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Regulation of buccal mucosal calcium channel activity by salivary mucins
Int. J. Biochem. Vol. 25, No. 9, pp. 1281-1289, 1993 F’rinted in Great Britain. All rights reserved
0020-711X/93 $6.00 + 0.00 Copyright 0 1993 Pergamon Press Ltd
REGULATION OF BUCCAL MUCOSAL CALCIUM CHANNEL ACTIVITY BY SALIVARY MUCINS B. L. SLOMIANY,* Z. FEICETE,V. L. N. MURTY, M. GRABSKA, J. PIOTROWSKI,F. YOTWMOTO, A. CZAJKOWSKIand A. SLOMIANY Research Center, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, University Heights, 110 Bergen Street, Newark, NJ 07103-2400, U.S.A. [Tel. (201) 982-70521 (Received
3 March 1993)
Abstract-l. The effect salivary mucins on the activity of calcium channel isolated from buccal mucosal cell membranes was investigated. The uptake of 45CaZ+while only moderately (15%) affected by the intact low and high molecular weight mucin forms, was significantly inhibited, by the acidic low and high molecular weight salivary mu&s which evoked 64 and 60% inhibition, respectively. 2. The inhibitory effect of salivary mucins was associated with the sialic acid and sulfate ester groups of the carbohydrate chains, as the removal of either group caused partial loss in the glycoproteins inhibition, and the complete loss in the inhibitory effect occurred following desialylation and desulfation. 3. The channel in the presence of epidermal growth factor (EGF) and ATP responded by an increase in tyrosine phosphorylation of 55 and 170 kDa proteins, and the phosphorylated channels showed a 46% increase in “Ca*+ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein. 4. The binding of EGF to calcium channel receptor protein was inhibited by the low and high molecular weight acidic mucins, causing 41.2 and 36.1% reduction, respectively. This reduction in binding was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoproteins’ inhibitory capacity following removal of these groups. 5. The results for the first time demonstrate that salivary mucins actively participate in the modulation of the EGF-controlled buccal mucosal calcium channel activity expression, a process of importance to the preservation of oral tissue integrity.
INTRODUCIXON Although the importance of salivary mucins in the nonimmune defense of teeth against caries is well documented (Mandel, 1982; Slomiany et al., 1986; Tabak, 1990), considerably less is known about the role these glycoproteins play in the preservation of oral mucosal integrity. The available data, however, indicate that salivary mucins enter into a tenacious interaction with oral epithelial surfaces forming protective coating known as oral mucosal mucus coat (Slomiany et al., 1989). This coat constitutes the pre-epithelial element of oral mucosal defense by acting not only as passive physical obstacle, but also as a dynamic functional barrier capable of modulating the untoward effects of oral environment, including bacterial colonization. Salivary mucins of oral mucosal mucus coat, remaining in intimate association with the epithelial cell surfaces in the oral cavity, could also conceivably play an important role in affecting the processes occurring within the epithelial perimeter of oral mucosal defense. Primary among these processes is the maintenance of intracellular calcium level. Studies indicate that calcium is an important regulatory *To whom correspondence should he addressed.
element for many cellular functions, including contraction, cell differentiation and secretion, and that its influx potentiates mucosal injury caused by a variety of noxious agents (Hosey and Lazdunski, 1988; Kass et al., 1990; Tamawski et al., 1990). The calcium entry in most excitatory and secretory cells occurs through a carefully controlled process involving specific voltage or receptor-dependent channels (Hosey and Lazdunski, 1988; Kass et al., 1990; Liu et al., 1993). Recently, we demonstrated the presence in mucosal epithelial tissues of receptor-dependent calcium channels and have shown that epidermal growth factor (EGF), through stimulation of channel protein phosphorylation, enhances the calcium uptake (Liu et al., 1993; Slomiany et al., 1992a,c). In this report, we provide evidence that the low and high molecular weight acidic salivary mucus glycoproteins are actively involved in the modulation of the EGFinduced calcium channel activity in buccal mucosa.
MATERIALSAND METHODS Materials
Human submandibular/sublingual saliva used for mucus glycoprotein isolation was obtained from five adult 1281
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secretory with blood-group B. The salivary flow was stimulated with 2% citric acid and 25530mg of saliva was collected from each individual per session (Slomiany er al., 1986). Immediately following collection, the samples were treated with buffered EDTA (PH 7.0), dialyzed against distilled water and lyophilized (Slomiany ef al., 1986). Male Sprague-Dawley rats weighing 180-200 g were obtained from Taconic Farms Inc., Germantown, NY, ([4sCa]CaCl,, ( +)-[methyl-3H]PN200-l 10) from New England Nuclear, Boston, MA and [‘251]EGF from Amersham Corp., Arlington Heights, IL. BAY K8644, egg-yolk phosphatidylcholine, V. cholerae neuraminidase, bovine serum albumin, and mouse epidermal growth factor (EGF) were supplied by Sigma, St. Louis, MO, PN200-110 was a generous gift of Dr Houlihan, Sandoz Research Institute, E. Hanover, NJ, and Whatman filter GF/C (0.22 FM) was from Whatman International Ltd., Maidstone, U.K. Wheat germ agglutininSepharose, DEAE-Sephacel and Sephadex G-50 were obtained from Pharmacia, Piscataway, NJ, Chelex 100 (SO-100 mesh), and reagents for sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and BioGel A-50 (Xl-lOOmesh) were purchased from Bio-Rad, Rockville Centre, NY. 5-Bromo-4-chloro-3-indole phosphate and nitro blue tetrazolium were from Oncogene Science Inc., Manhasset, NY. Goat anti-mouse alkaline phosphatase conjugated IgG and O-phospho-l-tyrosine were obtained from Boehringer Mannheim Biochemicals, Indianapolis, IN, and tyrosine kinase inhibitor, genistein and anti-phosphotyrosine monoclonal IgG from Upstate Biotechnology In., Lake Placid, NY. Calcium channel bolation
Calcium channels were isolated form rat buccal mucosal cell membranes. The animals were anesthesized by i.p. injection with 25% urethane (1 ml/lOOg body wt), and buccal mucosal cells were collected by scraping the mucosa with a blunt spatula. Scrapings were placed in ice-cold buffer (2.5 mM Tri-HCI, pH 7.0, 250mM sucrose, 2.5 mM EDTA, 1 mM phenyl methyl sulfonyl fluoride (PMSF), lOOTIU/ml aprotinin and 1 pg/ml leupeptin), and homogenized for 1 min in a Polytron tissuemizer. The homogenate was centrifuged at 400g for 15 min at 4°C and NaCI/MgSO, was added to the supematant to form final concentrations of 0.1 mM and 0.2 mM respectively (Wang et al., 1992). After centrifugation of the supematant at 40,OOOg for 1 hr at 4°C the membrane pellet was resuspended in 0.1 M sodium phosphate buffer, pH 7.2. The preparation was centrifuged for 20min at 10,OOOg at 4”C, and the pellet solubilized using a buffer containing 40 mM Tri-HCl, pH 7.4, 0.5 M NaCl, 20 mM 3-[[fsl] (3-cholamidopropyl)dimethylammonio]1-propanesulfonate (CHAPS), 10% glycerol, 108 TIU/ml aprotinin, 1 pg/ml leupeptin, and 1 mM PMSF. After 1 hr at 4”C, the mixture was centrifuged at 105,OOOg for 1 hr and the resulting supematant collected, 1 nM of [3H]PN200-l10, a dihydropyridine calcium channel receptor antagonist was added and incubated at 4°C for 30min. The preparation was then applied to a column of Sepharose-bound wheat germ agglutinin, and the [‘H]PN200-110 labeled calcium channel protein was eluted with buffer containing 300 mM N-acetylglucosamine (Liu ef al., 1993). Salivary mucins preparation The lyophilized samples were dissolved at 5 mg/ml in 6 M urea/lOmM sodium phosphate buffer (PH 7.0) and chromatographed on a Bio-Gel P-100 column (Slomiany et al.,
1986). The eluted fractions were monitored for protein and carbohydrate, and the excluded mucus glycoprotein peak collected. After dialysis and lyophilization, the crude glycoprotein was delipidated by washing with chloroformmethanol (2: 1) followed by acetone, dried, dissolved in 6 M urea/O. 1 M acetate buffer, pH 4.2, and chromatographed on a Bio-Gel A-50 column, and the low and the high molecular weight mucus glycoprotein peaks collected (Slomiany et al., 1992b). After rechromatography, each glycoprotein form was subjected to equilibrium density gradient centrifugation in 42% (w/w) CsCl for 48 hr at 12°C and 45,000 rev/min in a Beckman 50Ti rotor (Slomiany ef al., 1986). The resultant gradient was fractionated into 1 ml fractions using a Beckman gradient recovery system, and the fractions containing mucus glycoprotein were pooled, dialyzed against distilled water and lyophilized. Such prepared low and high molecular weight salivary mucin forms were then used for the isolation of neutral and acidic mucus glycoprotein fractions. For this, each glycoprotein form was dissolved in 0.05 M Tris-HCl buffer, pH 6.8, containing 1 mM EDTA and applied to a DEAE-Sephacel column (Piotrowski et al., 1991). The glycoprotein fractions were eluted in a stepwise manner first with the above buffer (neutral mucus glycoprotein fraction) followed by buffer containing 0.2 M NaCl and 0.4 M NaCl (acidic mucus glycoprotein fraction). The neutral and acidic mucus glycoprotein fractions derived from the low and high molecular weight salivary mucin forms were collected, dialyzed against distilled water. and lyophilized. Effect of mucins on EGF binding
The effect of intact and modified salivary mucus glycoprotein preparations on the binding of EGF was assessed following preincubation of 200 ~1 aliquots of calcium channel preparation (250 pg protein/assay) with the glycoprotein sample (O-120 pg) at room temperature for 30 min. EGF binding assays were carried out by incubating the channel complex samples (200 pg protein/assay) with [ ‘2SI]EGF (0.18 nM) and 5mM Tris/HCI (pH 7.0),125mM sucrose, 75 mM NaCl, 0.5 mM CaCl,, 0.5% bovine serum albumin (BSA) in a final volume of 200 ~1 (Wang er al., 1992). Unlabeled EGF was added to the incubations to form a final concentration of 0.25 PM in the experiments where nonspecific binding was being estimated. Incubates were maintained for 1hr at room temperature, and then stopped by addition of 1 mM ice-cold buffer containing 10mM Tris/HCI (PH 7.0) and 0.5% BSA. Membrane-bound [ 12sI]EGF was separated from the unbound [ “‘I]EGF by centrifugation at lO,O@Ogfor 10 min at 4°C. The pellet was washed with 1 ml of ice-cold buffer, centrifuged, and counted in a gamma counter. Effect of mucins phosphorylation
on
EGF-stimulated
channel
protein
The solubilized buccal mucosal calcium channel preparations containing 200-25Opg protein were incubated at room temperature for 30 min in 50mM HEPES buffer, pH 7.6, containing 10 mM MgSO, and 1 mM PMSF, with 0 and 100 pg mucus glycoprotein or &40 pg/ml genistein, and 2 PM EGF. The phosphorylation was then initiated by the addition of a solution containing 1OpM ATP, 1 mM CTP, 8 mM MnCls, 20mM MgCl,, and 2 mM sodium vanadate (Slomiany et al., 1992a,c). Incubations were maintained for 10min at 4°C after which the calcium channel preparation was recovered using wheat germ agglu-
Calcium channel activity and salivary mucins
kDa
Fig. 1. SDS-PAGE of [3H]PN20&l 10 labeled buccal mucosal calcium channels purified from solubilized buccal mucosal cell membranes by affinity chromatography on Sepharose-bound wheat germ agglutinin. Pattern under reducing conditions visualized by silver stain.
kDa
1
2
Fig. 7. Effect of tyrosine kinase inhibitor, genistein, on EGF-stimulated buccal mucosal calcium channel protein phosphorylation. The channel preparations following 30 min preincubation with 30 pg/ml genistein (Lane 1) were incubated for 30min with 2pM EGF and then ATP (40pM) was added, and the reaction was stopped after 20 min. After SDS-PAGE, the proteins were transferred onto nitrocellulose membranes and probed with mouse anti-phosphotyrosine monoclonal IgG. Visualization was achieved using alkaline phosphatase conjugated goat anti-mouse IgG and 5-bromo-4-chloro-3-indole phosphate/nitro blue tetrazolin. Lane 2, phosphorylation pattern in the absence of genistein.
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were recovered as a white band at the top of 10% sucrose layer. Effect of mucins on 45Ca2+ uptake into vesicles
‘N 5I.7 o-
I
I
0
0.1
I
I
0.2
0.3
I
0.4
I 0.5
PN200-110 (@.l,A)
V
0
I 2
4
BAY
a a K8644 (~rM,o)
10
Fig. 2. Effect of PN200-110 and BAY K8644 on the uptake of 45Ca2+into vesicles containing the reconstituted buccal mucosal calcium channels. Uptake assays were conducted using vesicles preincubated for 20 min at room temperature with different concentrations of PN20&110 (o-0.8 PM) or BAY K8644 (&lOpM). Values represent the means + SD of five separate experiments performed in duplicate. tinin affinity chromatography (Liu et al., 1993). For SDSPAGE, the reaction mixture was treated with 170mM Tris-HCl buffer, pH 6.8, containing 10% SDS and 100 mM dithiothreitol, and heated at 100°C for 8 min. Samples were then run on a 7.5% gel. Following electrophoresis, the proteins were electrophoretically transferred onto 0.2 pm nitrocellulose membranes. Mouse-antiphosphotyrosine monoclonal IgG (10 pg/ml) was used as detecting antibody, and goat anti-mouse alkaline phosphatase conjugated IgG (0.1 pg/ml) as secondary antibody. Visualization was achieved using 5-bromo-4-chloro-3-indole phosphate and nitro blue tetrazolium (Bielanski et al., 1991). Reconstitution
of calcium channels
Liposomes were prepared by the method of Mimms et al. (1981). Purified [3H]PN20(rl 10 labeled buccal mucosal calcium channel samples in their intact form and following EGF-stimulated phosphorylation were solubilized with a buffer containing 20 mM CHAPS, 5 mM CaCl,, 50 mM Tris-HCl, pH 7.4, and 10% glycerol, and mixed with 1 ml of octylglucoside containing 1% egg yolk phosphatidylcholine. Detergent was removed by dialysis against buffered saline for 24 hr at 4°C thus yielding liposomes. For further purification, liposomes were suspended in 45% sucrose, overlaid with 2 ml of 30% sucrose and 1 ml of 10% sucrose, and then centrifuged at 4°C for 18 hr at 45,OOOg in a Beckman SW50 rotor (Sengupta et al., 1991). The liposomes containing [3H]PN200-1 10 labeled channel protein Table
The effect of salivary mucus glycoproteins on the 45Ca2+ the reconstituted, uptake into vesicles containing [3H]PN20(rl 10 labeled, buccal mucosal calcium channel was measured following vesicles (200 ~1) preincubation for 30 min at room temperature with different concentrations of the intact and modified mucin preparations (O-12Opg). The external divalent cations were removed on Sephadex G-SO column (Curtis and Catterall, 1986) the vesicles (100~1) were suspended in 0.34 M sucrose, 1OmM MOPS/tetramethylammonium, pH 7.0, and the calcium uptake was initiated by addition of 50 ~1 of 0.1 M CaCl, plus 2pCi of 45Ca2+ After 20min incubation at 37°C the reaction was terminated with 150mM MgCl, in 10 mM N- (2 - hydroxyethyl)piperazine - N’ - 2 - ethanesulfonic acid (HEPES)-Tris buffer, pH 7.4 and the samples were applied into GFjC Whatman filter (0.22 pm). Following filtration and washing with 5 mM HEPESTris buffer, pH 7.4, containing 0.3 M glucose and 5 mM lanthanum oxide, the filter was subjected to 45Ca2+measurement. Binding of 45Ca2f to the filter was determined by filtration of incubation media without vesicle protein (Ghishan et al., 1989). The effect of calcium channel receptor antagonist, PN20&110 and that of calcium channel activator, BAY K8644, on the 45Ca2t uptake was measured following vesicles preincubation at room temperature for 20 min with o-O.8 PM of PN200-110 or O-10 PM BAY K8644 (Slomiany el a1.,1992a). Analytical
methods
The protein content of salivary mucus glycoprotein preparations and that of buccal mucosal calcium channel samples was measured by the method of Lowry et al. (1951) and sulfate was determined turbidimetrically (Clarke and Denborough, 1971). The phenol-H,SO, method was used for monitoring carbohydrates (Dubois et al., 1956). The quantitative analysis of the content and composition of carbohydrates in various mucus glycoprotein fractions was carried out by gas-liquid chromatography after methanolysis, re-N-acetylation, and derivatization with silylating reagent (Slomiany et al., 1984, 1986, 1992b). Removal of the
I. Chemical salivary
composition of the low molecular weight human mucin and its neutral and acidic fractions
Mucin fraction Component (mg/lOO mg) Fucose Galactose N-Acetylgalactosamine N-Acetylglucosamine Sialic acid Sulfate Protein Each value represents
Intact mucin 12.3 24.9 11.8 17.6 3.9 3.5 21.8
+ k f f + f +
1.0 2.2 1.3 1.7 0.4 0.3 2.4
Neutral 12.5 + 25.6 k 12.7i 18.9& 0.3 f 0.2 f 23.2 +
3.2 2.4 1.3 1.8 0.2 0.1 2.5
the mean + SD of three analyses.
Acidic 10.9 23.5 11.3 16.1 8.1 6.7 20.1
+ I .2 f 2.5 + 1.2 + 1.6 ? 0.9 + 0.7 $- 2.4
Fig. 3. Effect of human salivary mucins on 45Ca2+uptake into vesicles containing the reconstituted buccal mucosal calcium channels. Uptake assays were conducted using vesicles preincubated for 30 min at room temperature with 0 or lOOpg/ml of intact low (A) and high (B) molecular weight mucus glycoproteins (I), and their neutral (::i:) and acidic (a) fractions. Control (m). Values represent the means f SD of five experiments performed in duplicate.
Calcium channel activity and salivary mucins
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Table 2. Chemical composition of the high molecular weight human salivary muck and its neutral and acidic fractions Mucin fraction Component (mg/lOOmg) FUUISZ Galactose N-Acetylgalactosamine N-Acetylghwsamine Sialic acid Sulfate Protein
Intact mucin
Neutral
13.6+ 1.1 26.8 f 2.3 13.7 f 1.0 23.4 f 1.8 3.8 + 0.4 3.6 IO.5 14.1 + 1.6
14.2 f 1.3 27. I + 2.6 14.5 * 1.5 25.3 f 2.0 0.3 + 0.1 0.3 IO.2 15.2 f 1.6
Acidic 13.0 f 24.2 + 12.6 + 22. I f 7.9 + 6.8 ; 13.2 +
1.2 2.1 1.2 2.2 0.9 0.8 1.5
Each value represents the mean + SD of three analyses. 1
A
a
C
D
E
Fig. 4. Effect of desulfation and sialic acid removal on salivary mucus glycoproteins inhibition of 45Ca2+ uptake into vesicles containing the reconstituted buccal mucosal calcium channels. Uptake assays were conducted using vesicles preincubated for 30 min at room temperature with
100pg of the low (8) and high @iI)molecular weight acidic mucus glycoprotein preparations. (A) control; (B) acidic mucin fraction; (C) acidic mucin following removal of sialic acid; (D) acidic mucin following desulfation; and (E) acidic mucin following desulfation and desialylation. Values represent the means &-SD of five experiments performed in duplicate.
sulfate ester groups from the glycoprotein preparations was accomplished by acid catalyzed solvolysis with 0.05 M HCl in dry methanol (Carter et al., 1988). The sialic acid was cleaved enzymatically with neuraminidase in 0.05 M sodium acetate buffer, pH 5.5 (Slomiany and Slomiany, 1978). All experiments were carried out in duplicate and the results are expressed as means f SD. Student’s r-test was used to determine significance, and P values of 0.05 or less were
considered significant.
the case of BAY K8644, maximal enhancement (58%) in 4sCa2+ uptake occurred at 6 PM (Fig. 2). To evaluate the effect of salivary mucins on 4sCa2+ uptake into vesicles containing the reconstituted calcium channels, the low and high molecular weight mucus glycoproteins were isolated from human saliva and subjected to fractionation into neutral and acidic fractions. The chemical composition of the low molecular weight mucus glycoprotein preparations is given in Table 1, and that of the high molecular weight mucus glycoprotein preparations in Table 2. In comparison to intact mucins, in both cases, the neutral glycoprotein fractions eluted from DEAESephacel with buffer alone were essentially free of sialic acid and sulfate, whereas the acidic glycoprotein fractions eluted with buffer containing 0.4 M NaCl were enriched 2-fold in sialic acid, and 1.9-fold in sulfate. Preincubation of the reconstituted buccal mucosal calcium channels with the intact high and low molecular weight mucins produced in each case about 15% inhibition in 4sCa2+ uptake, while the
RESULTS Epithelial cell membranes of buccal mucosa were used for the isolation of dihydropyridine-sensitive calcium channels. The solubilized membranes were prelabeled with [ 3H]PN20&110, a dihydropyridine calcium channel receptor antagonist, and purified by affinity chromatography on Sepharose-bound wheatgerm agglutinin. Elution of the column with medium buffer containing 300 mM N-acetylglucosamine yielded the [ 3H]PN20&l 10 labeled calcium channel protein complex which, following rechromatography, displayed on SDS-PAGE under reducing conditions four major protein bands migrating in the region of 55, 90, 130 and 170 kDa (Fig. 1). The functional performance of the isolated buccal mucosal channel preparation was assessed following incorporation of the channel protein into phosphatidylcholine vesicles. The reconstituted calcium channel complex responded in a concentration dependent manner to the addition of calcium channel antagonist, PN2W110, as well as to the specific calcium channel activator, BAY K8644 (Fig. 2). The maximal inhibitory effect was attained at 0.4pM PN200-110 which gave 71% decrease in calcium uptake, while in
n
1.6
0
20 MUCUS
40
60
60
100
130
glgcoprotein (ug/mt)
Fig. 5. Effect of the low and high molecular weight salivary mucus glycoproteins on the EGF binding to buccal mucosal calcium channels. Binding assays were conducted as described. in Materials and Methods, except that channel preparations prior to binding were preincubated at room temperature for 30 min with O-120 pg of the glycoprotein. Low molecular weight intact (A) and acidic (A) mucin. High molecular weight intact (0) and acidic (@) mucin. Values represent the means f of four experiments performed in duplicate.
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B. L. SLQMIANY et al.
R
a
C
D
Fig. 6. Effect of EGF-stimulated channel protein phosphorylation on ‘YZa2+uptake into vesicles containing the reconstituted buccal mucosal calcium channels. Pnrified channels were reconstituted into phospholipid vesicles either in their intact form (A) or following EGF-induced phosphorylation in the absence (B) and the presence (C,D) of tyrosine kinase inhibitor, genistein lOpg/ml (C) and 4O&ml (D). Values represent the means f SD of four experiments performed in duplicate.
corresponding neutral mucin fractions had no discernible effect at all (Fig. 3). However, the acidic low molecular weight mucin fraction at its optimal concentration (W-100 pg/ml) caused a 64% inhibition in 4sCa2+ uptake, and the high molecular weight acidic mucin fraction evoked about 60% inhibition at its optimal con~ntration of 8~l~~g/rnl (Fig. 3). Lanthanum displacement assays, furthermore, revealed, in both cases, that the acidic mucin fractions affected the intravesicular calcium level. Figure 4 illustrates the effect of sialic acid and sulfate ester group removal on the 4SCa2+uptake into vesicles containing the reconstituted buccal mucosal calcium channel. The enzymatic cleavage (> 95%) of sialic acid from the low and high molecular weight acidic mucus glycoprotein fractions caused a 21% decrease in the low molecular weight glycoprotein inhibitory effect on the 45Ca2C uptake, and a 17% decrease in the inhibitor effect occurred with the high molecular weight glycoprotein. Desulfation of the glycoproteins produced in the case of the low molecular weight mucin a 41% decrease in the inhibitory effect on 45Ca2+ uptake, while in the case of the high molecular weight acidic mucin, a 38% decrease in the inhibitory effect was observed. In both cases, nearly complete loss in the acidic glycoprotein inhibitory effect on the 45Ca2+ uptake occurred following the desialylation and desulfation. The data on the effect of salivary mucins on the EGF binding to buccal mucosal calcium channel preparations are presented in Fig. 5 and Table 3. The resuIts revealed that the low and high molecular weight neutral salivary mucin fractions over the concentration range (O-120 ,ug/ml) tested had virtually no effect on the receptor binding of EGF, and the intact mucins produced only S-7% decrease in the EGF binding. A significant reduction in the receptor
binding of EGF was, however, attained with the low and high molecular weight acidic mucin fractions. This inhibitory effect of the acidic low molecular weight mucin reached a value of 41.2% and that of the acidic high molecular weight mucin 3&i%, at the glycoproteins concentrations of 80-100 pg/rnl. Following removal of sialic acid, the inhibitory effect of both the low and high acidic mucus glycoproteins on the EGF binding decreased to about 28%, desulfation reduced the i~ibito~ eff& of the glycoproteins to 12-14%, while the desulfated and desiatyzed glycoproteins totally lost their inhibitory effect to the buccal mucosal receptor binding of EGF. The calcium uptake into vesicles containing the reconstituted intact and EGF-induced phosphoryIated buccal mucosal calcium channels is shown in Fig. 6. The results revealed that prein~ubation of the channel preparations with EGF resulted in phosphorylation of the channel proteins, particularly those in the region of 55 and 170 kDa (Fig. 7), and the vesicles containing the phosphorylated calcium channels exhibited a 46% greater 45Ca2f uptake. The calcium uptake was, however, inhibited by genistein, a known specific tyrosine kinase inhibitor (Akiyama et al., 1987), which also caused the inhibition in 55 and 170 kDa channel protein phosphorylation (Fig. 7). DISCUSSION
Salivary mucins are recognized as a major factor in the nonimmune defense of teeth and oral mucosa against mechanical, chemical and microbial insults (Mandel, 1982; Slomiany el al., 1986, 1989; Tabak, 1990). Until now, however, their role has been ascribed primarily to the preservation of oral mucosat integrity at the pre-epithelial level (Beachey, 1981; Slomiany et al., 1989; Tabak, 1990). Studies along these lines established that these large, highly glycosylated glycoproteins are responsible for the maintenance of viscoelastic, hydrophobic and lubricative properties of saliva, bacterial aggregation and clearance from the oral cavity, and formation of protective pellicle covering tooth enamel and oral mucosal surfaces (Beachey, 1981; Mandel, 1982; Rosan et a/., 1982; Slomiany et al., 1987, 1989). Furthermore, the Table 3. E&et of the low and high molecular weight salivary mu&s on EGF binding to buccai mucosal calcium channels
[ ‘2JI]EGFbound Type of preparation
Control Intact high mol. wt mucin Intact low mol. wt muck Neutral high mol. wt mucin fraction Acidic high mol. wt mucin fraction Neutral low mol. wt mu& fraction Acidic low mol. wt mucin fraction
(fmol/mg protein) 3.56 + 3.40 k 3.32 f 3.53 * 2.27 * 3.55 i 2.09 +
0.28 0.26 0.27 0.30 cl.21* 0.32 0.19*
Buccal mucosal calcium channel samples were preincubated at room temperature for 30min with 100 fig of intact and modified salivary mucin preparations. Each value represents the means k- SD of five experiments performed in duplicate. *P < 0.05
Calcium channel activity and salivary mucins
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acidic mucins were shown to modulate the enzymatic recordings (Peppelenbosch et al., 1991), the actiactivity of bacterial proteases and lipases and to vation of the EGF receptor triggers the receptorinterfere with bacterial colonization of the mucosa operated calcium channel response, and since it has (Hogg et al., 1981; Piotrowski et al., 1991; Slomiany been shown that phosphorylation of calcium channel and Slomiany, 1991). A decrease in acidic mucin protein triggered by EGF receptor activation encontent and a decline in sulfomucin synthesis have hances calcium uptake (Liu et uf., 1993; Slomiany been recognized for years as a signal for the onset of et al., 1992a,c), the effect of salivary mucus glycomany diseases affecting the alimentary tract. (Glass proteins on the phosphorylation event and the exand Slomiany, 1977; Murakami and Mori, 1984; pression of channel activity was investigated. The Kakei et al., 1984; Liau et al., 1992). results revealed that while the intact low and high The data obtained in the study presented herein molecular weight mucus glycoproteins had only negdemonstrate for the first time that the low and high ligible effect (5-7%) on the receptor binding of EGF, molecular weight acidic human salivary mucus glycothe corresponding acidic mucin fractions caused a proteins have the ability to affect directly the prosignificant reduction (41.2 and 36.1%) in the EGF cesses occurring within the epithelial cells of buccal binding. This effect of the low and high molecular mucosa, viz. the maintenance of intracellular calcium weight acidic glycoproteins apparently depends upon level. Using calcium channel complex, isolated from the presence in their carbohydrate chains of both buccal mucosal cell membranes and reconstituted sialic acid and sulfate ester groups, as the desulfated into phosphatidylcholine vesicles, it was found that and desialyzed glycoproteins totally lost the inhibisalivary mucins are capable of modulating the caltory effect on the buccal mucosal receptor EGF cium channel activity of soft oral tissue. Experiments binding. conducted with the low and high molecular weight The calcium channels on the EGF binding in the mucus glycoprotein, and their neutral and acidic presence of ATP responded by an increase in protein fractions revealed that the inhibitory effect of salivary tyrosine phosphorylation, reflected mainly in 55 and mucins on calcium uptake in buccal epithelium is 170 kDa proteins, and the vesicles containing such associated with the acidic glycoprotein fractions. phosphorylated channels showed about 46% higher While the intact mucins at their optimal concen45Ca2+ uptake. The uptake of calcium was, however, tration of 120/rg/ml caused only about 15% inhiinhibited by a known protein tyrosine kinase inhibibition in the uptake, and the neutral mucin fractions tor, genistein (Akiyama et al., 1987). This tyrosinespecific kinase inhibitor, furthermore, caused had virtually no inhibitory effect, the low and high molecular weight acidic mucin fractions at their inhibition in buccal mucosal 55 and 170 kDa channel optimal concentrations of 80pg/ml evoked 54 and protein phosphorylation. The finding that the low and high molecular weight 60% inhibition, respectively in the uptake of 45Ca2+ into the vesicles containing the reconstituted buccal acidic salivary mucins are capable of regulating the buccal mucosal calcium channel activity through the mucosal calcium channels. inhibition of EGF-stimulated calcium channel proThe demonstrated inhibitory effect of the low and tein tyrosine phosphorylation attests further to the high molecular weight acidic salivary mucus glycoimportance of salivary mucins in the regulation of proteins on buccal mucosal calcium channel activity functional expression of EGF in the oral cavity, a is apparently due to the presence of sialic acid process of paramount importance to the maintenance residues and sulfate ester groups on the carbohydrate of oral tissue integrity under normal physiological chains, as the removal of either sialic acid or sulfate conditions, and its repair following injury. caused partial loss in the glycoproteins inhibitory The schematic representation of the inhibitory effect. Furthermore, a complete loss in the inhibitory mechanism of acidic salivary mucus glycoproteins in effect on 4sCa2+ uptake occurred following desialylthe EGF-induced buccal mucosal calcium channel ation and desulfation of these glycoproteins. Based activation is depicted in Fig. 8. Based on the data on the results obtained with the desialyzed and presented in this study, the salivary mucus glycodesulfated low and high molecular weight glycoproteins, by modulating the EGF-controlled calcium proteins, it appears that sulfomucins exert about channel phosphorylation, appear to play a major role 3-fold greater inhibitory effect on calcium channel in oral mucosal calcium homeostasis. As unconactivity than the sialomucins. The regulatory effect on calcium channel activity has also been reported in trolled calcium influx is known to aggravate mucosal other systems for such acidic glycoconjugates as injury (Tamawski et al., 1990), our results provide for heparin and GM,-ganglioside (Knaus et al., 1990; the first time a direct evidence for the active participation of salivary mucins in the maintenance of the Slomiany et al., 1992a). Hence, the identified ability epithelial perimeter of oral mucosal defense. of acidic salivary mucins to modulate the buccal mucosal calcium channel activity could be viewed as yet another feature of the multifunctional role the REFERENCES salivary mucus glycoproteins play in the preservation Akiyama T., Ishida J., Nakagawa S., Ogawara H., Watanof oral mucosal integrity. abe S., Itoh N., Shibuya M. and Fukami Y. (1987) Since, as reported recently with patch clamp
Calcium channel activity and salivary mucins Genistein, a specific inhibitor of tyrosine-specific protein kinase. J. biol. Chem. 262, 5592-5595. Beachey E. H. (1981) Bacterial adherence: adhesin-receptor interactions mediating the attachment of bacteria to mucosal surfaces. J. Infect. Dis. 143, 325-345. Bielanski W. J., Keogh J. P., Wang S. L., Liu J., Konturek S. J., Slomiany A. and Slomiany B. L. (1991) Transforming growth factor-a binds to the epidermal growth factor receptor in gastric mucosa. Biochem. Int. 25, 419-427. Carter S. R., Slomiany A., Gwozdzinski K., Liau Y. H. and Slomiany B. L. (1988) Enzymatic sulfation of mucus glycoprotein in gastric mucosa. J. biol. Chem. 263, 11977-I 1984.
Clarke A. E. and Denborougb M. A. (1971) The interaction of concanavalin A with blood-group substance glycoproteins from human secretions. Biochem. J. 121, 811816. Curtis B. M. and Catterall W. A. (1986) Reconstitution of the voltage-sensitive calcium channel purified from skeletal muscle transverse tubules. Biochemistry 25, 3077-3083.
Dubois M., Gilles K. A., Hamilton J. K., Rebers P. A. and Smith F. (1956) Calorimetric method for determination of sugars and related substances. Analy?. Chem. 28, 350-356.
Ghishan F. K., Arab N. and Nylander W. (1989) Characterization of calcium uptake by brush border membrane vesicles of human small intestine. Gastroenrerology 96, 122-129. Glass G. B. J. and Slomiany B. L. (1977) Derangement of biosynthesis, production and secretion of mucus in gastrointestinal injury and disease. In: Mucus in Health and Disease (Edited by Elstein M. and Parke D. V.), pp. 322-247. Plenum Press, New York. Hogg S. D., Handley P. S. and Embery G. (1981) Surface fibrils may be responsible for the salivary glycoproteinmediated aggregation of the oral bacterium. Arch. Oral Biol. 26, 945-949.
Hosey M. M. and Lazdunski M. (1988) Calcium channels: pharmacology, structure, and regulation. J. membr. Biol. 104, 81-105. Kakei M., Ohara S., Ishihara K. and Hotta K. (1984) Sulfated glycoprotein biosynthesis in human gastric mucosal biopsies. Digestion 30, 59-64. Kass G. E. N., Llopis J., Chow S. C., Duddy S. K. and Orrenius S. (1990) Receptor-operated calcium influx in rat hepatocytes. J. biol. Chem. 265, 1748617492. Knaus H. G., Scheffauer F., Romanin C., Schindler M. G. and Glossmann H. (1990) Heparin binds with high alfinity to voltage-dependent L-type Ca2+ channels. J. biol. Chem. 265, 11156-l 1166. Liau Y. H., Slomiany A. and Slomiany B. L. (1992) Role of sulfation in posttranslational processing of gastric mucins. Inr. J. Biochem. 24, 1023-1028. Liu J., Fekete Z., Slomiany A. and Slomiany B. L. (1993) Activation of gastric mucosal calcium channels by epidermal growth factor. Inr. J. Biochem., 25, 29-35. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin-phenol reagent. J. biol. Chem. 193, 265-275. Mandel I. D. (1982) The functions of saliva. J. Dent. Res. 66, 623-635.
Mimms L. T., Zampighi G., Nozaki Y., Tanford C. and Reynolds J. A. (1981) Phospholipid vesicle formation
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and transmembrane protein incorporation using octylglucoside. Biochemistry 20, 833-840. Murakami S. and Mori Y. (1984) Changes in the incorporating activity of 35S-sulfate into gastric sulfated glycoproteins in the rat with erosion by restraint and water immersion. Jup. J. Phurmuc. 35, 279-286. Peppelenbosch M. P., Tertoden L. G. F. and deLaat S. W. (1991) Epidermal growth factor-activated calcium and potassium channels. J. biol. Chem. 266, 19938-19944. Piotrowski J., Slomiany A., Murty V. L. N., Fekete Z. and Slomiany B. L. (1991) Inhibition of H. pylori colonization by sulfated gastric mucin. Biochem. Int. 24, 749-756.
Rosan B., Appelbaum B., Golub E., Malamud D. and Mandel I. D. (1982) Enhanced saliva-mediated bacterial aggregation and decreased bacterial adhesion in cariesresistant versus caries-susceptible individuals. Infect. Immun. 38, 1056-1059. Sengupta S., Shovlin F. E., Slomiany A. and Slomiany B. L. (1991) Identification of laminin receptor in gingival tissue and its interaction with tooth cementum laminin. Int. J. Biochem. 23, 115-121.
Slomiany A. and Slomiany B. L. (1978) Structures of the acidic oligosaccharides isolated from rat sublingual glycoprotein. J. biol. Chem. 253, 7301-7306. Slomiany A., Zdebska E. and Slomiany B. L. (1984) Structures of the neutral oligosaccharides isolated from A-active human gastric mucin. J. biol. Chem. 259, 14743-14749. Slomiany A., Tamura S., Grzelinska E., Piotrowski J. and Slomiany B. L. (1992b) Mucin complexes: characterization of the “link” component of submandibular mucus glycoprotein. Inr. J. Biochem. 24, 1003-1015. Slomiany B. L. and Slomiany A. (1991) Role of mucus in gastric mucosal protection. J. Physiol. Phurmuc. 42, 149-163.
Slomiany B. L., Liu J., Yao P. and Slomiany A. (1992a) GM,-ganglioside regulation of EGF-induced gastric mucosal calcium channel activation. Gen. Phurmac. 23, 799-803.
Slomiany B. L., Fekete Z., Liu J., Murty V. L. N. and Slomiany A. (1992~) Activation of dihydropyridine-sensitive parotid salivary gland calcium channels by epidermal growth factor. Arch. Oral Biol. 37, 863-868. Slomiany B. L., Gwozdzinski K., Murty V. L. N., Slomiany A. and Mandel I. D. (1987) Buoyant density and viscosity behavior of human salivary mucins. Ann. N. Y. Acud. Sci. 494, 266-269.
Slomiany B. L., Murty V. L. N., Mandel 1. D., Zalesna G. and Slomiany A. (1989) Physicochemical characteristics of mucus glycoproteins and lipids of the human oral mucosal mucus coat. Archs Orul Biol. 34, 229-237. Slomiany B. L., Murty V. L. N., Slomiany A., Zielenski J. and Mandel I. D. (1986) Mucus glycoprotein of human saliva: differences in the associated and covalently bound lipids with caries. Biochim. biophys. Actu 882, 18-28. Tabak L. A. (1990) Structure and function of human salivary mucins. Oral biol. Med. 1, 229-234. Tamawski A., Brzozowski T., Gergely H., Sekkon S. and Krause W. J. (1990) The role of calcium in injury of isolated gastric gland cells. Acru Physiol. PO!. 41, 84-85.
Wang S. L., Jacober L., Wu-Wang C. Y., Slomiany A. and Slomiany B. L. (1992) Ethanol-induced structural and functional alterations of epidermal growth factor receptor in buccal mucosa. Inr. J. Biochem. 24, 85-90.
0020-711X/93 $6.00 + 0.00 Copyright 0 1993 Pergamon Press Ltd
REGULATION OF BUCCAL MUCOSAL CALCIUM CHANNEL ACTIVITY BY SALIVARY MUCINS B. L. SLOMIANY,* Z. FEICETE,V. L. N. MURTY, M. GRABSKA, J. PIOTROWSKI,F. YOTWMOTO, A. CZAJKOWSKIand A. SLOMIANY Research Center, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, University Heights, 110 Bergen Street, Newark, NJ 07103-2400, U.S.A. [Tel. (201) 982-70521 (Received
3 March 1993)
Abstract-l. The effect salivary mucins on the activity of calcium channel isolated from buccal mucosal cell membranes was investigated. The uptake of 45CaZ+while only moderately (15%) affected by the intact low and high molecular weight mucin forms, was significantly inhibited, by the acidic low and high molecular weight salivary mu&s which evoked 64 and 60% inhibition, respectively. 2. The inhibitory effect of salivary mucins was associated with the sialic acid and sulfate ester groups of the carbohydrate chains, as the removal of either group caused partial loss in the glycoproteins inhibition, and the complete loss in the inhibitory effect occurred following desialylation and desulfation. 3. The channel in the presence of epidermal growth factor (EGF) and ATP responded by an increase in tyrosine phosphorylation of 55 and 170 kDa proteins, and the phosphorylated channels showed a 46% increase in “Ca*+ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein. 4. The binding of EGF to calcium channel receptor protein was inhibited by the low and high molecular weight acidic mucins, causing 41.2 and 36.1% reduction, respectively. This reduction in binding was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoproteins’ inhibitory capacity following removal of these groups. 5. The results for the first time demonstrate that salivary mucins actively participate in the modulation of the EGF-controlled buccal mucosal calcium channel activity expression, a process of importance to the preservation of oral tissue integrity.
INTRODUCIXON Although the importance of salivary mucins in the nonimmune defense of teeth against caries is well documented (Mandel, 1982; Slomiany et al., 1986; Tabak, 1990), considerably less is known about the role these glycoproteins play in the preservation of oral mucosal integrity. The available data, however, indicate that salivary mucins enter into a tenacious interaction with oral epithelial surfaces forming protective coating known as oral mucosal mucus coat (Slomiany et al., 1989). This coat constitutes the pre-epithelial element of oral mucosal defense by acting not only as passive physical obstacle, but also as a dynamic functional barrier capable of modulating the untoward effects of oral environment, including bacterial colonization. Salivary mucins of oral mucosal mucus coat, remaining in intimate association with the epithelial cell surfaces in the oral cavity, could also conceivably play an important role in affecting the processes occurring within the epithelial perimeter of oral mucosal defense. Primary among these processes is the maintenance of intracellular calcium level. Studies indicate that calcium is an important regulatory *To whom correspondence should he addressed.
element for many cellular functions, including contraction, cell differentiation and secretion, and that its influx potentiates mucosal injury caused by a variety of noxious agents (Hosey and Lazdunski, 1988; Kass et al., 1990; Tamawski et al., 1990). The calcium entry in most excitatory and secretory cells occurs through a carefully controlled process involving specific voltage or receptor-dependent channels (Hosey and Lazdunski, 1988; Kass et al., 1990; Liu et al., 1993). Recently, we demonstrated the presence in mucosal epithelial tissues of receptor-dependent calcium channels and have shown that epidermal growth factor (EGF), through stimulation of channel protein phosphorylation, enhances the calcium uptake (Liu et al., 1993; Slomiany et al., 1992a,c). In this report, we provide evidence that the low and high molecular weight acidic salivary mucus glycoproteins are actively involved in the modulation of the EGFinduced calcium channel activity in buccal mucosa.
MATERIALSAND METHODS Materials
Human submandibular/sublingual saliva used for mucus glycoprotein isolation was obtained from five adult 1281
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B. L. SLoMIANYer al.
secretory with blood-group B. The salivary flow was stimulated with 2% citric acid and 25530mg of saliva was collected from each individual per session (Slomiany er al., 1986). Immediately following collection, the samples were treated with buffered EDTA (PH 7.0), dialyzed against distilled water and lyophilized (Slomiany ef al., 1986). Male Sprague-Dawley rats weighing 180-200 g were obtained from Taconic Farms Inc., Germantown, NY, ([4sCa]CaCl,, ( +)-[methyl-3H]PN200-l 10) from New England Nuclear, Boston, MA and [‘251]EGF from Amersham Corp., Arlington Heights, IL. BAY K8644, egg-yolk phosphatidylcholine, V. cholerae neuraminidase, bovine serum albumin, and mouse epidermal growth factor (EGF) were supplied by Sigma, St. Louis, MO, PN200-110 was a generous gift of Dr Houlihan, Sandoz Research Institute, E. Hanover, NJ, and Whatman filter GF/C (0.22 FM) was from Whatman International Ltd., Maidstone, U.K. Wheat germ agglutininSepharose, DEAE-Sephacel and Sephadex G-50 were obtained from Pharmacia, Piscataway, NJ, Chelex 100 (SO-100 mesh), and reagents for sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and BioGel A-50 (Xl-lOOmesh) were purchased from Bio-Rad, Rockville Centre, NY. 5-Bromo-4-chloro-3-indole phosphate and nitro blue tetrazolium were from Oncogene Science Inc., Manhasset, NY. Goat anti-mouse alkaline phosphatase conjugated IgG and O-phospho-l-tyrosine were obtained from Boehringer Mannheim Biochemicals, Indianapolis, IN, and tyrosine kinase inhibitor, genistein and anti-phosphotyrosine monoclonal IgG from Upstate Biotechnology In., Lake Placid, NY. Calcium channel bolation
Calcium channels were isolated form rat buccal mucosal cell membranes. The animals were anesthesized by i.p. injection with 25% urethane (1 ml/lOOg body wt), and buccal mucosal cells were collected by scraping the mucosa with a blunt spatula. Scrapings were placed in ice-cold buffer (2.5 mM Tri-HCI, pH 7.0, 250mM sucrose, 2.5 mM EDTA, 1 mM phenyl methyl sulfonyl fluoride (PMSF), lOOTIU/ml aprotinin and 1 pg/ml leupeptin), and homogenized for 1 min in a Polytron tissuemizer. The homogenate was centrifuged at 400g for 15 min at 4°C and NaCI/MgSO, was added to the supematant to form final concentrations of 0.1 mM and 0.2 mM respectively (Wang et al., 1992). After centrifugation of the supematant at 40,OOOg for 1 hr at 4°C the membrane pellet was resuspended in 0.1 M sodium phosphate buffer, pH 7.2. The preparation was centrifuged for 20min at 10,OOOg at 4”C, and the pellet solubilized using a buffer containing 40 mM Tri-HCl, pH 7.4, 0.5 M NaCl, 20 mM 3-[[fsl] (3-cholamidopropyl)dimethylammonio]1-propanesulfonate (CHAPS), 10% glycerol, 108 TIU/ml aprotinin, 1 pg/ml leupeptin, and 1 mM PMSF. After 1 hr at 4”C, the mixture was centrifuged at 105,OOOg for 1 hr and the resulting supematant collected, 1 nM of [3H]PN200-l10, a dihydropyridine calcium channel receptor antagonist was added and incubated at 4°C for 30min. The preparation was then applied to a column of Sepharose-bound wheat germ agglutinin, and the [‘H]PN200-110 labeled calcium channel protein was eluted with buffer containing 300 mM N-acetylglucosamine (Liu ef al., 1993). Salivary mucins preparation The lyophilized samples were dissolved at 5 mg/ml in 6 M urea/lOmM sodium phosphate buffer (PH 7.0) and chromatographed on a Bio-Gel P-100 column (Slomiany et al.,
1986). The eluted fractions were monitored for protein and carbohydrate, and the excluded mucus glycoprotein peak collected. After dialysis and lyophilization, the crude glycoprotein was delipidated by washing with chloroformmethanol (2: 1) followed by acetone, dried, dissolved in 6 M urea/O. 1 M acetate buffer, pH 4.2, and chromatographed on a Bio-Gel A-50 column, and the low and the high molecular weight mucus glycoprotein peaks collected (Slomiany et al., 1992b). After rechromatography, each glycoprotein form was subjected to equilibrium density gradient centrifugation in 42% (w/w) CsCl for 48 hr at 12°C and 45,000 rev/min in a Beckman 50Ti rotor (Slomiany ef al., 1986). The resultant gradient was fractionated into 1 ml fractions using a Beckman gradient recovery system, and the fractions containing mucus glycoprotein were pooled, dialyzed against distilled water and lyophilized. Such prepared low and high molecular weight salivary mucin forms were then used for the isolation of neutral and acidic mucus glycoprotein fractions. For this, each glycoprotein form was dissolved in 0.05 M Tris-HCl buffer, pH 6.8, containing 1 mM EDTA and applied to a DEAE-Sephacel column (Piotrowski et al., 1991). The glycoprotein fractions were eluted in a stepwise manner first with the above buffer (neutral mucus glycoprotein fraction) followed by buffer containing 0.2 M NaCl and 0.4 M NaCl (acidic mucus glycoprotein fraction). The neutral and acidic mucus glycoprotein fractions derived from the low and high molecular weight salivary mucin forms were collected, dialyzed against distilled water. and lyophilized. Effect of mucins on EGF binding
The effect of intact and modified salivary mucus glycoprotein preparations on the binding of EGF was assessed following preincubation of 200 ~1 aliquots of calcium channel preparation (250 pg protein/assay) with the glycoprotein sample (O-120 pg) at room temperature for 30 min. EGF binding assays were carried out by incubating the channel complex samples (200 pg protein/assay) with [ ‘2SI]EGF (0.18 nM) and 5mM Tris/HCI (pH 7.0),125mM sucrose, 75 mM NaCl, 0.5 mM CaCl,, 0.5% bovine serum albumin (BSA) in a final volume of 200 ~1 (Wang er al., 1992). Unlabeled EGF was added to the incubations to form a final concentration of 0.25 PM in the experiments where nonspecific binding was being estimated. Incubates were maintained for 1hr at room temperature, and then stopped by addition of 1 mM ice-cold buffer containing 10mM Tris/HCI (PH 7.0) and 0.5% BSA. Membrane-bound [ 12sI]EGF was separated from the unbound [ “‘I]EGF by centrifugation at lO,O@Ogfor 10 min at 4°C. The pellet was washed with 1 ml of ice-cold buffer, centrifuged, and counted in a gamma counter. Effect of mucins phosphorylation
on
EGF-stimulated
channel
protein
The solubilized buccal mucosal calcium channel preparations containing 200-25Opg protein were incubated at room temperature for 30 min in 50mM HEPES buffer, pH 7.6, containing 10 mM MgSO, and 1 mM PMSF, with 0 and 100 pg mucus glycoprotein or &40 pg/ml genistein, and 2 PM EGF. The phosphorylation was then initiated by the addition of a solution containing 1OpM ATP, 1 mM CTP, 8 mM MnCls, 20mM MgCl,, and 2 mM sodium vanadate (Slomiany et al., 1992a,c). Incubations were maintained for 10min at 4°C after which the calcium channel preparation was recovered using wheat germ agglu-
Calcium channel activity and salivary mucins
kDa
Fig. 1. SDS-PAGE of [3H]PN20&l 10 labeled buccal mucosal calcium channels purified from solubilized buccal mucosal cell membranes by affinity chromatography on Sepharose-bound wheat germ agglutinin. Pattern under reducing conditions visualized by silver stain.
kDa
1
2
Fig. 7. Effect of tyrosine kinase inhibitor, genistein, on EGF-stimulated buccal mucosal calcium channel protein phosphorylation. The channel preparations following 30 min preincubation with 30 pg/ml genistein (Lane 1) were incubated for 30min with 2pM EGF and then ATP (40pM) was added, and the reaction was stopped after 20 min. After SDS-PAGE, the proteins were transferred onto nitrocellulose membranes and probed with mouse anti-phosphotyrosine monoclonal IgG. Visualization was achieved using alkaline phosphatase conjugated goat anti-mouse IgG and 5-bromo-4-chloro-3-indole phosphate/nitro blue tetrazolin. Lane 2, phosphorylation pattern in the absence of genistein.
1283
B. L.
1284
SLOMIANY et al.
were recovered as a white band at the top of 10% sucrose layer. Effect of mucins on 45Ca2+ uptake into vesicles
‘N 5I.7 o-
I
I
0
0.1
I
I
0.2
0.3
I
0.4
I 0.5
PN200-110 (@.l,A)
V
0
I 2
4
BAY
a a K8644 (~rM,o)
10
Fig. 2. Effect of PN200-110 and BAY K8644 on the uptake of 45Ca2+into vesicles containing the reconstituted buccal mucosal calcium channels. Uptake assays were conducted using vesicles preincubated for 20 min at room temperature with different concentrations of PN20&110 (o-0.8 PM) or BAY K8644 (&lOpM). Values represent the means + SD of five separate experiments performed in duplicate. tinin affinity chromatography (Liu et al., 1993). For SDSPAGE, the reaction mixture was treated with 170mM Tris-HCl buffer, pH 6.8, containing 10% SDS and 100 mM dithiothreitol, and heated at 100°C for 8 min. Samples were then run on a 7.5% gel. Following electrophoresis, the proteins were electrophoretically transferred onto 0.2 pm nitrocellulose membranes. Mouse-antiphosphotyrosine monoclonal IgG (10 pg/ml) was used as detecting antibody, and goat anti-mouse alkaline phosphatase conjugated IgG (0.1 pg/ml) as secondary antibody. Visualization was achieved using 5-bromo-4-chloro-3-indole phosphate and nitro blue tetrazolium (Bielanski et al., 1991). Reconstitution
of calcium channels
Liposomes were prepared by the method of Mimms et al. (1981). Purified [3H]PN20(rl 10 labeled buccal mucosal calcium channel samples in their intact form and following EGF-stimulated phosphorylation were solubilized with a buffer containing 20 mM CHAPS, 5 mM CaCl,, 50 mM Tris-HCl, pH 7.4, and 10% glycerol, and mixed with 1 ml of octylglucoside containing 1% egg yolk phosphatidylcholine. Detergent was removed by dialysis against buffered saline for 24 hr at 4°C thus yielding liposomes. For further purification, liposomes were suspended in 45% sucrose, overlaid with 2 ml of 30% sucrose and 1 ml of 10% sucrose, and then centrifuged at 4°C for 18 hr at 45,OOOg in a Beckman SW50 rotor (Sengupta et al., 1991). The liposomes containing [3H]PN200-1 10 labeled channel protein Table
The effect of salivary mucus glycoproteins on the 45Ca2+ the reconstituted, uptake into vesicles containing [3H]PN20(rl 10 labeled, buccal mucosal calcium channel was measured following vesicles (200 ~1) preincubation for 30 min at room temperature with different concentrations of the intact and modified mucin preparations (O-12Opg). The external divalent cations were removed on Sephadex G-SO column (Curtis and Catterall, 1986) the vesicles (100~1) were suspended in 0.34 M sucrose, 1OmM MOPS/tetramethylammonium, pH 7.0, and the calcium uptake was initiated by addition of 50 ~1 of 0.1 M CaCl, plus 2pCi of 45Ca2+ After 20min incubation at 37°C the reaction was terminated with 150mM MgCl, in 10 mM N- (2 - hydroxyethyl)piperazine - N’ - 2 - ethanesulfonic acid (HEPES)-Tris buffer, pH 7.4 and the samples were applied into GFjC Whatman filter (0.22 pm). Following filtration and washing with 5 mM HEPESTris buffer, pH 7.4, containing 0.3 M glucose and 5 mM lanthanum oxide, the filter was subjected to 45Ca2+measurement. Binding of 45Ca2f to the filter was determined by filtration of incubation media without vesicle protein (Ghishan et al., 1989). The effect of calcium channel receptor antagonist, PN20&110 and that of calcium channel activator, BAY K8644, on the 45Ca2t uptake was measured following vesicles preincubation at room temperature for 20 min with o-O.8 PM of PN200-110 or O-10 PM BAY K8644 (Slomiany el a1.,1992a). Analytical
methods
The protein content of salivary mucus glycoprotein preparations and that of buccal mucosal calcium channel samples was measured by the method of Lowry et al. (1951) and sulfate was determined turbidimetrically (Clarke and Denborough, 1971). The phenol-H,SO, method was used for monitoring carbohydrates (Dubois et al., 1956). The quantitative analysis of the content and composition of carbohydrates in various mucus glycoprotein fractions was carried out by gas-liquid chromatography after methanolysis, re-N-acetylation, and derivatization with silylating reagent (Slomiany et al., 1984, 1986, 1992b). Removal of the
I. Chemical salivary
composition of the low molecular weight human mucin and its neutral and acidic fractions
Mucin fraction Component (mg/lOO mg) Fucose Galactose N-Acetylgalactosamine N-Acetylglucosamine Sialic acid Sulfate Protein Each value represents
Intact mucin 12.3 24.9 11.8 17.6 3.9 3.5 21.8
+ k f f + f +
1.0 2.2 1.3 1.7 0.4 0.3 2.4
Neutral 12.5 + 25.6 k 12.7i 18.9& 0.3 f 0.2 f 23.2 +
3.2 2.4 1.3 1.8 0.2 0.1 2.5
the mean + SD of three analyses.
Acidic 10.9 23.5 11.3 16.1 8.1 6.7 20.1
+ I .2 f 2.5 + 1.2 + 1.6 ? 0.9 + 0.7 $- 2.4
Fig. 3. Effect of human salivary mucins on 45Ca2+uptake into vesicles containing the reconstituted buccal mucosal calcium channels. Uptake assays were conducted using vesicles preincubated for 30 min at room temperature with 0 or lOOpg/ml of intact low (A) and high (B) molecular weight mucus glycoproteins (I), and their neutral (::i:) and acidic (a) fractions. Control (m). Values represent the means f SD of five experiments performed in duplicate.
Calcium channel activity and salivary mucins
1285
Table 2. Chemical composition of the high molecular weight human salivary muck and its neutral and acidic fractions Mucin fraction Component (mg/lOOmg) FUUISZ Galactose N-Acetylgalactosamine N-Acetylghwsamine Sialic acid Sulfate Protein
Intact mucin
Neutral
13.6+ 1.1 26.8 f 2.3 13.7 f 1.0 23.4 f 1.8 3.8 + 0.4 3.6 IO.5 14.1 + 1.6
14.2 f 1.3 27. I + 2.6 14.5 * 1.5 25.3 f 2.0 0.3 + 0.1 0.3 IO.2 15.2 f 1.6
Acidic 13.0 f 24.2 + 12.6 + 22. I f 7.9 + 6.8 ; 13.2 +
1.2 2.1 1.2 2.2 0.9 0.8 1.5
Each value represents the mean + SD of three analyses. 1
A
a
C
D
E
Fig. 4. Effect of desulfation and sialic acid removal on salivary mucus glycoproteins inhibition of 45Ca2+ uptake into vesicles containing the reconstituted buccal mucosal calcium channels. Uptake assays were conducted using vesicles preincubated for 30 min at room temperature with
100pg of the low (8) and high @iI)molecular weight acidic mucus glycoprotein preparations. (A) control; (B) acidic mucin fraction; (C) acidic mucin following removal of sialic acid; (D) acidic mucin following desulfation; and (E) acidic mucin following desulfation and desialylation. Values represent the means &-SD of five experiments performed in duplicate.
sulfate ester groups from the glycoprotein preparations was accomplished by acid catalyzed solvolysis with 0.05 M HCl in dry methanol (Carter et al., 1988). The sialic acid was cleaved enzymatically with neuraminidase in 0.05 M sodium acetate buffer, pH 5.5 (Slomiany and Slomiany, 1978). All experiments were carried out in duplicate and the results are expressed as means f SD. Student’s r-test was used to determine significance, and P values of 0.05 or less were
considered significant.
the case of BAY K8644, maximal enhancement (58%) in 4sCa2+ uptake occurred at 6 PM (Fig. 2). To evaluate the effect of salivary mucins on 4sCa2+ uptake into vesicles containing the reconstituted calcium channels, the low and high molecular weight mucus glycoproteins were isolated from human saliva and subjected to fractionation into neutral and acidic fractions. The chemical composition of the low molecular weight mucus glycoprotein preparations is given in Table 1, and that of the high molecular weight mucus glycoprotein preparations in Table 2. In comparison to intact mucins, in both cases, the neutral glycoprotein fractions eluted from DEAESephacel with buffer alone were essentially free of sialic acid and sulfate, whereas the acidic glycoprotein fractions eluted with buffer containing 0.4 M NaCl were enriched 2-fold in sialic acid, and 1.9-fold in sulfate. Preincubation of the reconstituted buccal mucosal calcium channels with the intact high and low molecular weight mucins produced in each case about 15% inhibition in 4sCa2+ uptake, while the
RESULTS Epithelial cell membranes of buccal mucosa were used for the isolation of dihydropyridine-sensitive calcium channels. The solubilized membranes were prelabeled with [ 3H]PN20&110, a dihydropyridine calcium channel receptor antagonist, and purified by affinity chromatography on Sepharose-bound wheatgerm agglutinin. Elution of the column with medium buffer containing 300 mM N-acetylglucosamine yielded the [ 3H]PN20&l 10 labeled calcium channel protein complex which, following rechromatography, displayed on SDS-PAGE under reducing conditions four major protein bands migrating in the region of 55, 90, 130 and 170 kDa (Fig. 1). The functional performance of the isolated buccal mucosal channel preparation was assessed following incorporation of the channel protein into phosphatidylcholine vesicles. The reconstituted calcium channel complex responded in a concentration dependent manner to the addition of calcium channel antagonist, PN2W110, as well as to the specific calcium channel activator, BAY K8644 (Fig. 2). The maximal inhibitory effect was attained at 0.4pM PN200-110 which gave 71% decrease in calcium uptake, while in
n
1.6
0
20 MUCUS
40
60
60
100
130
glgcoprotein (ug/mt)
Fig. 5. Effect of the low and high molecular weight salivary mucus glycoproteins on the EGF binding to buccal mucosal calcium channels. Binding assays were conducted as described. in Materials and Methods, except that channel preparations prior to binding were preincubated at room temperature for 30 min with O-120 pg of the glycoprotein. Low molecular weight intact (A) and acidic (A) mucin. High molecular weight intact (0) and acidic (@) mucin. Values represent the means f of four experiments performed in duplicate.
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R
a
C
D
Fig. 6. Effect of EGF-stimulated channel protein phosphorylation on ‘YZa2+uptake into vesicles containing the reconstituted buccal mucosal calcium channels. Pnrified channels were reconstituted into phospholipid vesicles either in their intact form (A) or following EGF-induced phosphorylation in the absence (B) and the presence (C,D) of tyrosine kinase inhibitor, genistein lOpg/ml (C) and 4O&ml (D). Values represent the means f SD of four experiments performed in duplicate.
corresponding neutral mucin fractions had no discernible effect at all (Fig. 3). However, the acidic low molecular weight mucin fraction at its optimal concentration (W-100 pg/ml) caused a 64% inhibition in 4sCa2+ uptake, and the high molecular weight acidic mucin fraction evoked about 60% inhibition at its optimal con~ntration of 8~l~~g/rnl (Fig. 3). Lanthanum displacement assays, furthermore, revealed, in both cases, that the acidic mucin fractions affected the intravesicular calcium level. Figure 4 illustrates the effect of sialic acid and sulfate ester group removal on the 4SCa2+uptake into vesicles containing the reconstituted buccal mucosal calcium channel. The enzymatic cleavage (> 95%) of sialic acid from the low and high molecular weight acidic mucus glycoprotein fractions caused a 21% decrease in the low molecular weight glycoprotein inhibitory effect on the 45Ca2C uptake, and a 17% decrease in the inhibitor effect occurred with the high molecular weight glycoprotein. Desulfation of the glycoproteins produced in the case of the low molecular weight mucin a 41% decrease in the inhibitory effect on 45Ca2+ uptake, while in the case of the high molecular weight acidic mucin, a 38% decrease in the inhibitory effect was observed. In both cases, nearly complete loss in the acidic glycoprotein inhibitory effect on the 45Ca2+ uptake occurred following the desialylation and desulfation. The data on the effect of salivary mucins on the EGF binding to buccal mucosal calcium channel preparations are presented in Fig. 5 and Table 3. The resuIts revealed that the low and high molecular weight neutral salivary mucin fractions over the concentration range (O-120 ,ug/ml) tested had virtually no effect on the receptor binding of EGF, and the intact mucins produced only S-7% decrease in the EGF binding. A significant reduction in the receptor
binding of EGF was, however, attained with the low and high molecular weight acidic mucin fractions. This inhibitory effect of the acidic low molecular weight mucin reached a value of 41.2% and that of the acidic high molecular weight mucin 3&i%, at the glycoproteins concentrations of 80-100 pg/rnl. Following removal of sialic acid, the inhibitory effect of both the low and high acidic mucus glycoproteins on the EGF binding decreased to about 28%, desulfation reduced the i~ibito~ eff& of the glycoproteins to 12-14%, while the desulfated and desiatyzed glycoproteins totally lost their inhibitory effect to the buccal mucosal receptor binding of EGF. The calcium uptake into vesicles containing the reconstituted intact and EGF-induced phosphoryIated buccal mucosal calcium channels is shown in Fig. 6. The results revealed that prein~ubation of the channel preparations with EGF resulted in phosphorylation of the channel proteins, particularly those in the region of 55 and 170 kDa (Fig. 7), and the vesicles containing the phosphorylated calcium channels exhibited a 46% greater 45Ca2f uptake. The calcium uptake was, however, inhibited by genistein, a known specific tyrosine kinase inhibitor (Akiyama et al., 1987), which also caused the inhibition in 55 and 170 kDa channel protein phosphorylation (Fig. 7). DISCUSSION
Salivary mucins are recognized as a major factor in the nonimmune defense of teeth and oral mucosa against mechanical, chemical and microbial insults (Mandel, 1982; Slomiany el al., 1986, 1989; Tabak, 1990). Until now, however, their role has been ascribed primarily to the preservation of oral mucosat integrity at the pre-epithelial level (Beachey, 1981; Slomiany et al., 1989; Tabak, 1990). Studies along these lines established that these large, highly glycosylated glycoproteins are responsible for the maintenance of viscoelastic, hydrophobic and lubricative properties of saliva, bacterial aggregation and clearance from the oral cavity, and formation of protective pellicle covering tooth enamel and oral mucosal surfaces (Beachey, 1981; Mandel, 1982; Rosan et a/., 1982; Slomiany et al., 1987, 1989). Furthermore, the Table 3. E&et of the low and high molecular weight salivary mu&s on EGF binding to buccai mucosal calcium channels
[ ‘2JI]EGFbound Type of preparation
Control Intact high mol. wt mucin Intact low mol. wt muck Neutral high mol. wt mucin fraction Acidic high mol. wt mucin fraction Neutral low mol. wt mu& fraction Acidic low mol. wt mucin fraction
(fmol/mg protein) 3.56 + 3.40 k 3.32 f 3.53 * 2.27 * 3.55 i 2.09 +
0.28 0.26 0.27 0.30 cl.21* 0.32 0.19*
Buccal mucosal calcium channel samples were preincubated at room temperature for 30min with 100 fig of intact and modified salivary mucin preparations. Each value represents the means k- SD of five experiments performed in duplicate. *P < 0.05
Calcium channel activity and salivary mucins
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acidic mucins were shown to modulate the enzymatic recordings (Peppelenbosch et al., 1991), the actiactivity of bacterial proteases and lipases and to vation of the EGF receptor triggers the receptorinterfere with bacterial colonization of the mucosa operated calcium channel response, and since it has (Hogg et al., 1981; Piotrowski et al., 1991; Slomiany been shown that phosphorylation of calcium channel and Slomiany, 1991). A decrease in acidic mucin protein triggered by EGF receptor activation encontent and a decline in sulfomucin synthesis have hances calcium uptake (Liu et uf., 1993; Slomiany been recognized for years as a signal for the onset of et al., 1992a,c), the effect of salivary mucus glycomany diseases affecting the alimentary tract. (Glass proteins on the phosphorylation event and the exand Slomiany, 1977; Murakami and Mori, 1984; pression of channel activity was investigated. The Kakei et al., 1984; Liau et al., 1992). results revealed that while the intact low and high The data obtained in the study presented herein molecular weight mucus glycoproteins had only negdemonstrate for the first time that the low and high ligible effect (5-7%) on the receptor binding of EGF, molecular weight acidic human salivary mucus glycothe corresponding acidic mucin fractions caused a proteins have the ability to affect directly the prosignificant reduction (41.2 and 36.1%) in the EGF cesses occurring within the epithelial cells of buccal binding. This effect of the low and high molecular mucosa, viz. the maintenance of intracellular calcium weight acidic glycoproteins apparently depends upon level. Using calcium channel complex, isolated from the presence in their carbohydrate chains of both buccal mucosal cell membranes and reconstituted sialic acid and sulfate ester groups, as the desulfated into phosphatidylcholine vesicles, it was found that and desialyzed glycoproteins totally lost the inhibisalivary mucins are capable of modulating the caltory effect on the buccal mucosal receptor EGF cium channel activity of soft oral tissue. Experiments binding. conducted with the low and high molecular weight The calcium channels on the EGF binding in the mucus glycoprotein, and their neutral and acidic presence of ATP responded by an increase in protein fractions revealed that the inhibitory effect of salivary tyrosine phosphorylation, reflected mainly in 55 and mucins on calcium uptake in buccal epithelium is 170 kDa proteins, and the vesicles containing such associated with the acidic glycoprotein fractions. phosphorylated channels showed about 46% higher While the intact mucins at their optimal concen45Ca2+ uptake. The uptake of calcium was, however, tration of 120/rg/ml caused only about 15% inhiinhibited by a known protein tyrosine kinase inhibibition in the uptake, and the neutral mucin fractions tor, genistein (Akiyama et al., 1987). This tyrosinespecific kinase inhibitor, furthermore, caused had virtually no inhibitory effect, the low and high molecular weight acidic mucin fractions at their inhibition in buccal mucosal 55 and 170 kDa channel optimal concentrations of 80pg/ml evoked 54 and protein phosphorylation. The finding that the low and high molecular weight 60% inhibition, respectively in the uptake of 45Ca2+ into the vesicles containing the reconstituted buccal acidic salivary mucins are capable of regulating the buccal mucosal calcium channel activity through the mucosal calcium channels. inhibition of EGF-stimulated calcium channel proThe demonstrated inhibitory effect of the low and tein tyrosine phosphorylation attests further to the high molecular weight acidic salivary mucus glycoimportance of salivary mucins in the regulation of proteins on buccal mucosal calcium channel activity functional expression of EGF in the oral cavity, a is apparently due to the presence of sialic acid process of paramount importance to the maintenance residues and sulfate ester groups on the carbohydrate of oral tissue integrity under normal physiological chains, as the removal of either sialic acid or sulfate conditions, and its repair following injury. caused partial loss in the glycoproteins inhibitory The schematic representation of the inhibitory effect. Furthermore, a complete loss in the inhibitory mechanism of acidic salivary mucus glycoproteins in effect on 4sCa2+ uptake occurred following desialylthe EGF-induced buccal mucosal calcium channel ation and desulfation of these glycoproteins. Based activation is depicted in Fig. 8. Based on the data on the results obtained with the desialyzed and presented in this study, the salivary mucus glycodesulfated low and high molecular weight glycoproteins, by modulating the EGF-controlled calcium proteins, it appears that sulfomucins exert about channel phosphorylation, appear to play a major role 3-fold greater inhibitory effect on calcium channel in oral mucosal calcium homeostasis. As unconactivity than the sialomucins. The regulatory effect on calcium channel activity has also been reported in trolled calcium influx is known to aggravate mucosal other systems for such acidic glycoconjugates as injury (Tamawski et al., 1990), our results provide for heparin and GM,-ganglioside (Knaus et al., 1990; the first time a direct evidence for the active participation of salivary mucins in the maintenance of the Slomiany et al., 1992a). Hence, the identified ability epithelial perimeter of oral mucosal defense. of acidic salivary mucins to modulate the buccal mucosal calcium channel activity could be viewed as yet another feature of the multifunctional role the REFERENCES salivary mucus glycoproteins play in the preservation Akiyama T., Ishida J., Nakagawa S., Ogawara H., Watanof oral mucosal integrity. abe S., Itoh N., Shibuya M. and Fukami Y. (1987) Since, as reported recently with patch clamp
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