Purification and partial characterization of rat submaxillary mucin

Comp. Biochera. Physiol. Vol. 6915, pp. 605 to 609, 1981

0305-(M91/81/070605-05502.00/0 Copyright © 1981 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

PURIFICATION AND PARTIAL CHARACTERIZATION OF RAT SUBMAXILLARY MUCIN CAROL E. MALINOWSKIand ANTHONY HERP Department of Biochemistry, New York Medical College, Valhalla, NY 10595, U.S.A. (Received 30 September 1980)

Abstract--1. A simple method for the isolation and purification of rat submaxillary mucus glycoprotein is described; it involves water extraction of the minced glands, followed by recycling gel filtration chromatography on Sephacryl S-200. 2. Analysis of gross chemical composition indicates that threonine and serine account for 23 mol % of the total amino acids, while the basic amino acids lysine and histidine account for 33 mol %. Molar sugar ratios by gas-liquid chromatography were sialic acid:fucose: N-acetylgalactosamine:galactose:Nacetylglucosamine:glucose:mannose 1.0:1.0:2.7:2.7:1.0:0.67:0.5. 3. A comparison is made between this mucus glycoprotein and other purified mammalian mucins. This mucin shows striking similarities with human salivary mucus glycoproteins, and dissimilarities when compared to the rat sublingual glycoprotein.

INTRODUCTION

The molecules mainly responsible for the viscous nature of salivary secretions are negatively charged glycoproteins. Among the major salivary glands, the parotid contributes least, and the sublingual most to the viscosity. The submaxillary gland, composed of seromucous acini (Shackleford, 1968), is intermediate in this respect. The sublingual gland is rich in mucin which is easy to purify, and is obtained in high yield (Moschera & Pigman, 1975). In contrast, the mucus glycoprotein from rat submaxillary gland has been found to be extremely difficult to isolate, due to the small amount of sialic acid that it contains, and the abundance of contaminating proteins in the gland (Moschera & Pigman, 1975). For this reason, the physical and chemical properties of the rat sublingual mucin have been studied (Moschera & Pigman, 1975), but no such data are available concerning the rat submaxillary gland (RSM) of this species. It has been found that the usual methods of mucin preparation proved to be virtually ineffective for the purification of this mucin. These methods include those of Tettamanti & Pigman (1968) utilizing cetylpyridinium chloride, ethanol precipitation (Downs et al., 1976), and phenol extraction (Howe et al., 1972). The present paper describes a method of isolation and purification of the RSM mucus glycoprotein. The chemical composition and molecular weight of the purified mucin is reported, as well as a description of the purification procedure. A preliminary report has appeared concerning this material in abstract form (Malinowski & Herp, 1980). MATERIALS AND METHODS

All chemicals used were of commercial origin, and the highest quality obtainable. Sephacryl S-200 was chased from Pharmacia Fine Chemicals, Piscataway, Jersey. Rat submaxillary glands were obtained 6-month-old male Lewis rats (Rattus norvefficus).

were purNew from 605

Purification of rat submaxillary mucin The animals were killed by chloroform anaesthesia, the thoracic region opened and the submaxillary gland separated from the sublingual gland. The submaxillary gland was freed from connective tissue and fat and stored frozen at -20°C. The pooled glands from 80 rats were thawed, blotted dry, suspended in 3 vols of distilled water, and shaken for two days at 4°C. The extract was centrifuged to clarity, and the volume reduced with an Amicon microconcentrator to a volume of 20 ml. This crude gland preparation was brought to 100°C for 2-3 rain, and recentrifuged. This step removed much of the contamination by serum proteins, without any loss of sialic acid. Four-ml aliquots of the concentrated sample were applied to a Sephacryl S-200 column (2.6 x 100 cm), equilibrated and eluted with 0.005 M phosphate buffer containing 0.1 M NaCI, pH 7.0. Two-ml fractions were collected and the elution monitored by readings at 280 nm for protein and for sialic acid. The front half of the void volume (Vo) was collected (Fig. 1A) and rechromatographed on the same Sephacryl S-200 column, using the same buffer (Fig. 1B). The front of the void volume was again pooled, combined with the other elution Vo's, dialyzed, and lyophilized. The lyophilized material was dissolved in 1 ml of distilled water, and allowed to stand overnight in the cold. The small amount of insoluble material was removed by centrifugation, and the clear supernatant contained the purified mucin. The mucin preparation could be stored in the wet form at 4°C for 2-3 months without degradation or precipitation. Analytical procedures Protein was determined by the method of Lowry et al. (1951) with crystalline bovine serum albumin as standard, and also by calculation from summation of the amino acid composition of the hydrolysates. Amino acid analyses were performed on a Beckman Model 120C Amino Acid Analyzer after hydrolysis for 22 hr at 110°C in 6 N HCI in vacuo. Total neutral sugars were determined by the phenol-:sulfuric acid method (Dubois et al., 1956), sialic acid by the resorcinol method (Svennerholm, 1956). For the analysis of sialic acid in the crude extracts, samples were brought to pH 1.5 with H2SO4, and kept at 80°C for 1 hr. The hydrolyzate was passed through a column (0.5 x 6 era) of Dowex 1 x 8 anion exchange resin (acetate form). The column was washed with 4 ml of water, and the sialic acid

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Fig. 2. Sedimentation velocity pattern of the purified RSM glycoprotein. A sample of glycoprotein (5 mg/ml) in 0.5 M NaC1 was centrifuged at a speed of 47,060 rpm and a phase-plate angle of 60°. The photograph was taken after 32 min. eluted with 4 ml of 2 M sodium acetate buffer, pH 4.6. Sialic acid was then assayed as above. Polyacrylamide gel electrophoresis was performed on 5% acrylamide gels in the presence and absence of 0.1% sodium dodecyl sulfate (Weber & Osborn, 1969). Protein bands were stained with Coomassie Blue, and carbohydrates by the periodic acidSchiff fuchsin stain (Holden et al., 1971). Cellulose acetate electrophoresis was performed using the Beckman Microzone System, proteins stained with Ponceau S. Ultracentrifugai analysis was carried out in the Beckman Spinco Model C Analytical Ultracentrifuge, usin~ a double sector 12 mm center piece. Sedimentation velocity was carried out on a 5 mg/ml sample in 0.5 M NaCI at 47,060rpm using Schlieren optics. Molecular weight was determined by sedimentation equilibrium at a speed of 29,500rpm for 24 hr using a 1 mg/ml sample in 0.5 M NaCI with the Rayleigh interference optical system. A value of 0.633 (Creeth & Knight, 1967) for the partial specific volume was assumed to calculate the molecular weight. Gas-liquid chromatography (Perkin-Elmer Model 3920B, equipped with flame ionization detector) was performed according to Niedermeier (1971). RESULTS The water extract of the submaxillary glands of rats is not excessively viscous when compared to crude salivary gland extracts of other animals such as bovine, ovine and porcine, and considerably less viscous than the water soluble extract obtained from rat sublingual glands. However, the initial ratio of contaminating protein to actual mucus glycoprotein is far greater in the preparation from this gland (taken as the ratio of total protein to sialic acid content). For this reason, classical isolation procedures are ineffectual in purifying this giycoprotein. The large amount of extraneous proteins interferes with precipitation by quarternary ammonium salts, and tend to co-precipi-

tare with the mucin during alcohol precipitation. Since the extract was not prohibitively viscous, column treatment was chosen as the first purification step. Sephacryl S-200 was utilized, having a molecular weight exclusion limit of 250,000 for globular proteins and 80,000 for polysaccharides, with excellent nonadsorptive qualities. However, due to the large amount of contaminating proteins in the original extract, the sialic acid containing mucin peak was not well resolved from a nearby peak of slightly lower molecular weight impurities so that the front half of the void volume only was collected. After rechromatographing on the same gel with the same buffer system, the protein/sialic acid ratio was further improved (Table 1). The fractions that contained sialic acid and neutral sugars after the second chromatography were combined, dialyzed, and lyophilized. The dry residue was resuspended in a small volume of distilled water, and after centrifugation to remove insoluble material, the pure mucin remained in the supernatant. The purification scheme is shown in Table 1. An obvious advantage of this method of purification is that the Table 1. Purification of rat submaxillary mucin Treatment

Pr/SA*

Crude gland extract Extract after boiling After first chromatography After second chromatography After lyophilization, centrifugation

100 90 10 5

* On a weight basis.

1.5

Fold, purification -1.1 10 20 66.6

608

CAROLE. MALINOWSKIand ANTHONYHERP Table 2. Amino acid composition of RSM Amino acid

Mols/100 mols

Lys His Arg Asp Thr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe

24.87 8.34 1.59 7.27 9.84 13.28 6.60 8.64 5.30 5.90

mouse, and ovine mucus glycoproteins. However, the basic amino acids lysine and histidine account for an unusually high 33 mol ~ of the total amino acids, thus differentiating this glycoprotein from other mammalian preparations. In this respect, the rat submaxillary glycoprotein most closely resembles purified glycoproteins isolated from human saliva and sputum. Analysis of sugars was carried out by gas-liquid chromatography of the alditol acetate derivatives; sialic acid was determined colorimetrically. The molar ratios are shown in Table 3. The small amounts of glucose and mannose detected are not thought to be due to impurities, since they remained after extensive dialysis, and analytical ultracentrifugation did not reveal multiple peaks.

2.19 0.33 1.97 2.24 0.82 0.82

DISCUSSION

mucin is not subjected to harsh chemical conditions that may degrade or deform the glycoprotein. Purity of the mucin preparation was determined by three methods. Polyacrylamide gel electrophoresis showed only one protein band, just barely penetrating the top of the gel, with a corresponding carbohydrate staining band, both in the presence and absence of sodium dodecyl sulfate. Cellulose acetate electrophoresis also exhibited one protein band. Ultracentrifugal analysis was performed as described, and one hyperfine symmetrical peak appeared (Fig 2). This peak grew slightly broader after two hours, but remained highly symmetrical. The diffusion seen in the peak of the RSM mucin was considerably less pronounced than is seen with most mucins with a high negative charge density, such as the rat sublingual mucin. The S value calculated for this preparation was 4.05, with a molecular weight of 85,000. In addition, a linear Yphantis plot for In .c vs x 2 was obtained. This result indicates homogeneity with respect to molecular weight. The amino acid composition of the purified glycoprotein is given in Table 2. Serine and threonine account for 23 mol ~o of the total amino acids, with proline comprising another 8.6~. These values are quite typical for mucin preparations, similiar amounts of these amino acids being found in armadillo, canine, Table 3. Comparison of molar sugar composition of RSM, RSL*, and HSMt Sugar

RSM

Molar ratios RSL*

HSMt

Sialic acid Fucose N-acetylgal N-acetylglc Galactose Glucose Mannose

1.00 1.00 1.00 2.70 2.70 0.67 0.50

1.00 0.10 0.70 0.96 1.00 -0.20

1.00 1.00 1.00 2.00 2.40 0.50 0.35

* Rat sublingual glycoprotein (Mosehera & Pigman, 1975). t Human submandibular glycoprotein (Oemrawsingh, 1972).

The present paper describes a method of purification of the mucin of rat submaxillary glands which preserves the glycoproteins in a comparatively intact state due to the mild conditions employed. The submaxillary gland of the rat used in this study had a wet weight per pair of 450 + 20 mg. The sialic acid content was 0.6 mg/g wet tissue. This compares with a sialic acid content of rat sublingual gland of 7.4 mg/g wet tissue (Moschera & Pigman, 1975) and to hamster sublingual gland sialic acid content of 33.5 mg/g wet tissue (Downs & Herp, 1977). This indicates the difficulties encountered in attempting to isolate enough material for its physical and chemical characterization. Many studies that have been made concerning rat glands did not distinguish between the sublingual and submaxillary components because the small sublingual gland (approximately 20mg) is embedded within the much larger (approximataly 225 mg) submaxillary gland (Keryer et al., 1973). In this study, we carefully verified that no contamination by the sublingual gland occurred. In doubtful cases, the gland was not included for study. Since the extract of the crude gland was not excessively viscous, column treatment was chosen. After the first passage through the Sephacryl S-200, the ratio of protein to sialic acid decreases dramatically, with a slightly better ratio obtained after rechromatographing the sialic acid containing void volume. With subsequent lyophilization and centrifugation of the insoluble material, the mucin was determined to be homogenous by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and cellulose acetate electrophoresis. The purified mucin shows an unusually large amount of basic amino acids, especially lysine, when compared to mucins isolated from bovine (Tettamanti & Pigman, 1968), canine (Lombart & Winzler, 1972), murine (Roukema et al., 1976), ovine (Tettamanti & Pigman, 1968), and porcine (Herp et al., 1979) submaxillary glands. However, the values for serine, threonine and proline fall well within the normal range for mucus glycoproteins obtained from these mammals. These basic amino acids may play a physiological role in the ion-exchange properties of the mucin, or in the case of secretory glycoproteins,

Submaxillary mucin purification may assist in the buffering capacity of the secreted material. The sugar composition of this glycoprotein shows both similarities and dissimilarities when compared to various mucin preparations. The ratio of N-acetylgalactosamine to N-acetylglucosamine is greater than 2:1, while in most of the mucin preparations from mammals listed above, the ratio is similar, and for some--hamster submaxillary and armadillo submandibular (Wu & Pigman, 1977)--no N-acetylglucosamine is found at all. In contrast, a reversed hexosamine ratio is observed in human (Oemrawsingh, 1972) and monkey (Herzberg et al., 1979) saliva, and human bronchial secretions (Lamblin et al., 1979), where more N-acetylglucosamine is found than N-acetylgalactosamine. A comparison between RSM, rat sublingual and human submandibular mucins (Table 3) reveals that striking common features exist between RSM and human mucins, while the mucin from the adjoining sublingual gland is quite different in composition. This similarity with human mucin extends to the presence of greater than trace amounts of glucose and mannose (Oemrawsingh, 1972). The large amount of fucose found in the RSM mucin is also characteristic for the mucus glycoproteins from monkeys and humans. The presence of fucose, in substantial amounts, a major blood-group determinant, probably confers considerable serological activity on this glycoprotein. In conclusion, the RSM mucin, in its chemical composition, resembles more than any other hitherto studied salivary gland mucin that of its human counterpart. We therefore surmise that the rat is a particularly suitable animal model to study changes in mucin composition produced by stimuli such as drug and hormone action for extrapolation to humans. REFERENCES

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