature of the sulphate linkage can be studied by chromatographic comparison with authentic sulphated monosaccharides and infrared spectroscopy. We describe here a simplified approach that does not require sulphated standards and infrared spectroscopy.
METHODS
Materiuls
Mucin was purified from the extra-parotid saliva of the stumptail monkey (Macaca arctoides) as described by Herzberg et al. (1979). Xylosaminitol was prepared by Smith degradation (Smith and Unrau, 1959) of diN-acetylchitibiose isolated by preparative paper chromatography following the mild-acid hydrolysis of chitin (Arima and Spiro, 1972). Threosaminitol was prepared by Smith periodate oxidation of N-acetylchondrosinitol previously derived from N-acetylation and sodium borohydride (NaBH4) reduction of chondrosine (Spiro and Bhoyroo, 1974). Serinol was purchased from Vega Biochemicals, Tucson, AZ. Identijicntion
of sulphated
monosuccharide
Asialo-mucin was prepared as described by Levine er al. (1978) whereby approximately 90 per cent of the total siahc acids were released. Sulphated sugars were prepared by treating asialo-mucin (1 mg/ml) with 0.1 M HCI at 100°C for 90min (Embery and Ward, 1976). After neutralization with 0.1 M NaOH, hber-
ated constituents were separated from the parent material by dialysis. The lyophilized dialysate was passed through coupled columns of Dowex 50-X4-H+ (2mOO mesh) and Dowex l-X8 formate (200-400 mesh). Neutral sugars were identified and quantitated as described below. Hexosamines were desorbed from Dowex 50 with 2 M HCl. Sulphated sugars were desorbed from Dowex 1 with 2 M formic acid. An aliquot of the acidic material was desulphated with 2 M HCI at 100°C for 6 h. This hydrolysate was then passed through coupled Dowex columns and the neutral sugars and hexosamines obtained as described below.
Characterization
of sulphate linkage
An aliquot of the sulphated monosaccharide was reduced with sodium borohydride as follows: l&l5 nmol was incubated at 37°C for 24 h in 100 ~1 of 0.1 M NaOH with 1 M NaBH4. The reaction was then acidified to pH 5.0 with cold 4 M acetic acid and desalted at 4°C on a column (1.4 ml bed volume) of Dowex 50-X4-H+ (20&400 mesh). Materials were eluted with 0.01 M formic acid, lyophilized, and subjected to Smith periodate oxidation using NaBT4 to facilitate identification of the reaction product. A typical experiment was carried out as follows: a lo-fold molar excess of sodium metaperiodate (5 mM in 0.05 M sodium acetate, pH 4.5) was added to the sample and incubated at 4°C for 16 h in the dark. This reaction was terminated by the addition of ethylene glycol in a lo-fold excess over the periodate employed (5 h at 4°C). Following lyophilization, the dry residue was dissolved in 0.1 M tris, pH 9.0 (100 ~1) and reduced with NaBH, containing 0.5 &i (microNew England curies) NaBT, (300 mCi/mmol, Nuclear), using a 35 molar excess over the periodate used. After 4 h at 37°C excess NaBH, was destroyed by the addition of cold 4 M acetic acid to pH 5.0. Following lyophilization, samples were de-salted on Dowex 50 as described above, and then borate removed by co-distillation with methanol. The dried sample was then hydrolyzed with 2 M HCI for 6 h at 100°C. The hydrolyzate was applied to a column of Dowex 50-X4-H+ (2t%400 mesh) which was washed with 0.01 M formic acid, then eluted with 1.5 M NHLOH, and this eluate lyophilized. 315
316 Table
L. A. Tabak and M. J. Levine 1.
Component
Results
of mild-acid asialomucin”
Neutralb
Galactosamine Glucosamine Galactose Fucose
6’ 36’ 32 86
hydrolysis
of
2 M HCl and quantitated on a Beckman Model 120 C amino acid analyzer. Neutral sugar and hexosamine values were corrected for losses during hydrolysis.
Recovery of total (“/,) Basic’ Acidicd 0 4.6’ 0 0
RESULTS
0 1.6g 0 0
’ Treatment of 1 mg of material with 0.1 M HCI for 90min at 100°C. b Liberated dialyzable sugars which passed through columns of Dowex 50-Dowex 1. ’ Retained on Dowex 50. d Retained on Dowex 1. eN-acetylated. ’ Deacetylated. 8 Identified as glucosamine after elution from Dowex 1 and hydrolysis with 2 M I-ICI at 100°C for 6h. Reduced products were identified by descending paper chromatography (24 h) using a solvent system of n-butanol:acetic acid:water (4: 1:5). For 3Hlabelled samples, 1 cm strips were cut from the chromatograms, eluted with water, and the eluates examined by liquid scintillation spectrometry. Analytical procedures For the determination of neutral sugars and hexosamines, samples were hydrolyzed in 2 M HCI for 6 h at 100°C in sealed tubes. The hydrolysates were passed through coupled columns of Dowex 50-X4-H+ (2wOO mesh) and Dowex l-X8 formate (2oo-400 mesh). Neutral sugars in the effluent water wash were quantitated by automated borate-complex anion exchange chromatography (Lee et al., 1971). Amino sugars were eluted from the Dowex 50 columns with
AND
DISCUSSION
Mild acid hydrolysis (0.1 M HCl) has been utilized to release monosaccharide-0-SO,H derivatives in contrast to sulpho-amino (N-S03H) groups which are completely hydrolyzed from hexosamines in 0.04 M HCI at 90°C for 1 h (Lagunoff and Warren, 1962). The residues released by mild acid hydrolysis of asialo-monkey mucin are summarized in Table 1. The majority of liberated monosaccharides were neutral. Approximately 86 per cent of the total fucose in the asialo-mucin was released followed by substantial amounts of N-acetylglucosamine (36 per cent) and galactose (32 per cent). These findings probably reflect the marked microheterogeneity of the carbohydrate units of the mucin. Another 4.6 per cent of the glucosamine was released and also de-acetylated. The acidic materials retained on Dowex 1 were glucosamine derivatives because acid hydrolysis of this product yielded only free glucosamine. The sulphated glucosamine was then reduced to glucosaminitol sulphate. This material was oxidized with periodate, reduced with NaBT4, and then hydrolyzed with 2 M HCI. Following elution of the hydrolyzate from Dowex 50 with 1.5 M NHLOH, the [3H-labelled] derivative was identified by descending paper chromatography using co-chromatographed standard. The results (Fig. 1) revealed that glucosaminitol sulphate was converted to xylosaminitol, indicating that glucosamine was substituted at C-4. To date, few studies have provided information regarding the nature of the sulphated monosaccharide of salivary mucins. Lombart and Winzler (1974) indicated that canine submandibular mucin contained glucosamine substituted at C-3 or C-4. Embery and Ward (1976) found that a glycoprotein from rat whole
x
-
6
02
4
8
12
16
20
24
28
32
36
40’
Migration ( cm) Fig. 1. Paper chromatography (n-butanol :acetic acid: water; 4: 1: 5) of glucosaminitolsulphate following Smith periodate oxidation using sodium borotritide. Standards were detected by silver nitrate staining (Trevelyan er al., 1950).
Sulphated monosaccharides saliva contained a sulphate-hexosamine with the characteristics of galactosamine-6-sulphate. We have here characterized the sulphated monosaccharide of salivary mucin from the stumptail monkey as glucosamine-4-sulphate. Acknowledgements-This research was supported in part bv Public Health Service research grants 1 ROl-DE04971. l-R23-DE04518 and training gran; 5T32-DE-07034. Par; of this report was taken from a thesis submitted by L.A.T. to the State University of New York at Buffalo in partial fulfillment of the Ph. D. degree.
REFERENCES
Arima T. and Spiro R. G. 1972. Studies on the carbohydrate units of thyroglobulin. J. biol. Chem. 247, 1836-1848. Embery G. and Ward C. 1976. The nature of the sulphate grouping in a rat salivary sulphated glycoprotein. Archs oral Biol. 21, 627-629. Heifetz A., Kinsey W. H. and Lennarz W. J. 1980. Synthesis of a novel class of sulphated glycoproteins in embrvonic liver and hma. J. biol. Chem. 255.4528-4534. Herzbeig M. C., Levine h. J., Ellison S. A: and Tabak L. A. 1979. Purification and characterization of monkey salivary mucin. J. biol. Chem. 254, 1487-1494.
from salivary mucin
317
Lagunoff D. and Warren G. 1962. Determination of a 2-deoxy-2-sulphoaminohexose content of mucopolysaccharides. Archs Biochem. Biophys. 99, 39&401. Lee Y. C., Johnson G. S., White B. and Scocca J. 1971. An accelerated system for analysis of neutral sugars in complex carbohydrates. Analyt. Biochem. 43, 640-643. Levine M. J., Herzberg M. C., Levine M. S., Elhson S. A., Stinson M. W., Li H. C. and Van Dyke T. 1978. Specificity of salivary-bacterial interactions: role of terminal sialic acid residues in the interaction of salivary glycoproteins with Streptococcus sanguis and Streptococcus mutans. Infect. Immun. 19, 107-l 15. Lombart C. G. and Winzler R. J. 1974. Isolation and characterization of oligosaccharides from canine submaxillary mucin. Eur. J. Biochem. 49, 77-86. Pamer T., Glass G. B. J. and Horowitz M. I. 1968. Purification and characterization of sulphated glycoproteins and hyaluronidase resistant mucopolysaccharides from dog gastric mucosa. Biochemistry 7, 3821-3829. Slomiany B. L. and Meyer K. 1972. Isolation and structural studies of sulphated glycoproteins of hog gastric mucosa. J. biol. Chem. 247, 5062-5070. Smith F. and Unrau A. M. 1959. On the presence of 1+6 linkages in laminarin. Chemy Ind. p. 881. Spiro R. G. and Bhoyroo V. D. 1974. Structure of the . 0-glycosidically linkkd carbohydrate units of fetuin. J. biol. Chem. 249. 570&5717. Trevelyan W. E., Proctor D. P. and Harrison J. S. 1950. Detection of sugars on paper chromatograms. Nature l&444445.
OC03-9969/X1/040315-03102.00/O Pergamoo Press Ltd
CHARACTERIZATION OF SULPHATED MONOSACCHARIDES FROM STUMPTAIL MONKEY (MACACA ARCTOIDES) SALIVARY MUCIN L. A. Department
TABAK
and M. J.
LEVINE
of Oral Biology, School of Dentistry New York at Buffalo 14226
State University
of
Summary+_Jsing a combination of mild acid hydrolysis, ion-exchange chromatography on Dowex resins, and Smith periodate oxidation with sodium borotritide, the sulphated monosaccharide of monkey salivary mucin was identified as glucosamine-4-sulphate.
INTRODUCTION
Identification of sulphated monosaccharides and characterization of the linkage from glycoproteins can be laborious and is often impossible because material cannot be obtained. The latter problem can be partially avoided in animal species (Embery and Ward, 1976) or tissue culture (Heifetz, Kinsey and Lennarz, 1980) by utilizing 35S-labelled glycoproteins. However, when dealing with highly purified salivary mucins, the small amount of material available requires simple yet sensitive methods for identifying and characterizing the sulphated monosaccharide. Mild-acid hydrolysis had been utilized to cleave sulphated monosaccharides from the parent molecule (Pamer, Glass and Horowitz, 1968); Slomiany and Meyer, 1972; Embery and Ward, 1976) and the monosaccharide identified following complete hydrolysis. The nature of the sulphate linkage can be studied by chromatographic comparison with authentic sulphated monosaccharides and infrared spectroscopy. We describe here a simplified approach that does not require sulphated standards and infrared spectroscopy.
METHODS
Materiuls
Mucin was purified from the extra-parotid saliva of the stumptail monkey (Macaca arctoides) as described by Herzberg et al. (1979). Xylosaminitol was prepared by Smith degradation (Smith and Unrau, 1959) of diN-acetylchitibiose isolated by preparative paper chromatography following the mild-acid hydrolysis of chitin (Arima and Spiro, 1972). Threosaminitol was prepared by Smith periodate oxidation of N-acetylchondrosinitol previously derived from N-acetylation and sodium borohydride (NaBH4) reduction of chondrosine (Spiro and Bhoyroo, 1974). Serinol was purchased from Vega Biochemicals, Tucson, AZ. Identijicntion
of sulphated
monosuccharide
Asialo-mucin was prepared as described by Levine er al. (1978) whereby approximately 90 per cent of the total siahc acids were released. Sulphated sugars were prepared by treating asialo-mucin (1 mg/ml) with 0.1 M HCI at 100°C for 90min (Embery and Ward, 1976). After neutralization with 0.1 M NaOH, hber-
ated constituents were separated from the parent material by dialysis. The lyophilized dialysate was passed through coupled columns of Dowex 50-X4-H+ (2mOO mesh) and Dowex l-X8 formate (200-400 mesh). Neutral sugars were identified and quantitated as described below. Hexosamines were desorbed from Dowex 50 with 2 M HCl. Sulphated sugars were desorbed from Dowex 1 with 2 M formic acid. An aliquot of the acidic material was desulphated with 2 M HCI at 100°C for 6 h. This hydrolysate was then passed through coupled Dowex columns and the neutral sugars and hexosamines obtained as described below.
Characterization
of sulphate linkage
An aliquot of the sulphated monosaccharide was reduced with sodium borohydride as follows: l&l5 nmol was incubated at 37°C for 24 h in 100 ~1 of 0.1 M NaOH with 1 M NaBH4. The reaction was then acidified to pH 5.0 with cold 4 M acetic acid and desalted at 4°C on a column (1.4 ml bed volume) of Dowex 50-X4-H+ (20&400 mesh). Materials were eluted with 0.01 M formic acid, lyophilized, and subjected to Smith periodate oxidation using NaBT4 to facilitate identification of the reaction product. A typical experiment was carried out as follows: a lo-fold molar excess of sodium metaperiodate (5 mM in 0.05 M sodium acetate, pH 4.5) was added to the sample and incubated at 4°C for 16 h in the dark. This reaction was terminated by the addition of ethylene glycol in a lo-fold excess over the periodate employed (5 h at 4°C). Following lyophilization, the dry residue was dissolved in 0.1 M tris, pH 9.0 (100 ~1) and reduced with NaBH, containing 0.5 &i (microNew England curies) NaBT, (300 mCi/mmol, Nuclear), using a 35 molar excess over the periodate used. After 4 h at 37°C excess NaBH, was destroyed by the addition of cold 4 M acetic acid to pH 5.0. Following lyophilization, samples were de-salted on Dowex 50 as described above, and then borate removed by co-distillation with methanol. The dried sample was then hydrolyzed with 2 M HCI for 6 h at 100°C. The hydrolyzate was applied to a column of Dowex 50-X4-H+ (2t%400 mesh) which was washed with 0.01 M formic acid, then eluted with 1.5 M NHLOH, and this eluate lyophilized. 315
316 Table
L. A. Tabak and M. J. Levine 1.
Component
Results
of mild-acid asialomucin”
Neutralb
Galactosamine Glucosamine Galactose Fucose
6’ 36’ 32 86
hydrolysis
of
2 M HCl and quantitated on a Beckman Model 120 C amino acid analyzer. Neutral sugar and hexosamine values were corrected for losses during hydrolysis.
Recovery of total (“/,) Basic’ Acidicd 0 4.6’ 0 0
RESULTS
0 1.6g 0 0
’ Treatment of 1 mg of material with 0.1 M HCI for 90min at 100°C. b Liberated dialyzable sugars which passed through columns of Dowex 50-Dowex 1. ’ Retained on Dowex 50. d Retained on Dowex 1. eN-acetylated. ’ Deacetylated. 8 Identified as glucosamine after elution from Dowex 1 and hydrolysis with 2 M I-ICI at 100°C for 6h. Reduced products were identified by descending paper chromatography (24 h) using a solvent system of n-butanol:acetic acid:water (4: 1:5). For 3Hlabelled samples, 1 cm strips were cut from the chromatograms, eluted with water, and the eluates examined by liquid scintillation spectrometry. Analytical procedures For the determination of neutral sugars and hexosamines, samples were hydrolyzed in 2 M HCI for 6 h at 100°C in sealed tubes. The hydrolysates were passed through coupled columns of Dowex 50-X4-H+ (2wOO mesh) and Dowex l-X8 formate (2oo-400 mesh). Neutral sugars in the effluent water wash were quantitated by automated borate-complex anion exchange chromatography (Lee et al., 1971). Amino sugars were eluted from the Dowex 50 columns with
AND
DISCUSSION
Mild acid hydrolysis (0.1 M HCl) has been utilized to release monosaccharide-0-SO,H derivatives in contrast to sulpho-amino (N-S03H) groups which are completely hydrolyzed from hexosamines in 0.04 M HCI at 90°C for 1 h (Lagunoff and Warren, 1962). The residues released by mild acid hydrolysis of asialo-monkey mucin are summarized in Table 1. The majority of liberated monosaccharides were neutral. Approximately 86 per cent of the total fucose in the asialo-mucin was released followed by substantial amounts of N-acetylglucosamine (36 per cent) and galactose (32 per cent). These findings probably reflect the marked microheterogeneity of the carbohydrate units of the mucin. Another 4.6 per cent of the glucosamine was released and also de-acetylated. The acidic materials retained on Dowex 1 were glucosamine derivatives because acid hydrolysis of this product yielded only free glucosamine. The sulphated glucosamine was then reduced to glucosaminitol sulphate. This material was oxidized with periodate, reduced with NaBT4, and then hydrolyzed with 2 M HCI. Following elution of the hydrolyzate from Dowex 50 with 1.5 M NHLOH, the [3H-labelled] derivative was identified by descending paper chromatography using co-chromatographed standard. The results (Fig. 1) revealed that glucosaminitol sulphate was converted to xylosaminitol, indicating that glucosamine was substituted at C-4. To date, few studies have provided information regarding the nature of the sulphated monosaccharide of salivary mucins. Lombart and Winzler (1974) indicated that canine submandibular mucin contained glucosamine substituted at C-3 or C-4. Embery and Ward (1976) found that a glycoprotein from rat whole
x
-
6
02
4
8
12
16
20
24
28
32
36
40’
Migration ( cm) Fig. 1. Paper chromatography (n-butanol :acetic acid: water; 4: 1: 5) of glucosaminitolsulphate following Smith periodate oxidation using sodium borotritide. Standards were detected by silver nitrate staining (Trevelyan er al., 1950).
Sulphated monosaccharides saliva contained a sulphate-hexosamine with the characteristics of galactosamine-6-sulphate. We have here characterized the sulphated monosaccharide of salivary mucin from the stumptail monkey as glucosamine-4-sulphate. Acknowledgements-This research was supported in part bv Public Health Service research grants 1 ROl-DE04971. l-R23-DE04518 and training gran; 5T32-DE-07034. Par; of this report was taken from a thesis submitted by L.A.T. to the State University of New York at Buffalo in partial fulfillment of the Ph. D. degree.
REFERENCES
Arima T. and Spiro R. G. 1972. Studies on the carbohydrate units of thyroglobulin. J. biol. Chem. 247, 1836-1848. Embery G. and Ward C. 1976. The nature of the sulphate grouping in a rat salivary sulphated glycoprotein. Archs oral Biol. 21, 627-629. Heifetz A., Kinsey W. H. and Lennarz W. J. 1980. Synthesis of a novel class of sulphated glycoproteins in embrvonic liver and hma. J. biol. Chem. 255.4528-4534. Herzbeig M. C., Levine h. J., Ellison S. A: and Tabak L. A. 1979. Purification and characterization of monkey salivary mucin. J. biol. Chem. 254, 1487-1494.
from salivary mucin
317
Lagunoff D. and Warren G. 1962. Determination of a 2-deoxy-2-sulphoaminohexose content of mucopolysaccharides. Archs Biochem. Biophys. 99, 39&401. Lee Y. C., Johnson G. S., White B. and Scocca J. 1971. An accelerated system for analysis of neutral sugars in complex carbohydrates. Analyt. Biochem. 43, 640-643. Levine M. J., Herzberg M. C., Levine M. S., Elhson S. A., Stinson M. W., Li H. C. and Van Dyke T. 1978. Specificity of salivary-bacterial interactions: role of terminal sialic acid residues in the interaction of salivary glycoproteins with Streptococcus sanguis and Streptococcus mutans. Infect. Immun. 19, 107-l 15. Lombart C. G. and Winzler R. J. 1974. Isolation and characterization of oligosaccharides from canine submaxillary mucin. Eur. J. Biochem. 49, 77-86. Pamer T., Glass G. B. J. and Horowitz M. I. 1968. Purification and characterization of sulphated glycoproteins and hyaluronidase resistant mucopolysaccharides from dog gastric mucosa. Biochemistry 7, 3821-3829. Slomiany B. L. and Meyer K. 1972. Isolation and structural studies of sulphated glycoproteins of hog gastric mucosa. J. biol. Chem. 247, 5062-5070. Smith F. and Unrau A. M. 1959. On the presence of 1+6 linkages in laminarin. Chemy Ind. p. 881. Spiro R. G. and Bhoyroo V. D. 1974. Structure of the . 0-glycosidically linkkd carbohydrate units of fetuin. J. biol. Chem. 249. 570&5717. Trevelyan W. E., Proctor D. P. and Harrison J. S. 1950. Detection of sugars on paper chromatograms. Nature l&444445.