The murine sublingual and submandibular mucins their isolation and characterization

From the mouse sublingual and submandibular glands high-molecular weight glycoproteins (mucins) were isolated. These mucins appeared to be homogeneous in polyacrylamide gel electrophoresis and in the analytical ultracentrifuge. S_20,w values of 10.9 and 5.5 were found for the sublingual and submandibular mucin respectively. With sodium dodecyl sulfate or N-acetylcysteine no subunits could be detected.Both mucins consisted for about 1/3 of protein and 2/3 of carbohydrate. Their mucin character was also denoted by the high content of serine plus threonine. Respectively 42 mol% and 34 mol% of the protein core of the sublingual and submandibular mucins consisted of these amino acids. The main sugars in these mucins were sialic acid, galactosamine, galactose, glucosamine and mannose. The molar ratio for the sublingual and submandibular mucin being 1.00 : 1.03 : 1.08 : 0.26 : 0.23 and 1.00 : 0.71 : 1.10 : 0.65 : 0.53, respectively.The sialic acid content of both mucins was about 25%. Fucose and sulfate, on the other hand, were less than 1%. The presence of sulfate was also indicated by preliminary studies in vivo on the incorporation of [³⁵SO₄] sulfate.     

Localization of amylase and mucins in the major salivary glands of the mouse

Summary Antibodies against murine submandibular and sublingual mucins have been raised in rabbits. Both antisera appeared to be specific. Using these antibodies, the mucins were localized in the acinar cells of the submandibular and sublingual glands respectively.The dyed amylopectin method was used to estimate the activity of amylase in the salivary glands. The enzyme was localized either by a starch-substrate film method or with antibodies against purified parotid amylase. The activity of amylase in parotid homogenates is about 1000-fold higher than that in homogenates of either submandibular or sublingual glands, in which the activity was comparable. Amylase was localized in the acinar cells of the parotid gland with both localization techniques. In the sublingual gland, amylase was found predominantly in the stroma around the acini, and there was some evidence that amylase was present in the demilune cells as well. In the submandibular gland, contradictory results were obtained with both techniques. With the starch-substrate film method, amylase activity was found in the granular convoluted tubular cells, whereas immuno-reactive amylase could only be demonstrated in the acinar cells of this gland. It is concluded that in the submandibular gland amylase and mucin are present in the same cell type.     

Characterization of the major and minor mucus glycoproteins from bovine submandibular gland

Two mucins were isolated from bovine submandibular glands and termed major and minor on a quantitative basis. The major mucin representing over 80% of the total glycoprotein fraction contained 37% of its dry weight as protein in contrast to 62% for the minor mucin. Differences in the amino acid composition reflected the higher proportion of typically non-glycosylated peptide in the minor mucin. The molar ratio ofN-acetylgalactosamine to serine plus threonine was 0.82 in major and 0.65 in minor mucins, indicating a lower degree of substitution of potential glycosylation sites in the minor mucin.Differences in the carbohydrate composition were found largely related to the sialic acids, with higher relative amounts ofN-glycoloylneuraminic acid in the minor mucin. In addition, the proportion of di-O-acetylated sialic acids was higher in the major mucin. The rate of sialidase action on the two mucins could be correlated with the content ofN-glycoloylneuraminic acid in each glycoprotein. There was no difference in the type of oligosaccharide found in each mucin and the differences in relative proportions reflected the monosaccharide composition for the two mucins. Gel filtration on Sepharose CL 2B showed a lower molecular weight distribution for the minor in contrast to the major mucin which was partially excluded. Density gradient centrifugation reflected this variation. SDS-PAGE demonstrated a regular banding pattern for the major mucin with a lowest subunit size of 1.8×10⁵ Da and aggregates in excess of 10⁶ Da, while the minor mucin ranged from 3.0 × 10⁵ to 10⁶ Da. The chemical composition of the isolated mucins was compared with previous histochemical analysis of mucin distribution in bovine submandibular glands and indicates a possible cellular location for each mucin.Abbreviations PBS 0.01m sodium phosphate buffer, pH 7.3, containing 0.15m NaCl - Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycoloylneuraminic acid - GalNAc-ol N-acetylgalactosaminitol     

Mucin histochemistry of submandibular and parotid salivary glands of man: light and electron microscopy

Light-microscopy showed parotid serous acinar cells to contain neutral mucin, serous and mucous acinar cells of submandibular gland and intercalary ductal cells of both glands to contain acid and neutral mucins, and cells of striated ducts and excretory ducts to contain neutral mucin. Mucins were demonstrated ultrastructurally in a portion of the components of secretory granules of acinar cells and intercalary ductal cells, and in secretory granules of striated and excretory ductal cells. The mucins were all stained by techniques that reveal 1,2-glycols. Secretory granules of submandibular mucous and serous acinar cells and intercalary ductal cells were stained variably by the low iron-diamine technique for acid mucin, and those of mucous acinar cells by the high iron-diamine technique for sulphomucins mucin and possibly consisted of protein. The results suggest that one type of cell may be able to produce a range of secretory products and to package them variously into secretory granules.     

Purification and characterization of a human pancreatic adenocarcinoma mucin.

Pancreatic mucins consist of core proteins that are decorated with carbohydrate structures. Previous studies have identified at least two physically distinct populations of mucins produced by a pancreatic adenocarcinoma cell line (HPAF); one is the MUC1 core protein, which includes an oligosaccharide structure identified by a monoclonal antibody (MAb) recognizing the DU-PAN-2 epitope. In this study, we purified and characterized a second mucin fraction, which also shows reactivity with the DU-PAN-2 antibody, but which has an amino acid composition that is not consistent with the MUC1 core protein. This new mucin was purified by ammonium sulfate precipitation, molecular sieve chromatography, and density gradient centrifugation. It eluted in the void volume of a Sepharose 4B column together with an associated low molecular weight protein, which could be further resolved. The mucin is highly polyanionic due to numerous sulfated and sialylated saccharide chains. Carbohydrate analyses of the purified mucin showed the presence of galactose, glucosamine, galactosamine, and sialic acid, but no mannose, glucose, or uronic acid. The purified and deglycosylated mucin shows no reactivity with anti-MUC1 apomucin antibody, but reacts with antiserum against deglycosylated tracheal mucins and antiserum against the MUC4 tandem repeat peptide. Analysis of mucin expression in HPAF cells revealed high levels of MUC1 and MUC4 mRNA, and moderate levels of MUC5AC and MUC5B mRNA. The amino acid composition of the purified mucin shows a high degree of similarity to the MUC4 core protein.     

Microstereological and histochemical studies of the salivary glands of the giant rat (Cricetomys gambianus, waterhouse)

The histology and histochemistry of the parotid, submandibular and sublingual glands were studied. The submandibular gland contained only serous acini as in the guinea pig, but unlike in many other mammals. The parotid gland contained only serous acini while the sublingual gland was mixed, mucous acini being the predominant secretory tissue interspersed by a few serous acini. Serous demilunes also commonly formed caps on the mucous acini. The ducts of the gland contributed over 30% of the volume of the submandibular gland, while those of the parotid and sublingual glands formed about 12 and 10% of the gland, respectively. The secretions of the parotid gland, as judged by histochemical methods, contained neutral mucins and some sialomucins. Neutral mucins, sulphomucins and sialomucins were detected in both the submandibular gland and sublingual gland.     

Effects of rabbit gastrointestinal mucins and dextran on hydrochloride diffusion in vitro

We compared a viscous fingering formation of hydrochloric acid (HCl) in rabbit corpus, antral and duodenal mucins and with dextran under neutral and acidic conditions with respect to relative viscosity, molecular mass, and carbohydrate composition. The effect of desialyzation of duodenal mucin on the viscous fingering formation of HCl was also examined. HCl (0.1 N) was injected into 1% solutions of mucins and dextran and a subsequent viscous fingering formation was assessed based on an influx volume rate of HCl. A low influx volume rate indicates a high ability of the solutions to produce viscous fingers. The influx volume rate of HCl was lowest in duodenal mucin followed bl corpus mucin, antral mucin, and dextran at pH 7. The influx volume rate of HCl was inversely correlated with the relative viscosity of the solution. Maximum molecular masses were large in the order of corpus, antral, and duodenal mucins, and they were larger than dextran T2000. Rabbit gastrointestinal mucins were very polydisperse system. Duodenal mucin contains more sialic acid than gastric mucins; the influx volume rate of HCl increased in desialylated duodenal mucin. It is suggested that the higher ability of gastric mucins to prevent HCl diffusion than dextran were due to the differences in the molecular mass. The ability of duodenal mucin to prevent HCl diffusion was probably attributed to its high sialic acid content, which may reflect a physiological role of duodenal mucin in the duodenum that has to deal with HCl influx from the stomach.     

Interaction of the salivary low-molecular-weight mucin (MG2) with Actinobacillus actinomycetemcomitans

Periodontitis is associated with the presence of certain Gram-negative bacteria in the oral cavity, among these Actinobacillus actinomycetemcomitans. In order to determine which types of salivary components interact with A. actinomycetemcomitans two strains (HG 1175 and FDC Y4) were incubated with whole saliva and individual glandular secretions, viz. parotid, submandibular, and sublingual saliva. Immunochemical analysis by immunoblotting of bacteria-bound salivary proteins showed that IgA, the low-molecular mucin MG2, parotid agglutinin, and a 300 kDa sublingual and submandibular glycoprotein, were bound to the bacterial strains tested. In addition, adherence of A. actinomycetemcomitans to salivary proteins in a solid-phase was studied. After electrophoresis and transfer of salivary proteins to nitrocellulose membranes A. actinomycetemcomitans adhered only to MG2. In this assay periodate treatment, mild acid hydrolysis or neuraminidase digestion of the saliva glycoproteins abolished binding of two clinical isolates (HG 1175 and NY 664), suggesting that sialic acid residues on MG2 are involved in the binding. In contrast, adherence of the smooth laboratory strain Y4 was not affected by removal of sialic acid residues or even periodate treatment of MG2.Abbreviations S-IgA Secretory IgA - MG1 high-molecular-weight mucin - MG2 low-molecular-weight mucin - EP-GP extra parotid-glycoprotein - PRPs proline-rich proteins - SNA Sambucus nigra agglutinin - MAA Maackia amurensis agglutinin - PNA peanut agglutinin - UEA Ulex europaeus agglutinin     

Comparison of physicochemical properties of purified mucus glycoproteins isolated from respiratory secretions of cystic fibrosis and asthmatic patients

The major nonreduced mucus glycoproteins (mucins) from sputa of cystic fibrosis (CF) and asthmatic patients have been purified to electrophoretic homogeneity and subjected to physical and chemical characterization. The sputum specimens were solubilized in buffer containing 0.22 M KSCN and fractionated on Bio-Gel A-5m, followed by digestion with DNase, rechromatography on the same column, and chromatography on hydroxylapatite. Sodium dodecyl sulfate gel electrophoresis of purified mucins gave a single band. Carbohydrate analyses of the purified mucins showed no significant differences in the sugar components from the two mucins. However, the CF mucin contained substantially higher (11%) sulfate content than that observed for the asthmatic mucin (5.9%). Amino acid analyses indicated that the CF mucin had higher levels of serine plus threonine (35%) as compared to the asthmatic mucin (29%). In contrast, CF mucin contained a lower content of aspartate, glutamate, and glycine than that observed for the asthmatic mucin. Molecular weights of 3.8 X 10(6) and 3.5 X 10(6) were obtained for CF and asthmatic mucins, respectively, from light-scattering studies of mucins in the presence of 6 M guanidine hydrochloride. Reduction of the disulfide bonds of the two mucins did not alter their molecular weights. Liquid chromatographic studies on Sepharose CL2B showed that CF mucin forms aggregates sufficiently large to be excluded from the gel. As compared to the CF mucin, the asthmatic mucin formed fewer of these large aggregates under identical experimental conditions. Reduction and alkylation of the mucins resulted in their inability to form aggregates. The higher state of aggregation of CF mucin may influence the viscoelastic properties of the CF lung's mucus secretions.     

Identification of 9-O-acetyl-N-acetylneuraminic acid in normal canine pre-ocular tear film secreted mucins and its depletion in Keratoconjunctivitis sicca

O-Acetylated sialic acids have been reported in many sialoglycoproteins where they mediate a variety of immune and other biological events. We have previously demonstrated that the protective mucus barrier on the surface of the canine eye contains sialoglycoproteins. We have also investigated the occurrence of O-Acetylated sialic acids in these ocular mucins. Mucus aspirated from the surface of normal dog eyes and those with keratoconjunctivitis sicca (KCS) was fractionated into three pools by density gradient centrifugation. Sialic acids comprised 0.6–0.9% of the dry weight of the mucins isolated. The sialic acid profile in these pools was examined using HPLC. O-Acetylated sialic acids, mainly Neu5,9Ac₂, were detected in normal animals and made up 10–30% of the total sialic acids detected. A doubling of the sialic acid content was found in KCS mucins, but the level of 9-O-Acetylated sialic acid was reduced below 4% of total. Histological analysis of conjunctival tissue from normal and KCS dogs showed the presence of sialic acids, detected with the α(2–6) sialic acid-specific lectin Sambucus nigra, in the goblet cells and corresponding to the staining pattern for MUC5AC, the major ocular-secreted mucin gene product. In KCS animals a disruption of the normal pattern of conjunctival goblet cells was seen with preservation of the pattern of lectin binding observed in normal animals. Thus the data demonstrate the presence of mono-O-Acetylated sialic acids in normal canine ocular mucins and a loss of this population of sialic acids in dry eye disease in spite of a significant increase in total sialic acids in KCS mucin.     

Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product

The MG1 population of mucins was isolated from human whole salivas by gel chromatography followed by isopycnic density gradient centrifugation. The reduced and alkylated MG1 mucins, separated by anion exchange chromatography, were of similar size (radius of gyration 55-64 nm) and molecular weight (2.5-2.9 x 10(6) Da). Two differently-charged populations of MG1 subunits were observed which showed different reactivity with monoclonal antibodies to glycan epitopes. Monosaccharide and amino acid compositional analyses indicated that the MG1 subunits had similar glycan structures on the same polypeptide. An antiserum recognizing the MUC5B mucin was reactive across the entire distribution, whereas antisera raised against the MUC2 and MUC5AC mucins showed no reactivity. Western blots of agarose gel electrophoresis of fractions across the anion exchange distribution indicated that the polypeptide underlying the mucins was the product of the MUC5B gene. Amino acid analysis and peptide mapping performed on the fragments produced by trypsin digestion of the two MG1 populations yielded data similar to that obtained for MUC5B mucin subunits prepared from respiratory mucus (Thornton et al., 1997) and confirmed that the MUC5B gene product was the predominant mucin polypeptide present. Isolation of the MG1 mucins from the secretions of the individual salivary glands (palatal, sublingual, and submandibular) indicate that the palatal gland is the source of the highly charged population of the MUC5B mucin.     

Developmental changes of ferret tracheal mucin composition and biosynthesis

We characterized the chemical composition of mucins secreted by ferret tracheal explants and the activities of key mucin glycosyltransferases in ferret tracheal epithelium during a period of rapid postnatal maturation of the mucin-secreting structures. Ferret tracheal explants secrete three major groups of high molecular weight glycoconjugates: (1) those susceptible to bovine testicular hyaluronidase; (2) those resistant to hyaluronidase and exhibiting high density (p greater than or equal to 1.60 g/mL); and (3) those resistant to hyaluronidase and exhibiting low density (1.45 less than or equal to p less than 1.60 g/mL). The hyaluronidase-resistant, low-density glycoconjugates have typical mucin properties and constitute 36% of total glycoconjugates released in newborns but only 8% in adult ferrets. Mucin secretory rate per unit surface area of trachea progressively decreases with age. Mucin amino acid and total carbohydrate contents do not vary; however, the sialic acid content increases, and fucose content as well as blood group A activity of the mucins decreases with age. Four glycosyltransferases involved in mucin biosynthesis [Gal beta 3GalNAc:(GlcNAc-GalNAc)beta 6 N-acetylglucosaminyl-, GalNAc:beta 3 galactosyl-, Gal:alpha 2 fucosyl-, and GalNAc alpha 2----6 neuraminyltransferase] are present in tracheal epithelium of ferrets at all ages. Activities of all but the neuraminyltransferase decrease with age. The relatively greater neuraminyltransferase activity is consistent with increased incorporation of sialic acid into secreted mucins over the same age span. Conversely, diminution of fucosyltransferase relative to galactosyltransferase activity may contribute to the lower fucose content and lower blood group A activity of mucins secreted by mature ferret tracheas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of mucin isolated from rat tracheal transplants

Subcutaneous rat tracheal grafts yield several milligrams of secretions from which a homogeneous mucin fraction was isolated and purified. Histological evidence demonstrated that a normal mucociliary epithelium and mucous secretion were maintained for the 4-6 weeks of the experiment. The collected secretions were initially characterized by column chromatography on Sepharose CL-6B which separated the excluded high molecular weight mucins (unpurified mucin fraction) from most of the serum-type glycoproteins and proteins, including albumin. A reductive alkylation treatment of the unpurified mucin fraction followed by Sepharose CL-4B chromatography removed contaminating protein and most of the mannose-containing material from the mucin fraction. The void volume material from this column produced a single high molecular weight band upon sodium dodecyl sulfate agarose/acrylamide gel electrophoresis. The purified mucin fraction contained 16.5% protein and primarily galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid. This fraction also underwent beta-elimination in the presence of alkaline borohydride, demonstrating the presence of O-glycosidic linkages.     

Purification and immunofluorescent localization of rat submandibular mucin

Rat submandibular mucin (RSM) was purified by acid precipitation, then alcohol precipitation of the 30000g supernatant of gland homogenate, followed by column chromatography on Sephadex G-200. The mucin, which was eluted in the void volume, had an amino acid profile typical of a salivary mucus glycoprotein with high proportions of threonine, serine and proline (48.8% of total amino acids), and low proportions of aromatic and basic amino acids. It consisted of 63% (w/w) carbohydrate, which was shown by g.l.c. analysis to contain N-acetylglucosamine, N-acetylgalactosamine, galactose, sialic acid and fucose in the proportions 1.0:3.4:2.6:3.1:1.2. After staining of the mucin with periodic acid/Schiff reagent, analytical equilibrium ultracentrifugation in a CsCl density gradient produced a symmetrical peak of buoyant density 1.449g/ml, without evidence of protein contaminants. Sedimentation velocity centrifugation revealed a major periodate/Schiff-positive component (S⁰_20,w 5.06) with an associated shoulder of slower sedimenting material, suggesting polydispersity in the size of the mucin. Our findings suggest that the RSM purified in these studies has a molecular weight between 200000 and 1×10⁶. Antibody to RSM was prepared in a rabbit and produced a single precipitin line on immunoelectro-osmophoresis with the mucin. Immunofluorescence studies showed that the antibody localized only to submandibular acinar cells and confirmed that these cells were the source of RSM. The antibody was not directed towards the blood-group-A determinant (terminal N-acetylgalactosamine) present in the mucin.     

Reactivity of mucin-specific lectin from Sambucus sieboldiana with simple sugars, normal mucins and tumor-associated mucins. Comparison with other lectins.

Mucin-specific lectin from Sambucus sieboldiana (SSA-M) reacts in Western blotting and ELISA with mucins from porcine stomach, bovine and ovine submaxillary glands, the human milk fat globule membrane, in vitro human ovarian, breast and colonic tumor cell lines, and mucins produced in vivo in the ascites of patients with endometrial and ovarian tumors, but not with fetal bovine fetuin or human transferrin. Sialidase treatment of these mucins led to an increase in the binding of SSA-M, suggesting that sialic acid is not part of the binding site for this lectin. Furthermore, sialic acid did not inhibit lectin binding. Treatment of asialomucin with O-glycanase decreased the binding of SSA-M, confirming the reactivity of the lectin with an O-linked carbohydrate. Treatment of mucins with trifluoromethanesulfonic acid, which removes all but core carbohydrate, led to an increase in the binding of SSA-M, suggesting that the lectin reacts with O-linked core glycans. Indeed, the increased reactivity after sialidase treatment of ovine submaxillary mucin suggests the lectin reacts with peptide-linked N-acetylgalactosamine (GalNAc), since more than 98% of the glycan chains attached to this mucin are sialylated GalNAc. The binding of SSA-M to sialidase-treated porcine mucin was inhibited strongly by GalNAc and disaccharides containing galactose (lactose, melibiose, and N-acetyllactosamine) but not by free galactose (Gal), suggesting that the glycan for optimum binding is Gal beta(1-3)GalNAc. This pattern of inhibition was different to other core glycan-reactive lectins tested, indicating that SSA-M is distinct, and should be of use in the isolation and characterisation of mucins and O-linked glycans.     

Regulation of buccal mucosal calcium channel activity by salivary mucins

• 1.1. The effect salivary mucins on the activity of calcium channel isolated from buccal mucosal cell membranes was investigated. The uptake of ⁴⁵Ca²⁺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 mucins which evoked 64 and 60% inhibition, respectively.
• 2.2. 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.
• 3.3. 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 ⁴⁵Ca²⁺ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein.
• 4.4. 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.
• 5.5. 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.

     

Lectin histochemistry and identification of O-acetylated sialoderivatives in the horse sublingual gland.

This study was aimed at characterizing the glycoconjugates produced by the horse sublingual gland and, in particular, at discriminating between the sialoderivatives by means of differential oxidation and saponification combined with lectin histochemistry and enzymatic degradation. The results showed a predominance of sialoglycoconjugates with beta-galactose as acceptor sugar in the salivary mucins produced by the sublingual gland. Besides being the most represented terminal residue, sialic acid was also expressed in a great variety of derivatives distinguishable on the basis of acceptor sugars to the penultimate beta-galactose as well as linkage and acetylation degree of the pyranose ring and the polyhydroxyl side chain. A role in the protection of mucous membranes from physical, chemical and pathogenic agents can be hypothesized for the horse sublingual mucins.     

Sialic Acid Derivatives and their Distribution in Rat Sublingual Gland Acini during Pre- and Post-natal Development

Sialoglycoconjugates in rat sublingual gland acinar cells, at different stages of pre- and post-natal development, were investigated in situ with specific lectins and by the selective removal of terminal sialic acids. Cleavage of acetyl substituents sited in the pyranose ring and/or polyhydroxyl side chain was used as an additional means of characterising the glycoconjugates. The first expression of terminal sialic acid linked to -galactose was found at gestational day 17 and progressive different derivatives were observed. The terminal disaccharide sialic acid-N-acetylgalactosamine was constantly visualized in the sublingual gland from gestational day 18. In both terminal disaccharides, sialic acids were characterized by variable degrees of acetylation and were found to be highly packaged and responsible for the hydration coat. The complex data obtained indicated that the sublingual gland is characterized by a marked fluctuation of complex sialoglycoconjugates that differ from those in the submandibular gland of the same species.