Isolation and characterization of a mucin-glycoprotein from rat submandibular glands

ARCHIVES

OF BIOCHEMISTRY

Vol. 242, No. 2, November

AND BIOPHYSICS 1, pp. 383-392,1985

Isolation and Characterization of a Mucin-Glycoprotein from Rat Submandibular Glands LAWRENCE A. TABAK,**’ LILY MIRELS,t LAURA D. MONTE,* AMY L. RIDALL,? MICHAEL J. LEVINE,? RONALD E. LOOMIS,? FELICIA LINDAUER,* MOLAKALA S. REDDY,t AND BRUCE J. BAUMS Departments Investigations

of *Endodontics and t&-al Biology, and Patient Care Branch, National

SUNYAB, Institute

Received

May

Buffalo, of Dental

New York l&X4, and the $Clinical Research, Bethesda, Maryland 20205

22, 1985

A blood group A’ mucin-glycoprotein was purified from aqueous extracts of rat submandibular glands by sequential chromatography on columns of Sepharose CL-6B and Sephacryl S-300 in urea-containing buffers. Final purification was facilitated by reductive methylation which appeared to release contaminating (hydrophobic) peptides. Homogeneity of the purified mucin was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at varying concentrations of acrylamide, lectin affinity chromatography, and Western blot analysis. In contrast to previously described preparations, the purified mucin contained only trace amounts of N-acetylglucosamine and aromatic amino acids. In addition, only low levels of basic amino acids were present. 0 1985 Academic

Press, Inc.

Rat submandibular gland (RSMG)2 acinar cells are seromucous in nature, containing large quantities of neutral glycoproteins and lesser amounts of acidic glycoproteins. Many investigators have utilized this gland as a model to study the secretion of mucin-glycoproteins (l-6). Unfortunately, only limited and conflicting data concerning the chemical and physical properties of RSMG mucin are available (7-9). For example, Malinowski and Herp (7) report that lysine is the most abundant amino acid of RSMG mucin, accounting for 25% of the protein core. In contrast, Fleming et al (8) found that threonine, serine, and proline comprise almost 50% of their

mucin peptide. Differences also exist in the observed carbohydrate composition. Keryer et al (9) found relatively low levels of N-acetylgalactosamine (GalNAc) relative to N-acetylglucosamine (GlcNAc), whereas GalNAc:GlcNAc ratios of 2.7:l and 3.4:1 have been reported by Malinowski and Herp (7) and Fleming et al. (8), respectively. Finally, estimates of RSMG mucin size ranging from 85,000 (7) to 1 X lo6 (8) have been reported. Characterization of RSMG mucin is a prerequisite to using the RSMG as a model to study the regulation of mucin biosynthesis. In view of the many differences summarized above we have employed previously described methods (10,ll) to isolate a mucin from the submandibular glands of male Wistar rats. Homogeneity of the preparation was assessed by several complementary criteria. In addition, the mutin-glycoprotein was characterized with respect to apparent size, chemical composition, and blood group activity.

1 To whom correspondence should be addressed. ’ Abbreviations used: RSMG, rat submandibular gland; GalNAc, N-acetylgalactosamine; GlcNAc, Nacetylglucosamine; ANS, l-anilino-%naphthalene-lsulfonate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PAS, periodic acid/ Schiff. 383

0003-9861/85 Copyright All rights

$3.00

Q 1985 by Academic Press, Inc. of reproduction in any form reserved.

384

TABAK EXPERIMENTAL

PROCEDURES

Materials. Submandibular glands were obtained from (3-6 month) Wistar-derived rats (Charles River Laboratory, Wilmington, Mass.). Sepharose CL-GB, Sephacryl S-300, and Dolichos b&rrus lectin were purchased from Pharmacia P-L Biochemicals (Piscataway, N. J.). Bovine serum albumin (fatty acid free), Escherichia coli fi-galactosidase, A. hypogae agglutinin, and D. &tloru.s-agarose were obtained from Sigma Chemical (St. Louis, MO.). Reagents for gel electrophoresis, Bio-Gel P-2 (200-400 mesh), and AG 1X-8 and 50X-4 were purchased from Bio-Rad Laboratories (Richmond, Calif.). ‘l-Protein A (30 Ci/g) was obtained from ICN Biomedicals (Irvine, Calif.). [“ClFormaldehyde (50 Ci/mmol) and Enhance were purchased from New England Nuclear (Boston, Mass.). i4C-labeled molecular weight standards were obtained from Bethesda Research Laboratories (Bethesda, Md.). [‘HISodium borohydride (10 Ci/mmol) was purchased from Research Products Int. (Mount Prospect, Ill.). Nitrocellulose filters (HAHY) were obtained from Millipore Inc. (Bedford, Mass.). Complete Freunds adjuvant was purchased from GIBCO (Grand Island, N. Y.). The hydrophobic probe, l-anilino-&naphthalene-1-sulfonate (ANS) was obtained from Aldrich (Milwaukee, Wise.). All other chemicals used were reagent grade. Analytical procedures. Protein was determined by the method of Lowry et al (12). Neutral and amino sugars were determined after hydrolysis with 2 M HCl for 6 h at 100°C and passage of the hydrolyzate through coupled columns of AG 50 and 1. Neutral sugars were determined as alditol acetates by gasliquid chromatography, and hexosamines, amino acids, and sialic acids as described previously (11). Purijkation of mucin. Male Wistar rats (3-6 months) were lightly anesthesized with ether and killed by cardiac puncture. Submandibular glands were excised, and connective tissue, superficial fat, and sublingual glands were removed. For extraction, 2-3-mm segments of tissue were suspended in 3 vol (w/v) of ice-cold 0.1 M Tris-HCl buffer, pH 7.5, with 2% NazEDTA and 2 mM phenylmethanesulfonyl fluoride, and stirred at 4°C for 24 h. Insoluble residue was recovered by centrifugation at 12,000g for 10 min and reextracted as described above. The resulting supernatants were combined, dialyzed successively against 0.2% NazEDTA and distilled water, and lyophilized (yielding approximately 10% (w/w) of the original gland material extracted). Extract was resuspended by stirring at 4°C at a concentration of 20 mg/ml in 0.1 M Tris-HCI buffer, pH 7.5, with 6 M urea (Tris-urea). The undissolved material was removed by centrifugation at 12,OOOg for 20 min at 4°C. After being warmed to room temperature, samples were applied to columns (1.5 X 120 cm) of Sepharose CL-GB, equilibrated with Tris-urea

ET

AL.

buffer. Fractions (2.0 ml), collected at room temperature, were monitored for protein and pooled as indicated in Fig. 1A. Following dialysis and lyophilization, each peak was analyzed for hexosamine and sialic acid as a measure of mucin glycoprotein content. On this basis, materials in peak 2 (Fig. 1A) were identified as crude mucin. Further purification was achieved by recycling the crude mucin on columns (1.5 X 120 cm) of Sephacryl S-300 equilibrated with Tris-urea buffer. Fractions (2.5 ml), collected at room temperature, were monitored for protein and pooled as indicated in Figs. lBD. Following dialysis and lyophilization, aliquots of each peak were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) for the presence of periodic acid/Schiff (PAS)-staining components, and for amino acid and carbohydrate content. Based upon these criteria, materials in peak 2B-2 (Fig. 1D) were identified as partially purified RSMG mucin. Previous studies (10,ll) suggested that the c-amino group of lysine residues contribute to the formation of heterotypic complexes between mucins and other constituents. To determine if such complexes were present in the partially purified RSMG mucin, peak 2B-2 (Fig. 1D) was subjected to reductive methylation using [‘*C]formaldehyde as the methyl donor as previously described (11). Unreacted products were removed by gel filtration on columns (1 X 45 cm) of BioGel P-2 equilibrated with 0.1 M pyridine acetate buffer, pH 5.1. Void volume materials were rechromatographed on columns of Sephacryl S-300; fractions (2 ml), collected at room temperature, were monitored by liquid scintillation spectrometry. The major peak, 2B-2C (Fig. lE), represented the purified [“‘CIRSMG mucin. Fluorescence spectroscopy. To evaluate the effects of reductive methylation on RSMG mucin, fluorescence emission spectra were obtained on a Perkin-Elmer Model 650-40 fluorescence spectrophotometer interfaced through a Cyborg Model 42A ISACC to an Apple IIE computer. The excitation wavelength was 365 nm and the spectrum was recorded over the range 400600 nm. All spectra were corrected to Rhodamine B; background subtractions were performed with a Perkin-Elmer Model 650-0265 ordinate data processor. The sample, dissolved (0.1 rig/ml) in 0.01 M potassium phosphate, 0.04 M potassium chloride, 0.003 M sodium azide buffer, pH 7.0, was maintained at 30°C in a lcm2 cuvette. The hydrophobic probe, ANS, was used in selected experiments at a concentration of 5 nmol. For analysis, spectra generated with the mucin in the presence of ANS were digitally subtracted from the ANS spectrum. Electrophoretic procedures. SDS-PAGE was performed according to the method of Laemmli (13). Glycoproteins were stained with Coomassie blue for protein and with the PAS reagent for carbohydrate

CHARACTERIZATION

OF

A RAT

SUBMANDIBULAR

GLAND

MUCIN

385 B

om

025

020

m7oonn

0.15

0.10

0.05

0.W

600aoo, SW0

”

so00

”

2540

”

moo

”

cm

I

WQ~~ looo

"

Fmcllm

No

FIG. 1. (A) Chromatography by gel filtration of RSMG extract (200 mg) on Sepharose CL-6B (2.0ml fractions); (B) chromatography by gel filtration of Peak 2 (A) (79 mg) on Sephacryl S-300 (2.5ml fractions); (C) rechromatography by gel filtration of Peak 2A (B) (10.3 mg) on Sephacryl S-300 (2.6-ml fractions); (D) rechromatography by gel filtration of Peak 2B (B) (20 mg) on Sephacryl S-300 (2.6-ml fractions); (E) rechromatography by gel filtration of desalted [%]RSMG mucin (0.9 mg) on Sephacryl S-300 (2.0-ml fractions).

(14). The apparent molecular weight of [“CIRSMG mucin was determined by varying the concentration of polyacrylamide as suggested by Sergrest and Jackson (15). Radiolabeled materials were visualized by fluorography after impregnation of the gel with Enhance and exposure to Kodak XAR film at -70°C. Molecular weights were estimated from plots of the log molecular weight versus relative mobility of standard

proteins (16), which included: [‘4C]myosin (205K), E. coli fl-galactosidase (116K), [“Clphosphorylase B (97.413), [“C]Bovine serum albumin (68K) and [“Cjovalbumin (43K). Immunological procedures. Antisera to partially purified rat mucin (peak 2B-2, Fig. 1D) were prepared in rabbits. Immunogen (1 mg/ml in sterile phosphatebuffered saline) was emulsified with complete

386

TABAK

Freund’s adjuvant (l:l, v/v) and distributed to three sites subcutaneously. After 14 days, a second immunization was performed. Two weeks later, serum was collected and tested by immunodiffusion as previously described (17). For Western transfer, proteins were separated on SDS-PAGE (13) and then transferred (1.9 A for 8 h) onto a nitrocellulose filter according to methods described previously (18). The filters were incubated with a 1:lOOO dilution of rabbit anti-mucin serum overnight at 4°C. Following washes, cross-reactive proteins were localized with ‘l-protein A, nitrocellulose filters were exposed to Kodak XAR film at -70°C. Reactivity with lectins. Purified lectins were dissolved in 0.154 M NaCl at a concentration of 0.5% (w/ v). Wells were cut in plates of 0.8% agarose buffered with barbital acetate, pH 8.2. A total of 50 pg of lectin or [14C]RSMG mucin was added to each well and diffusion was allowed to proceed for 24 h at 4°C in a moist chamber. The [i4C]RSMG mucin was chromatographed on columns (0.5 X 5 cm) of agarose-D. bi~?omcs lectin equilibrated with phosphate-buffered saline, pH 7.2. After extensive washing, the column was eluted with 0.01 M GlcNAc-phosphate-buffered saline, followed by 0.01 M GalNAc-phosphate-buffered saline. Fractions (1.0 ml) were collected at 4°C and were monitored by liquid scintillation spectrophotometry. Release of RSMG muck sH-laMed oligosoccharides Alkaline labile oligosaccharides from [“CIRSMG mutin (1.8 mg) were released (and tritiated to permit detection) by incubation with 0.05 M NaOH, 1.0 M NaBH, (0.5 ml) containing 0.64 nmol NaBaH at 45°C for 16 h. The released oligosaccharides were passed through columns of AG 50W-X8(Hf) to remove peptide, and following lyophilization of the water effluent, borate was removed by codistillation with methanol. The products obtained from the hydrolysis of the oligosaccharide alditols were analyzed by descending paper chromatography (n-butanol:acetic acid:water, 4:1:5) to localize radioactivity.

RESULTS

Puti$cation of RSMG mu&. Figure IA shows the elution profile following chromatography of solubilized RSMG extract on columns of Sepharose CL-6B. Material eluting in the void volume (peak 1) contained considerable amounts of 2-deoxyribose as ascertained by the thiobarbituric acid assay (0D532,549 = 1.7) and relatively small amounts of hexosamine and sialic acid. Crude mucin was localized in peak 2 which was enriched in both hexosamine

ET

AL.

(35-fold) and sialic acid (&fold) content when compared to the RSMG extract. Fractionation of the crude mucin on columns of Sephacryl S-300 yielded two partially resolved peaks (2A and 2B, Fig. 1B). To improve separation, the materials recovered from each peak were recycled through columns of Sephacryl S-300 (Figs. 1C and D). This led to resolution of four peaks, i.e. 2A-1, 2A-2 (Fig. 1C) and 2B-1, 2B-2 (Fig. 1D). Glycoproteins were localized by PAS staining of samples separated by SDS-PAGE. Two major glycoproteins were identified (Fig. 2A), having relative mobilities of 0.29 (peak 2B-2) and 0.37 (peaks 2A-1, 2A-2, and 2B-1). In addition, trace amounts of a third discrete glycoprotein (relative mobility, 0.16) were localized in pools 2A-1 and 2A-2. Materials from peaks 2A-1,2B-1, and 2B2 were obtained in sufficient quantity to permit chemical analysis. Table I summarizes the amino acid and carbohydrate composition of each. Peak 2A-1 was characterized by high levels of glutamic acid and glycine and low levels of proline. In contrast, materials from peaks 2B-1 and 2B-2 displayed amino acid profiles which were mucin-like in nature, i.e., high levels of threonine, serine, and proline were found. The partially purified mucin localized in Peak 2B-2 was further purified after reductive methylation and rechromatography on columns of Sephacryl S-300. The major peak, 2B-2C (Fig. 1E) represented the purified [14C]RSMG mucin. Approximately 1.1 mg of the [14C]RSMG mucin was obtained from 200 mg of RSMG extract, which represents a yield of 0.6%. Characterization of pur$ed rat SMG mu&. The homogeneity of the purified mucin was assessed by several criteria. A single band of material was observed by fluorography when the purified mucin was subjected to SDS-PAGE using a range of polyacrylamide concentrations. The asymptotic minimal molecular weight of the mucin (corrected for the presence of 60% carbohydrate) was estimated to be 103K (Fig. 2B). Electrophoresis in presence of the reducing agent, P-mercaptoethanol,

CHARACTERIZATION

OF

A RAT

SUBMANDIBULAR

GLAND

MUCIN

387

“t OJ 3.5

I 3

6.3

I

9.5

II

% Acrylmll6l

FIG. 2. (A) Gel electrophoresis in 5% polyacrylamide of peaks 2A-1 (lane l), 2B-1 (lane 2), and 2B2 (lane 3). The sample (100 pg) was run in the presence of P-mercaptoethanol. The gel was stained by the PAS reagent. (B) Observed molecular weight of [i4C]RSMG mucin calculated by electrophoretic mobility relative to standards as described in Ref. (16); (C) Western transfer of peak 2A-1 (lane 1) and peak 2B-2 (lane 2). The nitrocellulose filter was probed with rabbit antiserum to the partially purified RSMG mucin; cross-reactive species were visualized with ‘%I-protein A.

failed to alter the mobility of the molecule, indicating that the mucin contains neither disulfude-linked subunits nor detectable contaminants. The [14C]RSMG mucin failed to stain with Coomassie blue; additionally, no other proteins were detected on either 5 or 10% gels (data not shown). Precipitating antibody was raised against the partially purified mucin (peak 2B-2, Fig. 1D) in rabbits. This antiserum cross-reacted with the three glycoproteins separated by SDS-PAGE and identified by PAS staining. When the purified [14C]RSMG mucin was probed with this antiserum however, a single species was

noted, indicating immunochemical homogeneity (Fig. 2C). The [14C]RSMG mucin also displayed homogeneity when chromatographed on colAll sample umns of D. tijorus-agarose. remained adsorbed to the column after elution with phosphate-buffered saline, pH 7.0, or 0.01 M GlcNAc-phosphate-buffered saline. Quantitative recovery (>9’7%)of the mucin applied was obtained with elution with 0.01 M GalNAc-phosphate-buffered saline (Fig. 3). The effect of the reductive methylation step employed during the purification scheme was assessed by both chemical

TABAK

TABLE CHEMICAL

I

COMPOSITION OF RAT SUBMANDIBULAR GLAND MUCIN FRACTIONS Residues/1999

Constituent Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Fucose Mannose Gala&me N-Acetylgalactosamine N-Acetylghrcosamine Sialic acid”

residues

2A-1

2B-1

2B-2

2B-2C

84

90 139 90 123 91

98 169 98 135 89

99 231 117 90 129

65 73 18 50

63 71 13 48 7 31 50 16 21 14 62

48 75

67 77 133 29

107 86 25 64 12 43 93 29 40 21 59 36

19 13 30 25 7 -=

a Not detected. b Trace amounts detected. c Including 20 residues of monomethyl d Determined as N-acetylneuraminic *Not determined.

11 37 74

19 27 16 63 14 33 7 61 67 6 -

18 38 17 66

181 14

nd” 24 tr* 39 24 tr nd nd 51” 15 113 tr 192 521 tr 108

lysine. acid.

analysis and fluorescence spectroscopy. Based upon the specific activity of the methyl donor employed, incorporation was approximately 38% of the theoretical maximum. This compared favorably with the quantity of monomethyl lysine determined (40% of total lysine present) by amino acid analysis. A comparison of the amino acid composition before (Table II, Peak 2B-2) and after (Table II, Peak 2B-2C) reductive methylation indicated that there was a significant loss of hydrophobic amino acids (Cys, Val, Met, Leu, and Tyr) and glutamic acid. The lack of aromatic amino acids in the purified mucin was corroborated by the absence of intrinsic fluorescence (data not

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AL.

shown). To ascertain if the loss of hydrophobic amino acids exposed hydrophobic binding sites on the purified mucin, the binding of ANS was studied (Fig. 4). The ANS spectrum had a maximum intensity of 10.5 at 535 nm. The difference spectra for the RSMG mucin samples had intensities of 1.2 and 3.1 at 460 and 455 nm for peak 2B-2 and the [14C]RSMG mucin, respectively. Thus, the methylated mucin displayed a 2.6-fold enhancement in the binding of this hydrophobic probe and a characteristic red shift in the observed lambda maximum (19). The chemical composition of the purified RSMG mucin is typical of mucin-glycoproteins. Amino acid analysis (Table I) indicated high levels of threonine, serine, and proline; together these three residues accounted for greater than 50 mol% of the total amino acids. Carbohydrate accounted for approximately 64% of the recoverable weight of the mucin. Analysis of the monosaccharide content revealed the presence of galactose, fucose, sialic acid, and GalNAc, with only trace quantities of GlcNAc and mannose detected. The molar ratio of GalNAc to Ser + Thr was 1.3, which is consistent with the blood group A activity of this molecule.

Frrctm

No. (1.2

ml)

FIG. 3. Lectin affinity chromatography of [‘“CIRSMG muck (2.1 x 10’ cpm) on D. bifEwu.s-Agarose (1.2-ml fractions; 1.6 mg lectin/ml agarose). Following a phosphate-buffered saline wash (PBS), the column was equilibrated with (1) 0.01 M GlcNAc-PBS and (2) 0.01 M GalNAc-PBS.

CHARACTERIZATION

OF

A RAT

SUBMANDIBULAR

TABLE REACTIVITY Lectin D. br&rus agglutinin Peanut agglutinin

OF RSMG

GLAND

389

MUCIN

II MUCIN

WITH LECTINS

Specificity

Intact

GalNAc Galfll,3GalNAc

Preliminary characterization of RSMG mucin oligosaccharides. Reaction of the purified mucin with lectins (Table II) confirmed the blood group A reactivity of this molecule. Partial deglycosylation (by mild acid hydrolysis, 0.1 M HCl at 100°C for 90 min) of the mucin abolished this reaction and led to interaction with peanut agglutinin. This suggests that a Gal/31,3GalNAc sequence is made accessible by the partial deglycosylation (20). To verify that the carbohydrate sidechains of this molecule were O-linked to the peptide core, the RSMG mucin was subjected to p elimination in the presence of NaB3H4. Following desalting, aliquots of the released oligosaccharide alditols were hydrolyzed and the products obtained were subjected to paper chromatography. The 3H label was localized exclusively into the 2-acetamido-

WAVELENGTH

Deglycosylated -

+ -

+

2-deoxy-D-galactitol residue, indicating that the released chains were O-glycosyllinked to the apomucin. DISCUSSION

A mucin-glycoprotein was isolated from aqueous extracts of RSMG by sequential chromatography on columns of Sepharose CL-6B and Sephacryl S-300. In view of the marked polydispersity exhibited by mucinglycoproteins, the purity of our preparation was confirmed by electrophoretic, immunochemical, and lectin affinity methods. The variation reported in the size of RSMG mucins appears to be due, in part, to the percentage of acrylamide employed for molecular weight determination. Denny and Denny (21) have previously addressed this issue with regard to the size of mouse submandibular gland mucin. Our estimate

(nm)

FIG. 4. The relative fluorescence emission spectra of (A) ANS plus [‘“CjRSMG muein (solid line) and ANS plus peak 2B-2 (dotted line). In each case the ANS spectrum has been subtracted out. (B) ANS spectrum.

390

TABAK

ET

AL.

of the minimum asymptotic weight (103 have recently reported that RSMG mucous kDa) of RSMG mucin may still represent glycoproteins undergo fatty acid acylation. an overestimate since proline containing However, the relationship of this covaproteins are known to run aberrantly in lently bound fatty acid to ANS binding is SDS systems, yielding values which are as as yet unknown. much as 100% larger than their true size It has been assumed that much of the (22). These considerations suggest that the heterogeneity associated with mucins is size of RSMG mucin may approach the es- due to incomplete post-translational modtimate of 85,000 made by Malinowski and ification resulting in variable lengths of Herp (7) for their preparation of RSMG oligosaccharides. To assess the homogemucin. neity of the carbohydrate side chains, Significant differences were noted in the samples were chromatographed on colamino acid composition of our preparations umns of D. tijorus-Agarose. This lectin and those previously reported (7-9). In has been shown to react with both A1 and particular, we find an absence of aromatic A2 blood group substances, but not with amino acids and relatively low levels of ba- blood groups H or B (25). All material adsic amino acids. These differences may re- sorbed to this lectin column, and quantiflect the failure of previous approaches to tative recovery of the radiolabeled mucin, remove low-molecular-weight contamiwas achieved by elution with GalNAc. nants, which may function as “link” pro- Thus, at least some blood group A activity teins. Previously, we have shown that both was found in each molecule of the human salivary (10) and tracheobronchial [14C]RSMG mucin. The blood group A spec(11) mucins interact with small peptides ificity of the RSMG mucin is consistent by electrostatic bonds, and that these het- with previous histochemical observations erotypic complexes can be dissociated by made by Schulte and Spicer (26) and sesubjecting the mucin to reductive methrological analysis presented by Fleming ylation. Similar results have been noted by et ah (8). However, whereas we have Hill et al. (23), who removed lysine-confound only trace quantities of GlcNAc taining contaminants from ovine submaxin our preparations, previous studies of illary mucin by reaction of the mucin with RSMG mucin reported significant levels of dansyl chloride. GlcNAc. As pointed out by Malinowski and The changes in the amino acid compoHerp (7), Keryer et al. (9) did not report sition that occurred concomitant with re- the removal of the sublingual gland, which ductive methylation were accompanied by is embedded in the RSMG. Studies by a loss of intrinsic fluorescence and an Slomiany and Slomiany (27) have shown enhancement of the binding of the hydrothat the oligosaccharides of rat sublingual phobic probe, ANS, to the mucin. While it gland mucin are greatly enriched in could be argued that the incorporation of GlcNAc. Thus it is possible that much of the GlcNAc reported by Keryer et al. (9) is CH3 groups is responsible for this apparent sublingual increase in hydrophobicity, we have ob- derived from contaminating served that reductively methylated lower gland mucin. The relatively high levels of molecular weight human salivary mucin mannose reported by Malinowski and Herp fails to bind ANS (R. E. Loomis, A. Pra(7) suggest that their mucin either contains kobphol, M. J. Levine, M. S. Reddy, and N-glycosidic linkages [which have been reL. A. Tabak, manuscript in preparation). ported in mouse submandibular gland muTherefore, it is tempting to speculate that tin by Denny and Denny (21)] or contamthe reductive methylation uncovers hydroinating N-linked glycoproteins. Compariphobic binding sites on the RSMG mucin son of the carbohydrate analyses presented which normally serve as attachment sites by Fleming et al. (8) with those reported for the released hydrophobic amino acid- herein suggests that their preparation containing peptides. Slomiany et al. (24) (galactose:fucose:galactosamine ratio of

CHARACTERIZATION

OF

A RAT

1.0:0.5:1.3) more closely resembles the materials found in peak 2B-1 than those characteristic of peak 2B-2 (galactose:fucose: galactosamine ratio of 1.0:0.5:1.1 and 1.0: 0.6:2.7, respectively). While our data argue for the homogeneity of the [14C]RSMG mucin preparation, the results obtained suggest that there are additional mucin-like species in the RSMG. Materials in peak 2A-1 contain significant levels of carbohydrate, threonine, and serine, but rather low levels of proline (Table II). The apparent molecular weight of this fraction by gel filtration is greater than the [14C]mucin, making it unlikely that it is a breakdown product. We have also partially purified a second mucin-like species (peak 2B-1) which appears similar to the [i4C]RSMG mucin except that it has significantly less GalNAc. It is not yet known if this represents a biosynthetically incomplete version of the blood group A active mucin or a separate species. Histochemical (28), immunochemical (29), functional (30), and biochemical (31, 32) studies have suggested that mucousproducing cells may synthesize multiple mucin glycoproteins. Recent work indicates that these observations may be extended to include the RSMG. Wilson (33) reported that two polypeptides, molecular weights 190K and 150K, were secreted by rats in response to a P-adrenergic agonist. Baum et al. (34) observed the secretion of two glycoproteins, 225K and 160K (as ascertained by 3.5% SDS-PAGE) from RSMG dispersed cell aggregates. These findings, coupled with the present work, suggest that at least part of the reported heterogeneity of RSMG mucin is due to the existence of multiple forms. ACKNOWLEDGMENTS We acknowledge Mr. Hector Velasco for the photography and Dr. E. J. Bergy for performing some of the carbohydrate analysis. This work was supported in part by Public Health Service Research Grants DE 06970, DE 04971, and DE 04518. L.A.T. is supported by Career Development Award DE 00132. L.M. and A.L.R. are supported by Training Grant DE 07034. Parts of this report were taken from a thesis to be

SUBMANDIBULAR

GLAND

391

MUCIN

submitted by L.M. to the SUNYAB ment of the Ph.D. degree.

in partial

fulfill-

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