THE JOURNAL OP Bm.oc~crr. CHEMISTRY Vol. 252, No. 11, Issue of June 10, pp. 3791-3798, 1977

Printed LR II.S.A

Purification, Composition, Molecular Weight, and Subunit Structure of Ovine Submaxillary Mucin*

(Received for publication, December 13, 1976)

HOYLE D. HILL, JR.,+ JACQUELINE A. REYNOLDS, AND ROBERT L. HILL

From The Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Ovine submaxillary mucin was isolated by modifications of published methods to give preparations with properties similar to those reported earlier. The last step in purifica- tion is the removal of small amounts of contaminating pro- tein by gel filtration of mucin previously reacted with dansyl (5-dimethylaminonaphthalene-1-sulfonyl) chloride. The contaminating protein contained all of the protein-bound fluorescent dansyl groups. Asialomucin and apomucin were prepared from mucin by similar means after treatment with protease-free glycosidases. Each form of mucin contained a blocked NH,-terminal residue and was devoid of amino acids reactive with dansyl chloride. Threonine, serine, proline, glycine, and alanine accounted for about 75% of the amino acid content of each type of mucin. The composition of mucin suggests that the hydroxyl group of each threonine and serine residue in the molecule is in 0-glycosidic linkage with N-acetylgalactosamine and that about 86% of the N- acetylgalactosaminyl groups are in glycosidic linkage with sialic acid.

The molecular weights of mucin, asialomucin, and apo- mucin were examined by sedimentation-equilibrium in the ultracentrifuge. Apomucin in 0.5 M sodium chloride behaved as a pure protein with a molecular weight of 58,300. Mucin and asialomucin appeared to aggregate in the same solvent, suggesting that aggregation is dependent upon the carbohy- drate content of mucin. A more detailed ultracentrifugal analysis of mucin in 2 M sodium chloride indicates that mucin forms a self-associating system in which the extent of association is a function of ionic strength and protein con- centration. It is concluded that association occurs among molecules of M,. = 154,000, which is close to the expected size of apomucin containing sialyl-cu2 + &N-acetylpalactosa- mine in 0-glycosidic linkage at each of the threonyl and seryl residues in the molecule.

Mucin is the predominant sialic acid-containing glycopro- t&n in the mucous secretions of submaxillary glands. Its function, along with similar mucoproteins found in the respi-

* These studies were supported by grants from the National Heart and Lung Institute, National Institutes of Health (HL-06400), and from the National Science Foundation (GB-29334).

$ Predoctoral Trainee, Biochemistry Training Grant (GM-002331 from the National Institutes of General Medical Sciences, National Institutes of Health. Present address, Abbott Laboratory, Diagnostic Division, North Chicago, Ill.

ratory (l), gastrointestinal (Z), and reproductive tracts (3), is to lubricate epithelial cells and protect them from intimate contact with the external environment. Unlike serum glyco- proteins, the physical properties and biological functions of mucins are dominated by their high carbohydrate content (60 to 70% by weight). Submaxillary mucins are highly asymmet- ric, rod-like molecules with a large sphere of hydration, which endows their solutions with a high viscosity (2, 4).

The carbohydrate prosthetic groups of bovine, ovine, and porcine submaxillary mucins have been studied in great detail (2). Ovine submaxillary mucin contains a disaccharide pros-

thetic group, N-acetylneuraminosyl cu2 --$ 6-N-acetylgalacto- samine, which is in rua-glycosidic linkage with serine or threonine in the polypeptide chain. Bovine mucin (2) contains a similar disaccharide prosthetic group differing in its sialic acid, whereas porcine mucin has a family of oligosaccharides related to a pentasaccharide containing N-acetylgalactosa- mine, galactose, fucoue, and sialic acid (5).

Although ovine submaxillary mucin has been studied in detail, considerable variation has been noted in some of its physicochemical properties. For example, molecular weights from 394,000 (6) to more than 1 x 10” have been reported (2), indicating considerable polydispersity. Gottschalk et al. (7) have used specific glycosidases for the removal of the carbohy- drate prosthetic groups, but the molecular weight of the fully deglycosylated apomucin has not been reported.

We wish to report here a method for purification of ovine submaxillary mucin that removes small amounts of non-mu- tin protein contaminants. The composition and molecular weight of the purified mucin as well as asialomucin and apo- mucin have been determined. Evidence is presented that mu- tin forms a self-associating system in aqueous solution result- ing from aggregation of a molecule with a molecular weight of about 154,000, which is formed by a polypeptide containing about 650 residues with disaccharide prosthetic groups on each threonine and serine residue.

EXPERIMENTAL PROCEDURES

Materials

Unless otherwise indicated, all chemicals were reagent grade, or the highest aualitv provided bv the supplier, and were used without further puriication: The follo&g materials were obtained commer- cially. Stock culture of Clostridium perfringens (ATCC 3626, Ameri- can “Type Culture Collection); ne&amin?dase (Worthington Bio- chemical Corp.); p-nitrophenyl-P-o-galactopyranoside, p-nitro- phenyl-2-acetamido-2-deoxy-/3-D-glucopyranoside, cetyltrimethyl-

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3792 Characterization of Ovine Submaxillary Mucin

ammonium bromide and phenylmethyl sulfonyl fluoride (Sigma Chemical Co.); 5-dimethylaminonaphthalene-1-sulfonyl chloride (Pierce Chemical Co.); n-glucose, o-galactose, L-fucose, 2-acetamido- 2-deoxy-n-glucose, 2-acetamido-2-deoxy-n-galactose, and N-acetyl- neuraminic acid (Pfanstiehl Laboratories, Inc.); bovine serum albu- min (Calbiochem, Inc.); bovine hemoglobin (Pentex); p-nitrophenyl- 2-acetamido-2-deoxy-a-D-galactopyranoside (Koch-Light Laborato- ries); hydroxylapatite HTP (Bio-Rad Laboratories); DEAE-cellulose (DE521 (Whatman Biochemicals, Ltd.); Sephadex, Sepharose, SP- Sephadex, CM-Sephadex, and Blue Dextran 2000 (Pharmacia Fine Chemicals); ovine submaxillary glands (St. Louis Serum Co.); 11,4- ‘VJsuccinic anhydride, and ~Zyczne-l-‘4C]glycine ethyl ester hydro- chloride (New England Nuclear); Instagel (Packard Instruments, Inc.).

Before use, the following were recrystallized: anthrone from ben- zene; p-dimethylaminobenzaldehyde, from 50% ethanol; sodium do- decyl sulfate and succinic anhydride from absolute ethanol; resorci- no1 from chloroform:methanol (2:1, v/v). Each recrystallized reagent was stored, protected from light, in tightly sealed containers.

Analytical Procedures

Total neutral sugars were estimated by the anthrone procedure (8) with gala&se as standard. Fucose was measured by the method of Dische (9). Sialic acid was measured by the resorcinol method of Svennerholm (10) or, after enzymatic release, by the method of Warren (ll), with N-acetylneuraminic acid as standard for both procedures. N-Acetylhexosamines, after enzymatic release, were es- timated by the method of Morgan and Elson (12) as modified by Ressig et al. (13) withN-acetylgalactosamine as standard. After acid hydrolysis (4 N HCl, loo”, 4 h), hexosamines were determined on the amino acid analyzer.

Mucin concentrations and those of other proteins were estimated qualitatively by the method of Lowry et al. (14) or by absorbance at 220 or 280 nm. Quantitative estimation of mucin concentrations was made using the extinction coefficient, Eq$, ’ m = 10.25.

Amino acid compositions were determined as described by Evans et al. (15) except that amino acid concentrations were determined by automatic integration with a Beckman System AA Computing Inte- grator (Beckman Instruments, Inc.). Proteins with a high carbohy- drate content were hydrolyzed under conditions (<lo0 +g of glyco- protein/ml of 6 N HCl) that minimize destruction of amino acids by sugar degradation products (16).

Glycosidases were assayed by adding enzyme to 50 ~1 of the appropriate substrate (3.5 rnM p-nitrophenylglycosides of /3-o-galac- tose, n-N-acetyl-n-glucosamine, or a-N-acetyl-n-galactosamine in 0.01 M sodium cacodylate, pH 6.0, containing 3 mivr CaClp), incubat- ing for 10 min at 37”, terminating the reaction with 1.0 ml of 0.5 M sodium carbonate, and measuring the adsorbance at 490 nm. Under these conditions p-nitrophenol had a molar extinction coefficient of 18,000 M-I cm-‘. One unit of activity is defined as the release of 1 pmol of p-nitrophenolimin. Neuraminidase and a-A-acetylgalac- tosaminidase were assayed with native or desialized porcine sub- maxillary mucin, prepared as described earlier (171, at a concentra- tion of 10 mg of muciniml of 0.01 M sodium cacodylate, pH 6.0, containing 3 rnM CaCl, and the formation of N-acetylneuraminic acid or N-acetylgalactosamine was measured with time by the spe- cific procedures for each monosaccharide. One unit of activity was defined as the formation of 1 pmol of monosaccharideimin under these assay conditions.

Proteolytic activity in glycosidase and mucin preparations was measured by the radioisotopic assay of Williams and Lin (181, with methyl[‘Y!]glycinyl or [14C]succinyl bovine hemoglobin as sub- strates.

Sedimentation equilibrium analyses were carried out in a Spinco model E ultracentrifuge equipped with a photoelectric scanner. Ab- sorbance was monitored at 232 nm as a function of the radial distance and data from at least three scans were used to minimize random errors. Molecular weight heterogeneity was analyzed as described previously (19). Partial specific volumes were calculated from the amino acid composition (20) and the carbohydrate content. The partial specific volumes of N-acetylneuraminic acid and N-acetylga- lactosamine used in these calculations were 0.606 and 0.666, respec- tively. Solvent densities were measured in an Anton Paar Densime- ter at 25 i 0.005”.

Preparation of ru-N-Acetyl-o-galactosaminidase

This enzyme was prepared by modifying the procedure of McGuire et al. (17). Culture filtrates of C. perfringens were collected, concen-

trated with ammonium sulfate, and then chromatographed succes- sively on Bio-Gel P-150 and on DEAE-cellulose as described by these workers. Fractions from the DEAE-column containing u-l\r-acetyl-n- galactosaminidase were pooled, dialyzed against 0.01 M sodium caco- dylate, pH 6, containing 3 rniw CaCl,,.and then chromatographed on a column (2.5 x 68 cm) of Whatman DE52 equilibrated with the same buffer. The column was developed at a flow rate of 1.2 mlimin with a linear gradient formed with 2 liters of the equilibration buffer and 2 liters of the same buffer containing 0.5 M NaCl. Fractions containing the enzyme were pooled, diluted with an equal volume of water, applied to a column (1 x 10 cm) of DE52 in equilibration buffer, and then eluted with the same buffer containing 1 M NaCl in a total volume of 2 to 3 ml. The concentrated enzyme was dialyzed against 0.01 M cacodylate buffer, pH 6, containing 3 rnM CaCl,, and stored at -20”. Some preparations contained proteolytic enzymes as judged by assay with radioactive hemoglobin substrates, but after treatment with phenylmethylsulfonyl fluoride (0.1 rnM final concentration) at 37” for 2 h and dialysis against the cacodylate/CaCl,, pH 6.0, buffer, no proteolytic activity was detected over 72 h under conditions where as little as 0.01 pg of trypsiniml were detected in 2 h in the hemoglo- bin assay.

Purification of Ovine Submaxillary Mucin

Ovine submaxillary mucin was isolated by the following proce- dure, patterned after that of Tettamanti and Pigman (6). All steps, unless otherwise noted, were performed at 4”.

Step 1 -Fresh frozen ovine submaxillary glands were thawed, rinsed in 0.01 M NaCl, and dissected free from blood clots, fat, and connective tissue. The glands were blotted dry, weighed, and homog- enized for 2 min with a Waring Blendor in 0.01 M NaCl (4 ml of solution/g of tissue). The suspension was centrifuged at 7200 x fi for 20 min and the supernatant removed. The precipitate was resus- pended (3 ml of solution/g of original tissue) in 0.01 M NaCl, rehomog- enized, and centrifuged as before. Both supernatants were com- bined and the precipitates discarded.

Step 2 -The stirred supernatants were adjusted to pH 4.7 by the slow addition of 2 M acetic acid and the resulting precipitate removed by centrifugation at 7200 x g for 20 min.

Step 3 -The supernatant from Step 2 was applied to a column of sulfopropyl-Sephadex C-25 (4 ml of settled volume/g of original tis- sue) which had been equilibrated with 0.05 M sodium acetate, pH 4.7. Fractions (0.1 column volume) were collected, analyzed for protein and sialic acid, and those fractions containing sialic acid were com- bined.

Step 4 - Mucin was precipitated from solution by addition of cetyl- trimethylammonium bromide (0.1 ml of a 10% w/v solution/g of original tissue) and collected by centrifugation at 7200 x fi for 20 min. The precipitate was dissolved in 4.5 M CaCl, (2 ml of solution/g of original tissue) and absolute ethanol added to the solution to give a final concentration of 60% ethanol. After 60 min the solution was centrifuged at 27,000 x g for 30 min, the precipitate discarded, and the supernatant was brought to 75% ethanol concentration. After 60 min, the precipitate was collected by centrifugation at 27,000 x g for 30 min. The supernatant was discarded and the precipitate dissolved in 1.0 M NaCl (1 ml/g of original tissue) by dispersion with a Teflon- glass homogenizer and stirring overnight. The solution was dialyzed for 24 h against three changes of distilled water (100 ml/ml of solution), and made 0.01 M with sodium phosphate, pH 6.8, by addition of 1 M phosphate buffer.

Step 5 -The solution from the preceeding step was applied to a column of hydroxylapatite (4 ml of settled volume/g of original tissue) equilibrated in 0.01 1~ sodium phosphate, pH 6.8. The column was eluted with the same buffer and fractions collected and analyzed for protein and sialic acid. Those fractions containing the mucin were detected by determining the sialic acid content, combined, dialyzed exhaustively against water, and lyophilized. Mucin was stored at -20” in a tightly sealed bottle.

The final step in purification was obtained by reaction of the mucin with dansyl’ chloride followed by gel filtration on Sepharose 4B as described under “Results.”

Deglycosylation of Ouine Much

Asialomucin’ was prepared by treatment of native or dansylated

I The abbreviation used is: dansyl, 5-dimethylaminonaphthalene- 1-sulfonyl.

2 Asialomucin refers to mucin prepared by enzymatic removal of sialic acid by neuraminidase and apomucin to mucin fully deglyco- sylated by treatment with neuraminidase and a-N-acetylgalactosa- minidase.

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Characterization of Ovine Submaxillary Mucin 3793

mu& with protease-free neuraminidase. Neuraminidase obtained commercially was freed of proteases by affinity chromatography of the enzyme on N-(p-aminophenyl)-oxamic acid coupled through azo linkage to agarose-bound tyraminylsuccinyldiaminodipropylamine as described earlier (21). This adsorbent was kindly provided by Dr. Joel Shaper, Department of Pharmacology, Johns Hopkins School of Medicine. The protease-free neuraminidase (0.1 unit) was added to 10 mg of mucin in 1 ml of 0.05 M sodium acetate, pH 5.5, incubated at 37” for 24 h and then dialyzed exhaustively against water. This sialic acid released was found to be equal to the original sialic acid content of the mucin, and no protein-bound sialic acid was found in the asialomucin. Apomucin, devoid of all carbohydrate, was prepared by treatment of asialomucin (1 ml, 10 mg) with a-A-acetyl-n-galactosa- minidase (0.1 unit) for 24 h at 37” in 0.01 M sodium cacodylate, pH 6, containing 3 rnM CaCl,, followed by exhaustive dialysis against the same buffer. The N-acetylgalactosamine released was equal to the N-acetylgalactosamine content of native and asialomucin. Apomu- tin was also prepared directly from mucin by incubation ofmucin (10 mgiml) with neuraminidase (0.3 unit) and a-N-acetylgalactosamine (0.1 unit) at 37” for 24 h in 0.01 M sodium cacodylate, pH 6, containing 3 rnM CaCl,.

Dansylation ofMu&, Asialomucin, and Apomucin

Protein (10 mgiml) in 0.5 M sodium bicarbonate, pH 9.8, was mixed with an equal volume of dansyl chloride in acetone (5 mgiml) and incubated at 37” for 20 min. The mixture was then adjusted to pH 4 with 90% formic acid and exhaustively dialyzed against distilled water in 0.01 M sodium cacodylate, pH 6, containing 0.5 M NaCl.

Analysis of dialysates revealed no carbohydrate, indicating that under conditions of dansylation, carbohydrate prosthetic groups were not removed from mucin and asialomucin by p elimination,

RESULTS

Purification ofMucin -Table I summarizes the purification of mucin through the first five steps and lists the sialic acid and protein contents of the material obtained at each step. Figs. 1 and 2 show the purification at Steps 3 and 5, respec- tively. The mucin obtained after Step 5 contained 28.5% N- acetylgalactosamine, 34.3% sialic acid, and 37.2% protein by weight, values in good agreement with those reported earlier (6). There was considerable variation in the lysine and histi- dine contents, which were found in small amounts compared to other amino acids from preparation to preparation.

It was extremely difficult to assess the heterogeneity of the mucin isolated since it was not amenable to many of the usual methods employed to judge purity, such as electrophoresis on gels. On gel filtration mucin emerged in the void volume from columns of Sepharose 4B in sodium cacodylate buffers, pH 6, at low ionic strength (Fig. 3). However, gel filtration in the same buffer containing 0.5 M NaCl showed some retention of mucin preparations. In order to obtain more information about

TABLE I

Purification ofovine suhmaxillar,y mucin

step Volume Protein’ Sialic acid” -Sialic acid/protein Total sialic acid

pnoliml .~

ml mglml g; 9% yield

1. Gland extract’ 480 8.78 3.51 0.40 1.684; 100

2. Precipitation at pH 4.7 460 3.89 2.99 0.77 1.375; 82 3. Chromatography on SP-Sephadex 800 1.12 1.86 1.66 1.488; 88 4. Ethanol fractionation 100 3.85 9.71 2.52 0.971; 53

5. Chromatography on hydroxylapatite 148 1.63 4.57 2.80 0.676; 40

” By the method of Lowry et al. (14). b By the method of Svennerholm (10). c From 65 g of submaxillary glands.

I I I I I I I

0.7 -

3.0 0.6-

. 0

2.5 ES $g 0.5-

w 2.0 6

2 0.4 ~

Y 3 5 g 1.5 E 0.3-

z

1.0 z

O.Z-

0.5 0.1 -

200 400 600 800 1000 I200 e-4 VOLUME (ml)

FIG. 1 (left). Chromatography of the supernatant solution from ovine submaxillary gland extracts obtained from Step 2. The solu- tion (460 ml) was applied to a column (3 x 42.5 cm) of SP-Sephadex C-25 equilibrated at 4” with 0.05 M sodium acetate, pH 4.7. The column was eluted with equilibration buffer (500 ml) followed by 500 ml of 0.05 M sodium acetate, pH 4.7, containing 1.0 M NaCl. The effluent was collected in 25-ml fractions and analyzed for protein (0) and sialic acid (0). The bar shows the fractions combined for further purification.

FIG. 2 (center). Chromatography of mucin from Step 4 on hydrox-

VOLUME (ml) FRACTION NUMBER

ylapatite. A solution of mucin (100 ml, 3.85 mg of protein/ml) in 0.01 M sodium phosphate, pH 6.8, was applied to a column (3 x 34 cm) of hydroxylapatite suspended in the same buffer. The column was eluted with the same buffer, and fractions (25 ml) were collected and analyzed for protein (0) and sialic acid (0). The bar shows the fractions combined for further purification.

FIG. 3 (right). Gel filtration of mucin (1.5 mg) on columns (1.5 x 62 cm) of Sepharose 4B in 0.01 M sodium cacodylate buffer, pH 6. The column was developed at 4” and fractions (2.5 ml) were collected and monitored at 220 nm.

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3794 Characterization of Ovine Submaxillary Mucin

the behavior of mucin on gel filtration, further studies were performed, but with mucin that had been treated with dansyl chloride. Originally, the rationale for dansylating mucin was to introduce a few fluorescent groups into the molecule in order to obtain a more sensitive, spectral means of locating mucin in column elutes. Surprisingly, however, it was found that the dansyl groups were not incorporated into mucin, but into a small amount of contaminating protein. Thus, after dansylation of mucin preparations and gel filtration, a small but significant amount of contaminant was removed and addi- tional purification was obtained to give mucin devoid of the small amounts of amino acids that were found persistently in material after Step 5 (Table I).

The elution profile of a preparation of mucin on Sepharose 4B after dansylation is shown in Fig. 4. All of the fluorescence in the preparation emerged in the void volume or in the included volume. The fluorescence in the included volume was due to dansyl sulfonic acid, as judged by thin layer chromatog- raphy of these fractions. That material, retarded slightly, contained both sialic acid as well as N-acetylgalactosamine in amounts close to those found in the original preparations. These results indicated that mucin obtained after Step 5 can be further purified to remove protein(s), that after dansyla- tion, emerge in the void volume of columns of Sepharose 4B; they also suggested that mucin is devoid of amino acids that react with dansyl chloride and has a blocked NH,-terminal residue. This is in accord with the observation that chromatog- raphy of extracts of acid hydrolysates of mucin fully dansyl-

0. I t

C E

6 2.4-

5:

: 2.0-

= :

1.6-

it? 9 1.2-

2 o.a-

0.4

t 0 I(

FRACTION NUMBER

FIG. 4. Gel filtration of mucin on Sepharose 4B. After dansyla- tion, 1.0 ml of mucin (1.5 mg of protein) in 0.01 M cacodylate, pH 6.0, containing 0.5 M NaCl was applied to a column (1.5 x 62 cm) of Sepharose 4B equilibrated and &ted with the same buffer at 4”. Fractions (2.5 ml) were collected (8 ml/h), analyzed for protein (A), N-acetylgalactosamine (B), fluorescence CC), and N-acetylneura- minic acid CD), and pooled as indicated by the bar.

3 I3

rl 0.6 0.7

E - 0.6 ki

5 k? -0.5 w

2

ated by the method of Gray (22) revealed no dansyl a-amino acids. Fig. 5 shows the elution profiles on Sepharose 4B of dansylated mucin from Step 5 (Table I) after treatment with neuraminidase to give asialomucin; Fig. 6, shows the profile after treatment with neuraminidase and cu-N-acetylgalactosa- minidase to give apomucin. As expected, asialomucin was retarded more than mucin and apomucin more than asialomu- tin. The apomucin (Fig. 6) was pooled, concentrated, reacted exhaustively with dansyl chloride, and rechromatographed on Sepharose 4B as shown in Fig. 7. From the spectral analyses shown in Figs. 6 and 7, it was estimated that less than one dansyl group per 5000 amino acid residues could be present in the preparation, using l -dansyl lysine as a fluorescent stand- ard. Analysis of acid hydrolysates of this preparation also revealed no dansyl a-amino acids. This suggests that amino groups were not produced during deglycosylation as the result of cleavage of peptide bonds and confirms the conclusion that mucin has a blocked NH,-terminal residue and is devoid of amino acids that react with dansyl chloride. Thus, after dan- sylation of mucin obtained after Step 5 (Table I), it was possi- ble to obtain native, asialo-, and apomucins, which subsequent analyses suggest are homogeneous preparations free from the small amounts of contaminating proteins that react with dan- syl chloride.

Amino Acid and Carbohydrate Compositions of Mucin Preparations-Table II gives the amino acid compositions of native mucin, asialomucin, and apomucin isolated after gel filtration as shown in Figs. 5, 6, and 7, respectively. The composition reported earlier (6) for ovine mucin is also given

I I C

5.6

h? 3 4.0-

= 2 3.2-

g 2.4-

3 LL 1.6-

L

1 .28

0.6 $

8 0.5 0

FRACTION NUMBER

FIG. 5. Gel filtration of asialomucin on Sepharose 4B. A sample of desialized, dansylated mucin (1.5 mg of protein) was chromato- graphed under the same conditions described in Fig. 4. Fractions were analyzed for protein (A), N-acetylgalactosamine (B), fluores- cence (C), andN-acetylneuraminic acid (II), and pooled as indicated by the bar.

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Characterization of Ovine Submaxillary Mucin 3795

FIG. 6. Gel filtration of apomucin on Sepharose 4B. Dansylated, apomucin (1.5 mg) was chromatographed on a column (5 x 86.5 cm) as described in Fig. 4 except that 40-ml fractions were collected at a flow rate of 42 ml/h. Fractions were analyzed for protein (Al, N- acetylgalactosamine (B), fluorescence (C), and N-acetylneuraminic acid (D), and pooled as indicated by the bar.

5 1.2

u" 5 m 0.9 B % a 0.6

0-0 FRACTION NUMBER

FIG. 7. Rechromatography of the apomucin after dansylation. A sample of apomucin from Fig. 6, was dansylated as described under “Experimental Procedures” and then rechromatographed under con- ditions identical with those described in Fig. 6. Fractions were analyzed for protein (0) and fluorescence (01, and pooled as indi- cated by the bar.

for comparison. There is generally good agreement between the compositions except for the absence of lysine and histidine in those preparations which had been treated previously with dansyl chloride. All preparations were devoid of cysteine, methionine, tyrosine, and tryptophan and about 75% of the

TABLE II

Amino acid composition of mucin, asialomucin, and apomucin

Duplicate samples were hydrolyzed for 24, 48, and 72 h and dupli- cate samples of each were analyzed to give the average values shown.

Amino acid Mu& Muck Mu& Asialo-

(Step 5) (Ref. 6) (Fig. 4) mucin Apomucin”

(Fie. 5) (Fig. 6)

Aspartic acid 16.9 Threonine” 141.1

Serine” 185.5 Glutamic acid 52.5

Proline 106.1 Glycine 193.1 Alanine 139.4 Valine 67.8

Isoleucine’ 11.4

Leucine 33.6

Phenylalanine 10.8

Lysine 3.6 Histidine 3.1

Arainine 35.2

17

148

175

53

96 198 136

60 13

52

16

3

widuesl1000 residues

15.0 19.7

142.3 141.3

180.4 186.6

53.4 49.0

110.9 103.5 199.2 196.7

139.1 137.4

66.7 69.5 11.3 13.7

35.4 37.4

13.8 10.1

15.4 (10)

136.9 (89)

176.6 (115) 57.5 (37)

115.4 (75) 200.5 (130)

139.6 (91) 65.9 (43) 10.5 (7)

36.5 (24)

13.9 (9)

36 32.4 35.1 31.3 (20)

Cz The numbers in parentheses are the number of residues per molecule assuming a molecular weight of 58,300.

h Values listed were extrapolated to zero time of hydrolysis. C Values listed were those obtained after 96 h hydrolysis.

TABLE III

Carbohydrate composition of mucin and asialomucin

Carbohydrate Mucin Mucin Mu& Asialomu- wep 5) (Ref. 6) (Fig. 4) tin (Fig. 5)

reszdues/lOOO amino acid residues

N-Acetylgalactosamine 312 272 301 347 N-Acetylneuraminic 268 255 257

acid

N-Acetylgalactosa- mine: serine + threo- nine content

N-Acetylneuraminic acid: N-acetylgalac- tosamine

0.96 0.84 0.93 1.06

0.86 0.94 0.86

composition was accounted for by threonine, serine, proline, glycine, and alanine. The number of residues of each amino acid per molecule was calculated for apomucin assuming a molecular weight of 58,300, obtained as described below.

Table III gives the carbohydrate content of the same mucin preparations listed in Table II. In accord with earlier reports (6), about 60% of the weight of the mucin molecule is ac- counted for by N-acetylgalactosamine and sialic acid. The ratio of the N-acetylgalactosamine to the serine plus threonine contents for native and asialomucin is near 1, suggesting that each serine and threonine in the molecule is glycosylated. The N-acetylneuraminic acid to N-acetylgalactosamine contents

suggest that about 85% of all N-acetylgalactosamine is glyco- sidically linked with sialic acid.

The partial specific volumes were calculated from the com- positions to be 0.663, 0.690 and 0.711 cc/g for mucin, asialomu- tin, and apomucin, respectively.

Ultracentrifugal Analysis of Mucin Preparation-Fig. 8 shows representative results of ultracentrifugal analyses of different mucin preparations obtained by sedimentation-equi- librium. The high viscosity of solutions of mucin in 0.5 M

sodium chloride necessitated analysis at initial protein con- centrations between 60 to 100 pg/ml. In addition, the protein

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3796 Characterization of Ovine Submaxillary Mucin

(RADIUS)’ (RADIUd

concentration throughout the cell was measured by absorb- ance at 232 nm, because mucins are devoid of chromophores that absorb at higher wavelengths. Tine maximum deviation in concentration at equilibrium from repetitive scans, how- ever, was i5% under these conditions.

Native mucin obtained after Step 5 (Table I) or after dansy-

lation and gel filtration (Fig. 4) gave nonlinear plots of In concentration versus radius2 at equilibrium when the initial mucin concentration was about 100 pg/ml (Fig. 8A). At lower initial protein concentrations (Fig. .a), a more linear relation- ship was observed, and the apparent weight average molecu- lar weight for mucin was calculated to be 547,000. Asialomucin (Fig. 8C) and apomucin (Fig. 80) gave linear In concentration uersus radius’ plots at equilibrium at initial protein concentra- tions between 140 to 175 pg/ml. From these analyses the weight average molecular weights of asialomucin and apomu- tin were estimated as 224,300 t 11,000 and 58,300 t 3,000, repectively.

These results suggest that mucin is composed of subunits that aggregate in aqueous solution. The polypeptide chain of mucin (M,. = 58,300) contains approximately 650 residues (Table II), including about 204 residues of threonine plus serine. If each hydroxyl group of threonine and serine is in O- glycosidic linkage with N-acetylgalactosamine and 86% of the

N-acetylgalactosamine groups are in ot2 + 6 linkage with N- acetylneuraminic acid (Table III), then a single glycosylated subunit is calculated to have a molecular weight of 154,150. Since the observed apparent molecular weight of mucin is estimated (Fig. 8B) to be well over 500,000, the glycosylated polypeptide subunits must aggregate in aqueous solution, in accord with the observations that in buffers of low ionic strength mucin emerges unretarded in the void volume of columns of Sepharose 4B (Fig. 31, but is included somewhat on gel filtration in 0.5 M sodium chloride (Fig. 4).

To assess the effect of ionic strength on the aggregation of mucin, its molecular weight was determined in 2 M sodium chloride by sedimentation equilibrium at two angular veloci-

FIG. 8. Sedimentation-equilibrium of mucin preparations. All samples were analyzed in 0.01 M sodium caco- dylate, pH 6.0, containing 0.5 M NaCl, with a column height of 4 mm. The preparations, initial protein concentra- tions and rotor speeds, were as follows. A. mucin after Steu 5. (Table I) 100 uel ml, 6000 rpm; B, n&in (Fig. 3j, 60 &$/ ml, 6000 rpm; C, asialomucin (Fig. 41, 140 pg/ml, 10,000 rpm; D, apomucin (Fig. 5), 175 pg/ml, 20,000 ‘pm.

ties and two initial protein concentrations. A representative plot of In concentration versus radius2 is shown in Fig. 9A. All data points have not been included for clarity but the results of two individual scans of the distribution at equilibrium show that the absorbance at 232 nm has a maximum deviation of ?O.Ol. The solid curve through the data points and the dotted lines corresponding to the molecular weights indicated were obtained by methods described earlier (19). The molecular weight of mucin is clearly a function of protein concentration, in accord with ultracentrifugal analysis in 0.5 M sodium chlo- ride (Fig. 8A). The molecular weights as a function of concen- tration were calculated from the following equation (19).

(1)

where c is protein concentration, r is the radial distance, U is the partial specific volume, w is the angular velocity, p is solvent density, and 7’ is the temperature. The calculated values are shown in Fig. 9B. These data show that mucin forms a rapid and reversible self-associating system at thermo- dynamic equilibrium since the dependence of molecular weight on concentration is independent of the initial mucin concentration and the angular velocity. The weight average molecular weight at the lowest concentration measurable (35

pgiml) is 155,000 2 9,000, in good agreement with the estimate that in monomeric mucin the weight average molecular weight of the polypeptide chain is 58,300 (Fig. 80) and that for the fully glycosylated chain is 154,150.

At equilibrium, the following equation describes the de- pendence of molecular weight on concentration (19).

(2)

where MV is the weight average molecular weight at concen- tration c, M,., the monomer molecular weight, X, the mole fraction of monomer at concentration c, and K, the ith associa- tion constant. Graphical integration of [(M,.&4,.) - 11/c versus

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Characterization of Ovine Suhmaxillar,y Mucin 3797

(RADIUS)Z id

CONCENTRATION (mg/ml)

sine, lysine, and histidine. Mucin prepared by the method of Tettamanti and Pi&man (6) gives two species, one in major amount (95%) and another in minor amount (5%). The major mucin is devoid of t,he small amounts of those amino acids foilnd by Bhargava and Gottschalk (23). except lysine, whereas the minor mucin contains tyrosine, histidlne, and lysine. The studies reported here indicate that ovine rnucin contains none of the amino acids previously observed in small amounts. These amino acids are associated with a small amount of contaminating protein which, after reaction of mu- tin preparations with dansyl chloride, can be removed by gel filtration. The contaminant represents no more than 2 to 8% of the weight of mucin prepared by the methods described here. Since the physical properties of the mucin before and after removal of the contaminant arc largely unchanged, the con- taminant does not appear to represent an integral part of the mucin st,ructure. The fact that apomucin behaves as a mono- disperise species on sedimentation equilibrium in the ultracen- trifuge also suggests that rnucin prepared a~ described here is highly purified.

-L- J 03 04 05 06

ABSORBANCE 232 nm

FIG. 9. A, sedimentation-equilibrium of mucin (Fig. 8) in 0.01 M sodium cacodylate, pH 6, containing 2 M sodium chloride at 10 000 rpm at an initial concentration of 88 p&l. The solrd cww is a computer fit to the data points and the doftrd iirlrs are computer- generated slopes corresponding to the molecular weights indicated. B, the weight average molecular weight of mucin as a fun&ion of’ protein concentration in 0.01 M sodium cacodylate, pH 6, containing 2 M sodium chloride. The rotor speeds and initial mucin concentra- tions are 0, 9000 rpm and 59 &ml; 0, lOZOO rpm and 59 &ml; n, 10,000 rpm and 88 b&ml. The point at zero conccntratlon. A, is the calculated monomer molecular weight ofthe fully glycotiylnted poly- peptide chain of mucin.

c gives values ofX, as a function of concentration. Plots of M,/ M,, X, versus X, c/M,, were found to be nonlinear, as expected for self-associating systems, and from the limiting slope of these plots, K,, the association constant for mucin rronomer ti dimer [K2 = (dimer)/(monomer)‘l was calculated to be 6 x 10’ liters/mol. Too few data were available to estimate the error in the calculated value ofK,, but it is of the order of magnitude of similar constants in other self-associating systems (19).

The mucin freed of contaminating protein has an amino acid composition much like that reported for the major component of Tettomanti and Pigman (6) except for lysinc, which persists in small amounts in their preparations. The mucin isolated here also has a carbohydrate cornposition similar to those reported earlier (6, 231, and cont.ains only sialic acid and N- acetylgalactosamine. The structure of the carbohydrate pros- thetic group has not been examined thoroughly, but it is probably the disaccharide N-acetylneurorninosyl cu2 + 6-N-

acetylgalactosamine in O-glycosidic linkage witch hpdroxyl groups on the side chains of serine and threonine, as reported earlier (2). The prosthetic groups are P-eliminated at alkaline pW in accord with expectations (2) and nsialomucin is an excellent substrate for a highly purified porcine CMP-sia- 1yl:mucin tramsferase. 1 More importantJy, the composition of mucin (Table III) suggests that each hydroxyl ~grottp of serine and threonine is glycosylated in rnucin, since within cxperi- mental error (&6’%), the ,~‘-acelylgalactasnmine content is

equal to the threonine plus serine content>.

Dissociafion of’Mucin h,~ Various &W is -Although mucin dissociates in solutions ofhigh ionic strength, other commonly used solvents for dissociating noncovalently bound protein subunits were ineffective. Gel filtration at low ionic strcngtb as shown in Fig. 3, indicates that mucin forms aggregates too large, or asymmetric in shape, to be included in Sepharose 4B. Columns equilibrated and developed with other solvents gave essentially the same results. This included 0.1% sodium dode- cyl sulfate or 8 M urea containing 0.01 M sodium cacodylate, pH 6, and 1 mM P-mercaptoethanol; 2%’ lactose in sodium cacodylate, pH 6; 7 M guanidine hydrochloride cont,aining 0.1 M EDTA and 0.1 M Tris hydrochloride, pH 8.5, and 50%’ ethylene glycol:l M NaCl (v/v).

The molecular weight of ovine mucin is markedly concentra- tion dcpendpnt (4) and has been reported to be as low as 394,000 in G.2 M sodium chloride (6) to as high as 1.3 x 10” at lower ionic strengths, a,csuming C’ = G 65 to 0.685 (2). The molecular wei$ts of the highly purified mucins reported here were examined in 0..5 and 2.0 M sodium chloride, since the molecular weight of mucin has a marked dependence on ionic strength, in accord with earlier studies on bovine and porcine mucins (24). Table IV summarizes the molecular weights esti- mated from the data in Fig. 8. The molecular weight of mucin cannol be calculated accurately rrom the nonlinear curves of lnc versus the square of the radial distance, but appear Lo be between 55G,OOG and 650,000. Asialomucin and apomucin, which give better linear plots, have apparent molecular weights of 224,300 and 58,300, respectively, using values for ii calculated from amino acid and carbohydrate compositions.

DISCUSSION

It is difficult to assess the purity of ovine submaxillary mucin by physical methods because of its large moiecular weight and high viscosity in solution at neutral pH. However, some insight into purity is obtained from comparison of the amino acid compositions reported for different preparations. Mucin isolated by the method of Rhargava and Gottschalk (23) contained small amounts of half-cysteine, methionine, tyro-

The molecular weights of asialomucin and apomucin were far lower than expected, and initially it was concluded that mucin had been de.yaded by contaminating proteolytic en- zymes in mucin itelf or in the glycosidases. This was ruled out, however, by bighly sensitive assays (18) for proteolytic activ-

ity. Incubation of mucin or the glycosidases with radioactively labeled hemoglobins for as long as 72 h under conditions of

” H. 11. Hill. Jr., and R. L. Hill, unpublished observation:s. ’ J. E. Sadler and R. L. Hill, unpublished observations.

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3798 Characterization of Ovine Submaxillary Mucin

TABLE IV

Molecular weights of mucin, asialomucin, and apomucin in 0.5 M

sodium chloride

Preparation Molecular weight

Experimental (Fig. 8) Calculated

Mucin 559,000-640,000 154,150 Asialomucin 224,300 108,000 Apomucin 58,300

deglycosylation gave no sign of proteolytic action. Moreover, free amino groups were not detected in deglycosylated mucins by reaction with dansyl chloride.

The molecular weights observed for mucin and the deglyco- sylated mucins give additional insight into the structure of ovine mucin. First, despite its apparent high molecular weight, mucin contains a polypeptide chain of about 650 resi- dues with a molecular weight of 58,300. There was no indica- tion of aggregation of apomucin, and evidence is presented in the following paper suggesting that it has a unique amino acid sequence (25). Secondly, if the hydroxyl group of each serine and threonine residue in the chain is 0-glycosidically linked with N-acetylgalactosamine, as in asialomucin, it should have a molecular weight of 108,000 (Table IV). The molecular weight of asialomucin was found to be 224,300, about twice the expected value. Thus it may associate noncovalently to form predominantly dimers. These results suggest that association of asialomucin is dependent on its N-acetylgalactosamine con- tent. Finally, the calculated molecular weight of mucin with its full complement of carbohydrate prosthetic groups should be 154,150 (58,300 for the polypeptide chain and 95,850 for prosthetic groups) assuming each hydroxyl group of serine and threonine is linked with N-acetylgalactosamine and 86% of these amino sugars are in cy2 + 6 glycosidic linkage with sialic acid. The calculated value, however, is about one-fourth the experimental value for mucin in 0.5 M sodium chloride, sug- gesting that under these conditions mucin associates to give a population of molecules containing a considerable fraction of tetramers of the fully glycosylated monomer of molecular weight 154,150. Again, association is dependent on the carbo- hydrate content of the molecule. Association to give even higher oligomers of the fully glycosylated mucin subunit at lower ionic strengths would account for molecular weights of the order of 10” reported by others (2, 4, 6, 24).

The association of mucin is clearly dependent on ionic strength, as shown by determination of its molecular weight in 2 M sodium chloride. These analyses indicate that mucin forms a self-associating system, since its molecular weight is independent of the initial protein concentration and angular velocity of the rotor, as other self-associating protein systems (19). Even in 2 M salt, however, mucin tends to associate as a function of protein concentration (Fig. 91, although the molec- ular weight at the lowest concentration of mucin was 155,000 + 9,000, in remarkably good agreement with the calculated molecular weight of 154,130 for fully glycosylated apomucin.

The nature of the noncovalent interactions that lead to association of mucin is unknown, but association must involve carbohydrate-protein or carbohydrate-carbohydrate interac- tions, or both, and is a function of ionic strength and mu&

From the studies presented here, ovine submaxillary mucin may now be defined as the sialoglycoprotein from submaxil- lary glands that (a) contains a blocked NH,-terminal residue; (h) contains neither half-cysteine, methionine, histidine, tryp- tophan, tyrosine, nor lysine; (c) contains only 12 amino acids with serine, threonine, glycine, proline, and alanine compris- ing 75% of the total amino acids; (~0 has each of its hydroxy amino acids in 0-glycosidic linkage with N-acetylgalactosa- mine, with about 86% of these amino sugars in cu2 + 6 glyco- sidic linkage with sialic acid, and (e) through self-association of fully glycosylated subunits with a molecular weight of about 154,000, forms higher oligomers as a function of ionic strength, protein concentration and carbohydrate content, with molecu- lar weights ranging from 0.5 to 1.0 x 10”.

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concentration. In addition, mucin does not appear to dissociate in solutions of urea, guanidine hydrochloride, or sodium dode- cyl sulfate, at concentrations of these agents that normally dissociate proteins into subunits.

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H D Hill, Jr, J A Reynolds and R L Hillsubmaxillary mucin.

Purification, composition, molecular weight, and subunit structure of ovine

1977, 252:3791-3798.J. Biol. Chem. 

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