Carbohydiate Research, 40 (1975) 53-67 0 BlsevierScientificPublishingCompany,Amsterdam-
Printedin Belgium
THE ISOLATION AND CHARACTERIZATION MUCUS-GLYCOPROTEIN* JOHNMOSCHERA ANLI New
York Medical
WARD
College,
OF RAT SUBLINGUAL
PIGhUN
Biochemistry
Departmen?,
Valhalla, New
York IO595 (U.
S. A.)
(ReceivedOctober 29th, 1974; acceptedin revisedform, December 16th, 1974)
ABSTRACT
A purified glycoprotein, designated RSL-major, was isolated from the rat sublingual gland by means of the procedure of Tettamanti and Pigman. It was found to be homogeneous by analytical ultracentrifugation, to have a mol. wt. of 2.2 x 106, and to contain 81% (w/w) of carbohydrate, which consists mainly of sialic acid, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, and D-galactose in the molar ratio of 1.4:1.4:1.0:1.5; small amounts of fucose and mannose Cl.2 and 2.8% (w/w), respectively] were also present. The sialic acid residues were resistant to the action of V. chokrae neuraminidase. This resistance was completely abolished by removal of the O-ace@ groups contained in the sialic acid. The sialic acid in RSL-major appeared to be a mixture of N-acetyl-4-O-acetyl- and _V-acetyl-4,7(8)-d& 0-acetylneuraminic acids. The carbohydrate to protein attachment of RSL-major was shown, by alkaline &elimination reaction, to consist of an 0-glycosyl linkage between 2-acetamido-2-deoxy-D-galactosyl residues in the oiigosaccharide chains and seryl and threonyl residues in the protein core. The average oligosaccharide, contained in RSL-major, was postulated to be a heptasaccharide. A second material, designated RSL-minor, and also isolated from the rat sublingual gland, was obtained as a mixture of glycoprotein(s) and hydroxylapatite gel, and was not purit?ed further. INTRODUCTION
The epithelial mucins are the glycoproteins responsible for the viscous nature of the mucus produced by the oral, respiratory, gastrointestinal, and reproductive tracts of higher animals. The best characterized of these materials are the mucus glycoproteins derived from bovine, ovine, and porcine submaxillary glands, human and porcine gastric mucosa, bovine cervix, and human saliva. Recent reviews of the epithelial mucus glycoproteins have been published’-4. Studies of the biosynthesis of mucus glycoproteins and of their physio1ogica.l function have been handicapped because sources of the most well characterized materials are either not readily available, or are derived from animaIs that cannot be conveniently handled in the laboratory. For this reason, we decided to investigate the * Dedicated to Professor Michael Heidelbergerin honor of his 87th birthday.
66
J. MGSCHEIU,
W. PIGMAN
evidence, D-galactose and D-mannose were arbitrarily assigned to the third position in the chain. It is evident, from the structure proposed, that the average oligosaccharide chain present in ML-major contains approximately 7 sugar residues. Because of the marked microheterogeneity observed in the oligosaccharide derived from porcine3’ and bovine” submaxillary mucins, it is probable that RSL-major contains smaller and larger chains as well. Recently, Keryer et al. 5 ’ reported the isolation of mucus glycoproteins from rat submaxillary glands derived from animals of various ages and sex. The materials obtained by them from adult, male and female animals was similar to our RSL products. In our study of the sublingual glycoproteins, careful consideration was given to the possibility of contamination arising from the submaxihary gland, which is located directly adjacent to the sublingual gland. In earlier work, we attempted to prepare the rat submaxillary glycoprotein but found that the amount of extractable, sialic acid-containing glycoproteins was too small for purification and characterization. This result is in agreement with histochemical studies that indicated that the rat submaxillary gland contains very little sialic acid. In our opinion, the products described by Keryer ef ~1.~’ were derived from the sublingual gland, since no mention was made of its existence or removal. REFERENCES 1 W. PrChiAN AND J. MOSCHERA, in J. BLANDAU AND K. MOGHISSI(Ed%), The Biology of the Cervix, University of Chicago Press, Chicago, 1973, pp. 143-172. 2 A. GOTTSCHALK, Glycoproteins, 2nd edn., Elsevier, Amsterdam, 1972. 3 R. G. SPIRO, .4nn. Rev. Biochem., 39 (1970) 599-637. 4 M. BERTOLINIAND F.BJZTTELHEIM,~~W.PIGMAN AND D. HORTON (Eds.)The Carbohydrates Vol. IIB, Academic Press, New York, 1970, pp. 677684. 5 S. S. SPICER AND L. WARREN, J. Histochem. Cytochem., 8 (1959) 135-137. 6 C. H. GANPER, A. A. Toccr, A. B. HOUSSAY, H. M. CURBELO, AXD J. J. DEVALLE, J. Dent. Res., 49 (1970) 346-349. 7 G. QUINTARELLI,S.TSUIKI,Y. HASHIMOTO,AND W.~?IGMAN, J. Histochem. Cytochem.,9 (1961) 176-183. 8 G. TRTAMAN~ AND W. PIGMAN, Arch. Biochem. Biophys., 124 (1968) 41-50. 9 0. H. LOWRY, N. J. ROSEBROUGH, A. L. FAFZI,AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265-275. 10 E DOWNS AND W. PIGMAN, Izt. J. Protein Res., 1 (1969) 181-184. 11 G. Bux, Acta Chem. Scund., 2 (1948) 467-473. 12 L. S~EN~~RHOL~~,Biochim. Biophys. Acta, 24 (1957) 604-61:. 13 L. SVENNERHOLM, Acta Chem. Stand., 12 (1958) 547-554. 14 S. HESTRIN, J. Biol. Chem., 180 (1949) 249-261. 15 L. WARREN, J. Biol. Chem., 234 (1959) 1971-1975. 16 L. SVEhWERHOLM, Actu Sot. Med. Upsal., 61 (1956) 75-85. 17 R. G. SPIRO, Methods Enzymol., 8 (1966) 3-26. 18 E.KI.ENKAND G.UHLENBRUCK,~. PhysioZ. Chem.,307 (1957) 266-271. 19 E. S~NNERHOLM AND L. SVENNERHOLM, Nature, 181 (1958) 1154. 20 W. E. TREVELYAN, D. P. PROCKXR, AND J. S. HARRISON, Nature, 166 (1950) 444445. 21 Z. DISCHEAND A. DANILCXENKO. Anal. Biochem., 21 (1967) 119. 22 Z. DISCHEAND L. B. SHEITLES,J. Biol. Chem., 195 (1948) 595-603. 23 W. NIEDERMEIER, Anal. Biochem., 40 (1971) 465475. 24 C. A. ANTONOPO~LOS, Actu Chem. Scar&., 16 (i962) 1521-1522.
RAT SUBLINGUAL MUCUS-GLYCOPROTEIN
67
25 2. DISCHE, J. BioZ. Chem., 171 (1947) 725-730. 26 N. PAYZA, M. ROBERT, AND A. HERP, ht. f. Protein Res., 2 (1970) 109-I 15. 27 J. M. CREETHAND C. G. KNIGHT, Biochem. J., 10.5 (1962) 1135-1145. 28 W. PIGMAN, 3.MOSCHEXA,M. WEISS, AND G. TETTAMANTI. Eur. J. Biochem., 32 (1973) 148-154. 29 M. BERTOLINIANIJ W. PIGMAN, J. Biol. Chem., 242 (1967) 3776-3781. 30 F. DONS, A. HERP, J. MOSCHERA, AND W. PIGhfAN, Biochim. Biophys. Actu, 328 (1973) 182-192. 31 R. TANAKA AND W. FIGMAN, J. BioZ. Chem., 240 (1965) FC1487. 32 D. M. CARLSON,J. Biol. Chem., 243 (1968) 616-626. 33 J. W. MAYO AND D. M. CARLSON, Carbohyd. Res., 15 (1970) 300-303. 34 A. M. BELLA, JR. AND Y. S. Kfhf, L Chromafogr., 51 (1970) 314-315. 35 A. S. R. DONALD, J. Chromatogr., 35 (1968) 106-107. 36 D. AMINOFF, Biochem. J., 81 (1961) 384-392. 37 M. DE SALEGUI AND H. PLONSKA, Arch. BZochem. Biophys., 129 (1969) 49-56. 38 N. PAYZA, L. MARTINEZ, AND W. PIGhrAN, Anim. Hood Groaps Biochem. Gener., I (1970) 195-206. 39 M. I. HOROWITZ,L. MARTINEZ, AND V. L. N. M’JRTY, Biochitn. Biophys. Acra. 83 (1964) 305-317. 40 Y. HASHIMOTO, S. &.WlhfOTO, AND W. PIGMAN, Arc/~. Biochem. Biophys., 104 (1964) 282-291. 41 \rJ. hGhfAN AND G. Tmw, in E. ROSSI AND E. STOU (Eds.), 4r11Irrr. Conf. Cystic Fibrosis Pancreas (Mrtcoviscidosis), Part II, Karger, Basel/New York, 1968, pp. 117-140. 42 C. LohfsART AND R. J. WINZLER, Biochem. J., 128 (1972) 975-977. 43 V. R. SCHAUER AND H. FAILLARD, Z. Physiol. Chem., 349 (1968) 961-968. 44 G. B. PAERELT AND J. SCHUT, Biochem. J., 96 (1965) 757-792. 45 V. R. SCHAUER, Angero. Chem., 12 (1973) 127-138. 46 R. W. JEANLOZ AND M. T&M&E, Fed. Proc., 15 (1956) 282. 47 K. 0. LLOYD, E. A. KABAT, AND E. LICERIO, Biochemistry, 7 (1968) 2976-2990. 4% P. WEBER AND R. J. WINZLER, Arch. Biochem. Biophys., 129 (1969) 534-538. 49 J. R. VERCELLOTII, R. FERNANDEZ, AND C. J. CHANG, Carbohyd. Res., 5 (1967) 97-101. 50 F. DOIVNSAND W. hGhwN, Bioclremisrry, 8 (1969) 1760-1766. 51 G. KERYER, G. HEFWW, AND B. RO~~IGNOL, Biochim. Biophys. Acra, 297 (1973) 186-191.
Printedin Belgium
THE ISOLATION AND CHARACTERIZATION MUCUS-GLYCOPROTEIN* JOHNMOSCHERA ANLI New
York Medical
WARD
College,
OF RAT SUBLINGUAL
PIGhUN
Biochemistry
Departmen?,
Valhalla, New
York IO595 (U.
S. A.)
(ReceivedOctober 29th, 1974; acceptedin revisedform, December 16th, 1974)
ABSTRACT
A purified glycoprotein, designated RSL-major, was isolated from the rat sublingual gland by means of the procedure of Tettamanti and Pigman. It was found to be homogeneous by analytical ultracentrifugation, to have a mol. wt. of 2.2 x 106, and to contain 81% (w/w) of carbohydrate, which consists mainly of sialic acid, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, and D-galactose in the molar ratio of 1.4:1.4:1.0:1.5; small amounts of fucose and mannose Cl.2 and 2.8% (w/w), respectively] were also present. The sialic acid residues were resistant to the action of V. chokrae neuraminidase. This resistance was completely abolished by removal of the O-ace@ groups contained in the sialic acid. The sialic acid in RSL-major appeared to be a mixture of N-acetyl-4-O-acetyl- and _V-acetyl-4,7(8)-d& 0-acetylneuraminic acids. The carbohydrate to protein attachment of RSL-major was shown, by alkaline &elimination reaction, to consist of an 0-glycosyl linkage between 2-acetamido-2-deoxy-D-galactosyl residues in the oiigosaccharide chains and seryl and threonyl residues in the protein core. The average oligosaccharide, contained in RSL-major, was postulated to be a heptasaccharide. A second material, designated RSL-minor, and also isolated from the rat sublingual gland, was obtained as a mixture of glycoprotein(s) and hydroxylapatite gel, and was not purit?ed further. INTRODUCTION
The epithelial mucins are the glycoproteins responsible for the viscous nature of the mucus produced by the oral, respiratory, gastrointestinal, and reproductive tracts of higher animals. The best characterized of these materials are the mucus glycoproteins derived from bovine, ovine, and porcine submaxillary glands, human and porcine gastric mucosa, bovine cervix, and human saliva. Recent reviews of the epithelial mucus glycoproteins have been published’-4. Studies of the biosynthesis of mucus glycoproteins and of their physio1ogica.l function have been handicapped because sources of the most well characterized materials are either not readily available, or are derived from animaIs that cannot be conveniently handled in the laboratory. For this reason, we decided to investigate the * Dedicated to Professor Michael Heidelbergerin honor of his 87th birthday.
66
J. MGSCHEIU,
W. PIGMAN
evidence, D-galactose and D-mannose were arbitrarily assigned to the third position in the chain. It is evident, from the structure proposed, that the average oligosaccharide chain present in ML-major contains approximately 7 sugar residues. Because of the marked microheterogeneity observed in the oligosaccharide derived from porcine3’ and bovine” submaxillary mucins, it is probable that RSL-major contains smaller and larger chains as well. Recently, Keryer et al. 5 ’ reported the isolation of mucus glycoproteins from rat submaxillary glands derived from animals of various ages and sex. The materials obtained by them from adult, male and female animals was similar to our RSL products. In our study of the sublingual glycoproteins, careful consideration was given to the possibility of contamination arising from the submaxihary gland, which is located directly adjacent to the sublingual gland. In earlier work, we attempted to prepare the rat submaxillary glycoprotein but found that the amount of extractable, sialic acid-containing glycoproteins was too small for purification and characterization. This result is in agreement with histochemical studies that indicated that the rat submaxillary gland contains very little sialic acid. In our opinion, the products described by Keryer ef ~1.~’ were derived from the sublingual gland, since no mention was made of its existence or removal. REFERENCES 1 W. PrChiAN AND J. MOSCHERA, in J. BLANDAU AND K. MOGHISSI(Ed%), The Biology of the Cervix, University of Chicago Press, Chicago, 1973, pp. 143-172. 2 A. GOTTSCHALK, Glycoproteins, 2nd edn., Elsevier, Amsterdam, 1972. 3 R. G. SPIRO, .4nn. Rev. Biochem., 39 (1970) 599-637. 4 M. BERTOLINIAND F.BJZTTELHEIM,~~W.PIGMAN AND D. HORTON (Eds.)The Carbohydrates Vol. IIB, Academic Press, New York, 1970, pp. 677684. 5 S. S. SPICER AND L. WARREN, J. Histochem. Cytochem., 8 (1959) 135-137. 6 C. H. GANPER, A. A. Toccr, A. B. HOUSSAY, H. M. CURBELO, AXD J. J. DEVALLE, J. Dent. Res., 49 (1970) 346-349. 7 G. QUINTARELLI,S.TSUIKI,Y. HASHIMOTO,AND W.~?IGMAN, J. Histochem. Cytochem.,9 (1961) 176-183. 8 G. TRTAMAN~ AND W. PIGMAN, Arch. Biochem. Biophys., 124 (1968) 41-50. 9 0. H. LOWRY, N. J. ROSEBROUGH, A. L. FAFZI,AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265-275. 10 E DOWNS AND W. PIGMAN, Izt. J. Protein Res., 1 (1969) 181-184. 11 G. Bux, Acta Chem. Scund., 2 (1948) 467-473. 12 L. S~EN~~RHOL~~,Biochim. Biophys. Acta, 24 (1957) 604-61:. 13 L. SVENNERHOLM, Acta Chem. Stand., 12 (1958) 547-554. 14 S. HESTRIN, J. Biol. Chem., 180 (1949) 249-261. 15 L. WARREN, J. Biol. Chem., 234 (1959) 1971-1975. 16 L. SVEhWERHOLM, Actu Sot. Med. Upsal., 61 (1956) 75-85. 17 R. G. SPIRO, Methods Enzymol., 8 (1966) 3-26. 18 E.KI.ENKAND G.UHLENBRUCK,~. PhysioZ. Chem.,307 (1957) 266-271. 19 E. S~NNERHOLM AND L. SVENNERHOLM, Nature, 181 (1958) 1154. 20 W. E. TREVELYAN, D. P. PROCKXR, AND J. S. HARRISON, Nature, 166 (1950) 444445. 21 Z. DISCHEAND A. DANILCXENKO. Anal. Biochem., 21 (1967) 119. 22 Z. DISCHEAND L. B. SHEITLES,J. Biol. Chem., 195 (1948) 595-603. 23 W. NIEDERMEIER, Anal. Biochem., 40 (1971) 465475. 24 C. A. ANTONOPO~LOS, Actu Chem. Scar&., 16 (i962) 1521-1522.
RAT SUBLINGUAL MUCUS-GLYCOPROTEIN
67
25 2. DISCHE, J. BioZ. Chem., 171 (1947) 725-730. 26 N. PAYZA, M. ROBERT, AND A. HERP, ht. f. Protein Res., 2 (1970) 109-I 15. 27 J. M. CREETHAND C. G. KNIGHT, Biochem. J., 10.5 (1962) 1135-1145. 28 W. PIGMAN, 3.MOSCHEXA,M. WEISS, AND G. TETTAMANTI. Eur. J. Biochem., 32 (1973) 148-154. 29 M. BERTOLINIANIJ W. PIGMAN, J. Biol. Chem., 242 (1967) 3776-3781. 30 F. DONS, A. HERP, J. MOSCHERA, AND W. PIGhfAN, Biochim. Biophys. Actu, 328 (1973) 182-192. 31 R. TANAKA AND W. FIGMAN, J. BioZ. Chem., 240 (1965) FC1487. 32 D. M. CARLSON,J. Biol. Chem., 243 (1968) 616-626. 33 J. W. MAYO AND D. M. CARLSON, Carbohyd. Res., 15 (1970) 300-303. 34 A. M. BELLA, JR. AND Y. S. Kfhf, L Chromafogr., 51 (1970) 314-315. 35 A. S. R. DONALD, J. Chromatogr., 35 (1968) 106-107. 36 D. AMINOFF, Biochem. J., 81 (1961) 384-392. 37 M. DE SALEGUI AND H. PLONSKA, Arch. BZochem. Biophys., 129 (1969) 49-56. 38 N. PAYZA, L. MARTINEZ, AND W. PIGhrAN, Anim. Hood Groaps Biochem. Gener., I (1970) 195-206. 39 M. I. HOROWITZ,L. MARTINEZ, AND V. L. N. M’JRTY, Biochitn. Biophys. Acra. 83 (1964) 305-317. 40 Y. HASHIMOTO, S. &.WlhfOTO, AND W. PIGMAN, Arc/~. Biochem. Biophys., 104 (1964) 282-291. 41 \rJ. hGhfAN AND G. Tmw, in E. ROSSI AND E. STOU (Eds.), 4r11Irrr. Conf. Cystic Fibrosis Pancreas (Mrtcoviscidosis), Part II, Karger, Basel/New York, 1968, pp. 117-140. 42 C. LohfsART AND R. J. WINZLER, Biochem. J., 128 (1972) 975-977. 43 V. R. SCHAUER AND H. FAILLARD, Z. Physiol. Chem., 349 (1968) 961-968. 44 G. B. PAERELT AND J. SCHUT, Biochem. J., 96 (1965) 757-792. 45 V. R. SCHAUER, Angero. Chem., 12 (1973) 127-138. 46 R. W. JEANLOZ AND M. T&M&E, Fed. Proc., 15 (1956) 282. 47 K. 0. LLOYD, E. A. KABAT, AND E. LICERIO, Biochemistry, 7 (1968) 2976-2990. 4% P. WEBER AND R. J. WINZLER, Arch. Biochem. Biophys., 129 (1969) 534-538. 49 J. R. VERCELLOTII, R. FERNANDEZ, AND C. J. CHANG, Carbohyd. Res., 5 (1967) 97-101. 50 F. DOIVNSAND W. hGhwN, Bioclremisrry, 8 (1969) 1760-1766. 51 G. KERYER, G. HEFWW, AND B. RO~~IGNOL, Biochim. Biophys. Acra, 297 (1973) 186-191.
