Int. J. Peptide Protein Res. 10, 1917, 229-234 Published by Munksgaard, Copenhagen, Denmark

*Nopart may be reproduced by any process without written permission from the [email protected])

CHEMICAL STUDIES ON A HAMSTER SUBLINGUAL GLYCOPROTEIN *FRED DOWNS and ANTHONY HERP

*Herbert Lehman College, C.U.N. Y., Department o f Chemistry, Bronx, New York, and New York Medical College, Department of Biochemistry, Valhalla, New York, U.S.A.

Received 24 January, accepted for publication 19 April 1977 A secretory glycoprotein was isolated from the hamster sublingual gland. I t contains 65% carbohydrates, the remainder being protein. The principal type o f sialic acid was identified as N-acetylneuraminic acid with about If4 o f the molecules O-acetylated. The hexosamine occurs mainly as N-acetylgalactosamine; other sugars present are galactose and fucose. The carbohydrate side chains are linked to the protein core by an 0-glycosyl linkage between seryl and threonyl residues and N-acetylgalactosamine. The glycoprotein has an apparent molecular weight of 330,000. The sialoglycoprotein may act as a biological antifreeze. Relevant differences in the chemical composition o f the secretory glycoproteins of the sublingual and submaxillav salivary glands of this species are briefly discussed.

Key words: freezing point depression-glycoprotein-hamster-sublingual

The epithelial glycoproteins are responsible for the viscous nature of the secretions by the bral, respiratory, gastrointestinal and reproductive tracts of higher animals. Interest in these mucus glycoproteins is partly explained .by their property to exhibit blood-group activity and, consequently, knowledge of their chemical structure may assist in an understanding of the metabolism of blood-group substances and other glycoproteins. Most work on the isolation and characterization of mucus glycoproteins of the oral tavity has been done on the submaxillary glands from the bovine, ovine and porcine species (Pigman et al., 1973). More recently, information on the mouse and hamster submaxillary glycoproteins has been reported '(Roukema et al., 1976; Downs et al., 1976). In &he present study, we report on the composition of the secretory product of the hamster sublingual salivary gland.

MATERIAL

All chemicals used in this work were of reagent grade. Palladium chloride (Eastman Kodak Company, Rochester, NY), was purified and converted into the water soluble palladium chloride dihydrate, as described by Tanaka & Pigman (1 965). Female hamsters (Syrian Golden Hamsters, Charles River, Lakeview, NJ), 6 months old, were housed in groups of three to a cage in animal quarters, maintained at constant temperature and humidity, and given Purina Lab mice and rat chow diet and tap water, ad lib. Isolation procedure. The animals were killed by

chloroform anesthesia; the thoracic region was opened, and the sublingual gland on each side carefully separated from the adhering submaxillary gland, freed from any extraneous tissue, weighed individually, pooled, and stored frozen 229

F. DOWNS and A. HERP

at -20' before use. The pooled sublingual glands were thawed, cut in half and thoroughly homogenized by means of a D u d glass homogenizer (Kontes Glass Company, Vineland, NJ) with 15 vol. of 0.05 M Tris buffer, pH7.4 containing 1 mM CaC12. The homogenate was centrifuged at 4' at 20,000 r.p.m. (42,800g) in a Sorvall RC-2 centrifuge, and the clear supernatant dialyzed for a period of 4 8 h at 4' against repeatedly changed distilled water. The precipitate, which formed in the nondialyzable, was removed by centrifugation, and the clear Supernatant lyophilized. The lyophilized material was suspended in 50% aqueous CaC12 (1 gm/lOOml) stirred overnight at 4" and any insoluble material removed by centrifugation for 30min at 20,000 r.p.m. To the clear supernatant, an equal volume of cold absolute ethanol was added. Then, the volume of the ethanolic solution was diluted by the addition of an equal volume of distilled water; absolute ethanol was added under constant stirring until turbidity appeared (at approximately 70% ethanol, v/v). The turbid solution was left in the cold overnight (or for about 2 h in the freezer at -20'). The precipitated glycoprotein was collected by centrifugation for 30min at 10,000r.p.m. (12,OOOg) at 4", dissolved in distilled water (same volume as aqueous CaCI2 originally used) by stirring for about 1 h , and centrifuged for 30 min at 20,000 r.pm. at 4". The clear supernatant containing the soluble glycoprotein was adjusted to pH 5.6 with 0.5 M NaH2 PO4 and an equal volume of cold absolute ethanol was added. A gel-like precipitate ensued, which was centrifuged for 1 to 2 min at 1,000 r.p.m., using glass centrifuge tubes. The low-speed centrifugation avoids contamination of the glycoprotein, which adheres to the wall of the centrifuge tube as a transparent layer with the extraneous proteins suspended in the supernatant. The supernatant was poured off and water was added to the tubes. The glycoprotein was scraped into the water by means of a glass rod, and the suspension stirred for about 20min in order to dissolve the glycoprotein, and centrifuged at 20,000 r.pm. for 1 h at 4" to eliminate any particulate matter. The protein to sialic acid ratio of the clear 23 0

solution was determined, and again subjected to the alcoholic precipitation step at pH 5.6, as described above. The procedure is repeated until a constant protein to sialic acid ratio is obtained. Usually, the alcoholic precipitation at pH5.6 has to be carried out a total of three times. Analytical methods. The protein content was measured by the method of Lowry er ul. (195 l),

using bovine serum albumin as the standard, and by calculation of the weight contribution of amino acids, as determined by amino acid analysis, after hydrolysis of the glycoprotein in 6 M HCl for 22 h at 110". Amino acids were analyzed on a model 120B Amino Acid Analyzer (Beckman Instruments, Inc., Fullerton, CA), using a Beckman 50A resin at 55", according to Spackman e l ul. (1958). Results were corrected for destruction of serine and threonine, as described earlier (Downs & Pigman, 1969). Total hexosamines were determined by. the Elson-Morgan reaction, as modified by Boas (1953) on samples hydrolyzed for 4 h at 1 loo in 6 M HCl, and dried in vucuo. For the differential analyses of D-ghcosamine and D-galacto-' samine, the vacuum-dried hydrolyzates were dissolved in 0.2 M citrate buffer pH 2.2. Aliquots containing between 0.1 to 0.5 pmol of hexosamine were placed on a 0.9 x 20 cm column of Beckman 50A resin, maintained at 55" and eluted with a 0.38M citrate buffer at pH5.2& on the Beckman Amino Acid Analyzer, using a flow rate of 60 ml/h (Downs & Pigman, 1976). Total sugar analysis was performed by the phenol-sulfuric acid method of Dubois et al. (1956). D-Galactose and L-fucose were quantitatively determined by the respective methods of Dische & Danilchenko (1967) and Dische & Shettles (1948). Samples were also analyzed by, gas-liquid chromatography, according to Niedermeier (1971) through the cooperation of Dr. W. Niedermeier (University of Alabama Medical Center, Birmingham, AL). Sialic acid (quantitation). Total sialic acid was

determined by the resorcinol method of Sven, nerholm (1 956). Sialic acid (identification). Gly c oly1 con t en&

was assayed by the method of Klenk & Uhlen-

CHEMICAL STUDIES ON A HAMSTER SUBLINGUAL GLYCOPROTEIN

bruck (1957). Quantitative information on the Oacetyl content of the sialic acids was obtained by the alkaline hydroxylamine method of Hestrin (1949). For chromatographic identification, sialic acids were liberated from the glycoprotein by hydrolysis at a pH of 1-5,using H2 SO4 at 80" for 1 h. When cold, the hydrolyzates were applied to a column (0.5 x 6.0cm) of Dowex 1 x 8 anionexchange resin, 200-400 mesh, in the acetate form (Herp & Pigman, 1968). The column was washed with 5.Oml of water, and the sialic acids were eluted with 10ml of 2 M sodium acetate buffer, pH4.6. The eluant was desalted by passage through a column (0.5 x 5.0cm) of Dowex 5 0 x 8 (200-400 mesh, H* form) and lyophilized. Chromatography of the sialic acids was done o n Whatman paper No. 1 and on thin-layer cellulose plates ("Q-2", Quantum Industries, Fairfield, NJ), pretreated with the appropriate developing solvent to improve resolution. Aqueous samples containing between 50-1 00 c(g of sialic acid were developed with the following solvents: 1-butanol: 1-propanol:O.l M HCI (1:2:1, by vol.; or 1-butanb1:acetic acid: H 2 0 (4:1:5, by vol.). The sialic acids were detected with either the Ehrlich or the resorcinol spray reagents, according to Svennerholm & Svennerholm (1 958). Alkaline treatment. This is a means of investi-

two pipets, 2 m l of N a B a (0.66M in 0.1 M NaOH) and 2 ml of PdC12 (0.016 M in 0.8 M HCI) were then added simultaneously, under constant stirring. The contents of each tube were hydrolyzed by the addition of 6.1 ml of concentrated HCI for 2 2 h at 110", dried in a rotary flash evaporator, dissolved in an appropriate amount of sodium citrate buffer (0.2 M, pH2.2), filtered and analyzed for amino acid composition. Ultracentrifugal analysis. Sedimentation velocity was carried out on 5 m g of dried glycoprotein dissolved in l ml of aqueous l M NaCl or 6 M urea, at 52,640 r.p.m. The molecular weight was determined by high-speed sedimentation equilibrium at a speed of 4609 for 48 h at 20°, using a 1 mg/ml sample dissolved in 1 M NaCl. The analyses were run in the Beckman Model E analytical ultracentrifuge, using a double-sector 12 mm center-piece.

RESULTS AND DISCUSSION

The sublingual gland of the hamsters (6 to 7 months old) used in this work had a wet weight, per pair, of 53mg 9. The sialic acid content was 33.5mg 4.3/g wet tissue. This represents a sialic acid content of about 100 c(mol/g wet tissue, and is over three times the value found for the rat sublingual gland (Moschera & Pigman, 1975). By comparison, the submaxillary glands of hamsters used in this work had a wet weight of 313mg +- 38 per pair and a sialic acid content of 9.65mg +1 9 / g wet tissue. Thus, of the two major submandibular glands, the sublingual gland contributes, on a mg/g basis, a considerably larger proportion to the secretory sialoglycoproteins. This observation is similar to those reported for the rat, in which the principal gland for the secretion of sialoglycoproteins is the sublingual gland (Moschera & Pigman, 1975).

*

*

gating the nature of the linkage between the karbohydrate side chains and the core-protein. The alkaline &elimination and reduction reactions were performed under the conditions which were found to be optimal for the release of the carbohydrate moiety from the protein core of other submandibular glycoproteins (Downs er al., 1973). Samples of dry, lyophilized glycoprotein (5 mg) were dissolved in 1 ml of aqueous 0.1 M NaOH containing 0.3 M N a B h and incubated at 4S0 in culture tubes, sealed with a Teflonlined screw cap for 5 and 1 0 h , respectively. After the times indicated, a magnetic stirring Isolation. The purification of the secretory bar and a drop of l a c t a n o l was added to each glycoprotein is based on selective solubilization tube and cooled to room temperature. With in 50% aqueous CaC12 and its precipitations by vigorous stirring, 1 ml of 0.4M HCI was added ethanol (-70%, v/v), at pH 5.6, using KH2P04. to each tube, immediately followed by 0.1 ml Substitution of other acidic compounds to of an aqueous 0.08M WC12 solution. Using lower the pH to 5.6 gave unsatisfactory results.

23 1

F. DOWNS and A. H E W TABLE 1 Chemical composition of hamster sublingual gly coprotein

2.

I.

Protein Serine and threonine Sialic acid

8-

I

6.

N-Acet ylhexosaminea

0 0

Galactose Fucose

I

0

4-

g/lOOg dry wt.

mo1/100mol amino acids

33.3 39.0 39.2 16.2 5.3 1.6

(100.0) 36.4 39.0 27.3 9.0 3.0

'N-Ace tylgalac tosamine TABLE 2 Amino acid composition of hamster sublingual gly coprotein

2-

r 2 in cm

FIGURE 1 Semi-logarithmic plot of sedimentation equilibrium run. Purified hamster sublingual glycoprotein (1 mg/ ml) was dissolved in 1 M NaCl. The experiment was performed at 4908 r.p.m., 20" for 24 h in a doubleseqor 12-mm interference center-piece; the Rayleigh interference optical system was used to record positions of fringes, which were measured by means of a Nikon microcomparator. Centrifugation, at this step, should be performed at the lowest possible speed in which case the glycoprotein coats the walls of glass centrifuge tubes as a transparent gel. The extraneous proteins remain in solution which has a turbid appearance. The procedure allows for the recovery in a high yield of the purified product. Based on sialic acid values, the purified mucus glycoprotein contained close to 40% of the sialic acid present in the intact gland. The purified material was monodisperse in the ultracentrifuge, showing a single symmetrical, sharp schlieren peak throughout the entire run. In the sedimentation equilibrium studies, plots of log c vs r2 gave a straight line (Fig. 1). In both 1 M NaCl and 6 M urea, the apparent molecular weight of the purified glycoprotein was calculated to be 937,000, based on a

23 2

Amino acid

mmo1/100 g

Mo1/100 mol amino acid

LYS His Arg ASP Thr Ser

14.1 3.3 8 .O 16.8 68.8 53.3 32.3

4.2 1.o

Glu Pro

G~Y Ala Val Ile Leu TYI Phe CYS

30.1

35.0 28.5 15.4 6.9 12.5 4.7 4.1 5.7

2.4

4.4 205 15.9 9.6 8.9 10.4 8.5 4.6 2.1 3.7 1.o 1-0 1.7

A correction factor of 1.10 for serine and 1.027 for threonine has been used for computation t o account for destruction of these two amino acids during acid hydrolysis (Downs & Pigman, 1969).

partial specific volume of 0.633 g/ml, according to Creeth & Knight (1967). Other methods for verifying homogeneity of the product, such as polyacrylamide gel electrophoresis are not applicable to these mucus glycoproteins, because of their viscous nature' and high molecular weight. However, on cellulose acetate electrophoresis only one line was obtained, when stained for protein andcarbohydrate.

CHEMICAL STUDIES ON A HAMSTER SUBLINGUAL GLYCOPROTEIN

Chemical composition

The gross composition of the sublingual glycoprotein is shown in Table l . Based on a dry weight, the protein represents 33% of the material, as determined by both the method of Lowry et al. (1951), and by computation of the sum of the anhydrous amino acids. The remainder of the material is accounted for by carbohydrates, sialic acid being the predominant sugar (40%). Amino acids. The amino acid composition of the hamster sublingual glycoprotein is given in Table 2. Serine and threonine, which together account for 36% of the protein core of the glycoprotein, are the two most abundant amino acids. This is similar to the data found for bovine, ovine and porcine submaxillary and for the rat sublingual glycoproteins (Pigman et al., 1973; Moschera & Pigman, 1975). However, it differs substantially from the amino acid composition of the hamster submaxillary glycoprotein, which contains almost twice as much threonine (50% of the protein core) but only half as much serine. In addition, on a mo1/100mol basis, the glycine content of the submaxillary glycoprotein is only 1/10 of that found in the sublingual material (Downs et ul., 1976).

Carbohydrutes. Sialic acid accounts for over

39% of the weight of the hamster sublingual glycoprotein. The nature of the sialic acid component was determined by glycolyl analysis, 0-acetyl content, and chromatographic mobility. Glycolyl analysis was negative, eliminating the possibility of N-glycolylneuraminic acid. On direct analysis of 0-acetyl groups, the glycoprotein contained 0.25 mol of 0-acetyl groups/ mol sialic acid. After hydrolysis of the glycoprotein at 80" and pH 1.5 for 1 h, abut 95% of the sialic acid was liberated. Both paper and thin4ayer chromatographies revealed N-acetylneuraminic acid as the principal sialic acid and a fainter spot having the mobility of N-acetyl-0-acetylneuraminic acid. Therefore, the sialic acids of the hamster sublingual gland are N-acetylneuraminic acid with about 25% of the molecules being 0-acetylated. By comparison, the hamster secretory submaxillary glyCOprOtein

contains N-acetylneuraminic acid as the sole sialic acid. Resolution by the amino acid analyzer showed that the sublingual glycoprotein contains N-acetylgalactosamine and less than 5% of N-acetylglucosamine. N-Acetylgalactosamine (Table 1) accounted for 15% of the dry weight of the glycoprotein, as opposed to 28% in that of the submaxillary gland of the hamster (Downs et ul., 1976). Other carbohydrates are galactose and fucose, 5.3% and 1.6%, respectively. No other neutral sugars were detectable in the purified sublingual glycoprotein. The alkaline catalyzed &elimination reaction showed that the linkage of the carbohydrates to the protein core in the hamster sublingual glycoprotein is an 0-glycosyl one. Samples of glycoprotein, which were treated with alkaline sodium borohydride for periods of 5 and IOh, were then reduced in the presence of colloidal palladium, followed by acid hydrolysis. This treatment resulted in a substantial loss of the hydroxyamino acids serine and threonine and in a corresponding increase in alanine and aProtein-carbohydrufe linkage.

TABLE 3 Alkaline @-eliminationand reduction reactions of the hamster sublingual glycoprotein.a Data as molJ100 mol Amino acid

Time

0

5h

10h

Threonine Loss of threonine Recovered as ABAb Recovery %

20.5

Serine Loss of serine Recovered as alanine Recovery %

15.9

11.2 9.3 9.3 100% 6.9 9.o 9.o 100%

7.5 13.0 12.8 99% 6.O 9.9 9.6 97%

5 mg/ml solutions of hamster sublingual glycoprotein in aqueous 0.1 M NaOH and 0.3 M NaBH, were incubated at 45" for the times indicated. This was followed by the simultaneous additions of 0.66 M NaBH, in 0.1 M NaOH and 0.016M PdCl, in

0.8 M HCl. ABA, aaminobutyric acid.

233

F. DOWNS and A. HERP

aminobutyric acid, respectively, as shown in Table 3. After 1 0 h , 63% of threonine and serine had been destroyed. This represents a combined loss of 22.9 mol hydroxyamino acids/lOOmol. From these results, it is concluded that the principal protein to carbohydrate linkage is via an 0-glycosyl link to seryl and threonyl residues. These data would assume that some of the oligosaccharide side chains contain more than one sialic acid residue, as the molar proportions of sialic acid: Nacetylhexosamine:galactose:fucose are 1.4 :1 .O: 0.3 :0.1, respectively (Table 1). In comparing the chemical composition of the secretory glycoproteins derived from the sublingual and submaxillary glands of the hamster, it is surprising to find such significant differences in both amino acid and carbohydrate constituents. Freezing p o i n t depression e f f e c t . It was observed

that a 1 mg/ml solution of the purified glycoprotein in l M Nacl could be kept in the freezer at -20" for several months without freezing. Due to the small amounts of materials, it was not possible to investigate this phenomenon thoroughly; however, in keeping with the hibernating habits of this species, it may be a biological requirement for the secretory glycoproteins of the oral cavity to act as antifreeze. No such effect was exhibited by the hamster submaxillary glycoprotein. ACKNOWLEDGMENT This work was supported by NIH grants Nos. 1-R 26 CA 1 7 1 6 8 4 2 and 5-ROIDE4408442. REFERENCES Boas, N.P. (1953) J. Biol. Chem. 204,553-563 Creeth, J.M. & Knight, C.C. (1967) Biochem. J. 105,1135-1145

234

Dische, Z. & Shettles, L.B. (1948) J. Biol. Chem. 195,595-603 Dische, Z. & Danilchenko, A. (1967) Anal. Biochem. 21,119-124 Downs, F. & Pigman, W. (1969) Znt. J. Protein Res. 1, 181-184 Downs, F., Herp, A., Moschera, J. & Pigman, W. (1973) Biochim. Biophys. Acta 328,182-192 Downs, F. & Pigman, W. (1976) Methods Carbohyd. Chem. 7,244-248 Downs, F., Harris, R. & Herp, A. (1976) Arch. Oral Biol. 21,307-311 Dubois, M., Gillis, K.A., Hamilton, J.K., Rebers, P.A. & Smith, F. (1956) Anal. Chem. 28,350-356 Herp, A. & Pigman, W. (1968) Biochim. Biophys. Acta 165,76-83 Hestrin, S . (1949)J. Biol. Chem. 180,249-261 Klenk, E. & Uhlenbruck, G. (1957) Hoppe-Seylerk Z . Physiol. Chem. 307,266-271 Lowry, O.H., Rosebrough, N.J., Farr, A. & Randall, R.J. (1951) J. Biol. Chem. 193,265-275 Moschera, J. & Pigman, W. (1975) Carbohyd. Res. 40,53-67 Niedermeier, W. (1971) Anal. Biochem. 40, 465475 Pigman, W., Downs, F., Herp, A., Moschera, J . & Wu, M.T. (1973) in Intern. C.N.R.S. (Paris), No. 221, VOI. 7,371-380 Roukema, P.A., Oderkerk, C.H. & Salkinoja-Salonen (1976) Biochim. Biophys. Aria 428,432-440 Spackman, D.H., Stein, W.H. & Moore, S. (1958) Anal. Chem. 30,1190-1206 Svennerholm, L. (1956) Acta Soc. Med. Upsalien. 61.75-85 Svennerholm, E. & Svennerholm, L. (1958) Nature (Lond.) 181,1154 Tanaka, K. & Pigman, W. (1965) J. Biol. Chem. 240, PC 1487 Address: Dr Anthony Herp New York Medical College Basic Science Building Department of Biochemistry Valhalla, New York 10595 USA.

Chemical studies on a hamster sublingual glycoprotein.

Int. J. Peptide Protein Res. 10, 1917, 229-234 Published by Munksgaard, Copenhagen, Denmark *Nopart may be reproduced by any process without written...
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