The gene encoding mouse Muc19: cDNA, genomic organization and relationship to Smgc.

We previously demonstrated expression of full-length transcripts for sublingual mucin apoprotein, Muc19, of approximately 24 kb (Fallon MA, Latchney LR, Hand AR, Johar A, Denny PA, Georgel PT, Denny PC, and Culp DJ. Physiol Genomics 14: 95-106, 2003). We now describe the complete sequence and genomic organization of the apomucin encoded by 43 exons. Southern analyses indicate a central exon of approximately 18 kb containing 36 tandem repeats, each encoding 163 residues rich in serine and threonine. Full-length transcripts are an estimated 22,795 bp in length that span 106 kb of genomic DNA. The transcriptional start site is 24 bp downstream of a TATA box and 42 bp upstream of the conceptual translational start codon. The putative apoprotein has an estimated mass of 693.4 kDa and contains 7,524 amino acids (80% serine, threonine, glycine, alanine, and proline). We present a model for rat Muc19 transcripts and compare the conceptually translated Muc19 proteins for mouse, rat, pig, and the 3' end of human Muc19. Conserved among these apoproteins are a signal peptide, a large tandem repeat region, von Willebrand factor type C and D domains, a trypsin inhibitor-like Cys-rich domain, and a COOH-terminal cystine knot-like domain. Southern blot analyses indicate transcripts for Muc19 and Smgc (submandibular gland protein C) are splice variants of a larger gene, Muc19/Smgc. Comparative Northern analyses between the major salivary glands demonstrate highly selective Muc19 expression in neonatal and adult sublingual glands, whereas Smgc is expressed in neonatal submandibular and sublingual glands. Regulation of Muc19/Smgc gene expression is discussed with respect to alternative splicing and mucous cell cytodifferentiation.

The sld mutation is specific for sublingual salivary mucous cells and disrupts apomucin gene expression.

NFS/N-sld mice harbor a spontaneous autosomal recessive mutation, sld (sublingual gland differentiation arrest) and histologically display attenuated mucous cell expression in sublingual glands (Hayashi et al. Am J Pathol 132: 187-191, 1988). Because altered serous demilune cell expression is unknown, we determined the phenotypic expression of this cell type in mutants. Moreover, we evaluated whether absence of glycoconjugate staining in 3-day-old mutant glands is related to disruption in apomucin gene expression and/or to posttranslational glycosylation events. Serous cell differentiation is unaffected, determined morphologically and by serous cell marker expression (PSP, parotid secretory protein; and Dcpp, demilune cell and parotid protein). Conversely, apical granules in "atypical" exocrine cells of mutant glands are PSP and mucin negative, but contain abundant SMGD (mucous granule marker). Age-related appearance of mucous cells is associated with expression of apomucin gene products, whereas SMGD expression is unaltered. "Atypical" cells thus appear specified to a mucous cell fate but do not synthesize mucin glycoproteins unless selectively induced postnatally, indicating the sld mutation disrupts apomucin transcriptional regulation and/or decreases apomucin mRNA stability.

G protein coupling to M1 and M3 muscarinic receptors in sublingual glands.

Rat sublingual gland M2 and M3 muscarinic receptors each directly activate exocrine secretion. To investigate the functional role of coreceptor expression, we determined receptor-G protein coupling. Although membrane proteins of 40 and 41 kDa are ADP-ribosylated by pertussis toxin (PTX), and 44 kDa proteins by cholera toxin (CTX), both carbachol-stimulated high-affinity GTPase activity and the GTP-induced shift in agonist binding are insensitive to CTX or PTX. Carbachol enhances photoaffinity labeling ([alpha-(32)P]GTP-azidoaniline) of only 42-kDa proteins that are subsequently tractable to immunoprecipitation by antibodies specific for Galpha(q) or Galpha(11) but not Galpha(12) or Galpha(13). Carbachol-stimulated photoaffinity labeling as well as phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis is reduced 55% and 60%, respectively, by M1 receptor blockade with m1-toxin. Galpha(q/11)-specific antibody blocks carbachol-stimulated PIP2 hydrolysis. We also provide estimates of the molar ratios of receptors to Galpha(q) and Galpha(11). Although simultaneous activation of M1 and M3 receptors is required for a maximal response, both receptor subtypes are coupled to Galpha(q) and Galpha(11) to stimulate exocrine secretion via redundant mechanisms.

Biochemical and immunological studies and assay of rat sublingual mucins.

Original studies of rat sublingual mucins raised questions as to the existence of a second mucin species as distinguished by binding to hydroxyapatite. The existence of multiple mucin species is of concern in pharmacological studies of mucous-cell secretion as each species could represent distinct mucous-cell populations that respond differently to secretagogues. Thus a separate hydroxyapatite-bound mucin pool expressed in rat sublingual glands was isolated and characterized. Biochemical comparison of hydroxyapatite-bound mucins to total and hydroxyapatite-unbound sublingual mucins demonstrated no substantial differences in either amino acid and carbohydrate contents or in size distributions. In addition, a radioimmunoassay was developed using antisera prepared previously against unbound mucins. The three mucin pools exhibited equal specificities in displacement of radiolabelled unbound mucin tracer in the radioimmunoassay. Thus, bound and unbound mucins are indistinguishable, both immunologically and in biochemical composition. The radioimmunoassay was then evaluated for use in pharmacological studies of acinar mucous-cell secretion. Measurement by radioimmunoassay of secretion from isolated acini in response to carbachol was concentration-dependent (EC50 approx. 0.3 microM and maximal stimulation at 1 microM carbachol). In immunolocalization studies the antiserum was highly selective for mucous cells, recognized all mucous cells within histological sections, and was localized subcellularly to mucous-cell secretion granules and trans-Golgi, further validating the radioimmunoassay as a method to detect exocrine secretion from the entire pool of acinar mucous cells. Moreover, the radioimmunoassay was compared and found equivalent to an acid-precipitation method to assess relative secretion, suggesting the acid-precipitation method is also valid for pharmacological studies of isolated acini.

Both M1 and M3 receptors regulate exocrine secretion by mucous acini.

We investigated the role of M1 and M3 receptors in regulating exocrine secretion from acini isolated from rat sublingual glands. In secretion experiments, we derived affinity values (KB) from Schild regression analysis for the antagonists pirenzepine (61.0 nM) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP; 1.06 nM). The KB for 4-DAMP is similar to its affinity value [equilibrium dissociation constant from competition studies (Ki); 1.81 nM] determined from radioligand competition experiments. In contrast, the KB for pirenzepine is between its high-affinity (17.6 nM) and low-affinity (404 nM) Ki values. In separate secretion experiments, we found that the M1 receptor antagonist, M1-toxin, induces a rightward shift in the concentration-response curve to muscarinic agonist and inhibits maximal secretion by 40%. The inhibitory effect of M1-toxin appears specific for M1 receptor blockade, since the toxin abolishes acinar high-affinity pirenzepine-binding sites and does not inhibit secretion induced by nonmuscarinic agents. Additional pharmacological studies indicate muscarinic receptors do not function through putative neural elements within isolated acini. Our combined results are consistent with both M1 and M3 receptors directly regulating mucous acinar exocrine secretion and indicate M3 receptors alone are insufficient to induce a maximal muscarinic response.

Composition of human airway mucins and effects after inhalation of acid aerosol.

Characterization of normal airway mucus is required to elucidate mechanisms protecting the airways and to understand changes associated with disease and environmental insult. Toward this goal, we collected bronchial washes (10 ml saline) from healthy human subjects to 1) evaluate the yield of high-density material (delta > or = 1.35 g/ml), and 2) characterize glycoconjugates associated with collected secretions. Samples were lipid extracted followed by CsCl density gradient centrifugation. The yield of high-density material from individual subjects was variable but sufficient to demonstrate that mucin glycoproteins are a major constituent of mucus from healthy airways and that proteoglycans are absent. Next, we investigated whether inhalation of H2SO4 aerosol (1,000 microgram/m3), an environmental insult associated with alterations in mucociliary clearance, changes the composition of high-density glycoproteins in airway secretions. In a paired, double-blinded study, high-density fractions of bronchial secretions from 12 subjects were collected 18 h after exposures of 2 h to aerosolized NaCl and H2SO4. In all cases the high-density material displayed characteristics of mucin glycoproteins. In addition, a unique 150-kDa glycoprotein was detected in most but not all samples and may represent a small mucin glycoprotein differentially expressed in humans. No differences were noted between the two exposure conditions in the profiles of the glycoproteins or proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Statistically, large changes with acid exposure in the composition of carbohydrates and amino acids were absent. Thus no substantial systematic changes in airway mucin glycoproteins or closely associated proteins and glycoproteins were correlated with H2SO4 exposure. Alternatively, statistical analysis of the differences between exposures in glycoprotein constituents among subjects denoted greater variability in carbohydrates compared with amino acids with repeated sampling, suggesting normal daily variations in the mucin composition of individual airway mucus.

Mucinlike glycoproteins from cat tracheal gland cells in primary culture.

In a recent study (D. J. Culp, D. K. P. Lee, D. P. Penney, and M. G. Marin. Am. J. Physiol. 263: L264-275, 1992), we reported that primary cultures of cat tracheal gland cells expressed histological, ultrastructural, and immunological characteristics of mucous cells when cultured on floating gels of rat tail collagen (released-gel cultures) compared with cells cultured on glutaraldehyde-fixed collagen gels (fixed-gel cultures). We therefore collected culture medium from gland cells grown under both culture conditions for determination and comparison of glycoconjugates with characteristics of mucin glycoproteins. Cells were cultured in the presence of [3H]glucosamine, and material of high molecular weight and density (HMD material) was isolated. HMD material from both culture conditions were each resistant to heparitinase and heparinase, whereas 72 and 25% of the radiolabel in released-gel and fixed-gel HMD material, respectively, was resistant to chondroitinase ABC. Material resistant to chondroitinase ABC was analyzed further. Both samples contained a single broad glycoprotein band [relative molecular weight (M(r)) > 250,000] after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had amino acid profiles similar to airway mucin. The sample from fixed-gel cultures had nearly equal amounts of carbohydrate and protein, was highly enriched in N-acetylglucosamine, contained mannose, displayed little blood group A immunoreactivity, and had few O-linked oligosaccharides. Conversely, the sample from released-gel cultures contained 80% carbohydrate, was composed of monosaccharides characteristic of airway mucins, displayed blood group A immunoreactivity, and contained oligosaccharides O-linked via N-acetylgalactosamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat sublingual gland as a model to study glandular mucous cell secretion.

To study the regulation of mucous cell secretion, we have developed an in vitro cell model consisting of enzymatically dispersed mucous acinar structures (cell aggregates) from rat sublingual glands. Histological and ultrastructural evidence demonstrates that the cell aggregates are highly enriched in mucous cells, retain the morphological and ultrastructural features observed in intact glands, and undergo transition to an extensive secretory state when stimulated by 10 microM carbachol. The secretory responsiveness of the cell aggregates was verified in pharmacological studies. Carbachol stimulated secretion in a dose-dependent manner with high affinity (concentration causing half-maximal response = 0.3 microM) and was completely inhibited by atropine. Secretion was also stimulated by vasoactive intestinal peptide and substance P but not by alpha- or beta-adrenergic agonists. Biochemical characterization of secretion during nonstimulated and carbachol-stimulated conditions (after preincubation in [3H]glucosamine) demonstrated that, in response to carbachol, cell aggregates synthesized and secreted mucins which were similar to mucin glycoproteins isolated from whole glands. Collectively, our results establish that the rat sublingual cell aggregate model is a viable and pharmacologically responsive cell system to study the regulation of mucous cell secretion.

Influenza virus infection of tracheal gland cells in culture.

Influenza virus-induced tracheobronchitis causes limited epithelial deciliation but markedly decreased mucociliary transport. This suggests that virus-induced alterations in airway mucus play a role in decreased mucociliary transport. Airway submucosal glands are a primary source of mucus. Therefore, we examined virus-gland cell interactions by exposing primary cultures of isolated feline tracheal gland cells to influenza A/Scotland/840/74 H3N2 virus for 1 h at a multiplicity of infection of 0.1. Virus production and release into the culture medium first occurred between 8 and 12 h postinfection and eventually reached a steady state that continued for at least 8 days. Virus which was produced and released by infected cells infected other monolayers, resulting in viral production similar to that after infection with stock virus. Hemadsorption assays conducted 24 h after infection demonstrated that most of the cells in a monolayer became infected. The infection was nonlytic according to cell morphology, trypan blue dye exclusion, and release of lactate dehydrogenase. Because lysis of a cell subpopulation could have been masked by subsequent cell division, we compared the uptake of [3H]thymidine by infected and control monolayers. There was no increase in uptake by infected monolayers. These results demonstrate that feline tracheal gland cells in primary culture undergo productive and nonlytic infection with influenza A virus. This model provides a unique system for the study of virus-gland interactions isolated from the influence of other tissues.

Tight divalent metal binding to Escherichia coli F1-adenosinetriphosphatase. Complete substitution of intrinsic magnesium by manganese or cobalt and studies of metal binding sites.

Tight divalent metal binding sites in Escherichia coli F1-adenosinetriphosphatase (F1-ATPase) were studied. Native enzyme contained two Mg per F1, confirming previous results. All of the Mg may be replaced by Co or Mn using a dissociation-repolymerization procedure. The substituted enzymes are homogeneous and contain two Mn per F1 or two Co per F1. They are fully active as ATPases, they rebind to F1-depleted membranes, and they catalyze ATP-driven proton pumping. N,N'-Dicyclohexylcarbodiimide-(DCCD) inactivated F1 retains all the intrinsic tightly bound Mg. Evidence is presented that DCCD affects at least two beta subunits in E. coli F1, and therefore, the tightly bound metals appear not to be bound at the DCCD-reactive glutamate residue on the beta subunit. However, the nature of the tightly bound metal (Mg, Mn, or Co) as well as the presence of added (2 mM) MgSO4, MnSO4, or CoSO4 affected the rate of DCCD inactivation, showing that metal binding changes the beta-subunit conformation. Isolated F1 alpha subunit bound Mg, Mn, or Co stoichiometrically and independently of ATP binding. Isolated F1 beta subunit bound only small amounts of Mg, and no Co or Mn. Therefore, it is possible, although not conclusively shown, that the alpha subunit is the site of tight metal binding in the intact F1.

Inhibition of Escherichia coli H+-ATPase by venturicidin, oligomycin and ossamycin.

The antibiotics venturicidin, oligomycin and ossamycin were investigated as potential inhibitors of the Escherichia coli H+-ATPase. It was found that venturicidin strongly inhibited ATP-driven proton transport and ATP hydrolysis, while oligomycin weakly inhibited these functions. Inhibition of the H+-ATPase by venturicidin and oligomycin was correlated with inhibition of F0-mediate proton transport. Both inhibitors were found to interfere with the covalent reaction between dicyclohexyl[14C]carbodiimide and the F0 subunit c (uncE protein). Ossamycin had no direct inhibitory effect on E. coli F0 or F1; rather, it was found to uncouple ATP hydrolysis from proton transport.

Specificity of the proton adenosinetriphosphatase of Escherichia coli for adenine, guanine, and inosine nucleotides in catalysis and binding.

Specificity of the Escherichia coli proton ATPase for adenine, guanine, and inosine nucleotides in catalysis and binding was studied. MgADP, CaADP, MgGDP, and MgIDP were each good substrates for oxidative phosphorylation. The corresponding triphosphates were each substrates for hydrolysis and proton pumping. At 1 mM concentration, MgATP, MgGTP, and MgITP drove proton pumping with equal efficiency. At 0.1 mM concentration, MgATP was 4-fold more efficient than MgITP or MgGTP. Nucleotide-depleted soluble F1 could rebind to F1-depleted membranes and block proton conductivity through F0; rebound nucleotide-depleted F1 catalyzed pH gradient formation with MgATP, MgGTP, or MgITP. This showed that the nonexchangeable nucleotide sites on F1 need not be occupied by adenine nucleotide for proton pumping to occur. It was further shown that no nucleotide was tightly bound in the nonexchangeable sites of F1 during proton pumping driven by MgGTP in these reconstituted membranes, whereas adenine nucleotide was tightly bound when MgATP was the substrate. Nucleotide-depleted soluble F1 bound maximally 5.9 ATP, 3.2 GTP, and 3.6 ITP of which half the ATP and almost all of the GTP and ITP exchanged over a period of 30-240 min with medium ADP or ATP. Also, half of the bound ATP exchanged with medium GTP or ITP. These data showed that inosine and guanine nucleotides do not bind to soluble F1 in nonexchangeable fashion, in contrast to adenine nucleotides. Purified alpha-subunit from F1 bound ATP at a single site but showed no binding of GTP nor ITP, supporting previous suggestions that the non-exchangeable sites in intact F1 are on alpha-subunits.

Defective proton ATPase of uncA mutants of Escherichia coli. 5'-Adenylyl imidodiphosphate binding and ATP hydrolysis.

The Escherichia coli uncA gene codes for the alpha-subunit of the F1 sector of the membrane proton ATPase. In this work purified soluble F1 enzymes from three mutant strains ( uncA401 , uncA447 , and uncA453 ) have been compared to F1 from a normal strain in respect to (a) binding of 5'-adenylyl imidodiphosphate (AMPPNP) to native enzyme in both the presence and absence of Mg, (b) high-affinity binding of MgATP to native enzyme, (c) total reloading of MgAMPPNP to nucleotide-depleted F1 preparations, (d, e) ability to hydrolyze MgATP at both high MgATP concentrations (d) (steady-state conditions) and low MgATP concentrations (e) where substrate hydrolysis occurs under nonsteady-state (" unisite ") conditions, and (f) sensitivity of steady-state ATPase activities to inhibitors of normal F1-ATPase activity. uncA mutant F1 showed normal stoichiometry of MgAMPPNP binding to both native (three sites per F1) and nucleotide-depleted preparations (six sites per F1). Native uncA F1 preparations showed lower-than-normal affinity for MgAMPPNP and MgATP at the first site filled. Binding of AMPPNP in the absence of Mg was similar to normal, except that no increase in affinity for AMPPNP was induced by aurovertin. The uncA F1-ATPases had low but real steady-state rates of ATP hydrolysis, which were inhibited by aurovertin but relatively insensitive to inhibition by AMPPNP, efrapeptin, and sodium azide. Non-steady-state ( unisite ) ATP hydrolysis rates catalyzed at low substrate concentrations by uncA F1-ATPases were similar to normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of F1-ATPase with impaired catalytic activity from partial revertants of Escherichia coli uncA mutant strains.

It is shown that F1-ATPase preparations having impaired catalytic rates may be purified from partial revertants of uncA mutant strains of Escherichia coli. Recovery of catalytic activity in the partial revertant F1 was accompanied by recovery of alpha in equilibrium beta intersubunit conformational interaction, supporting the hypothesis that such interaction is required for normal catalysis in F1. The specific ATPase activities of the partial revertant F1 preparations were in the range 1-29% of normal, and some of the preparations showed unusual insensitivity to inhibitors. The properties of a new uncA mutant F1 preparation (uncA498) which has approximately half of normal catalytic rate are also briefly described.

Properties of F1-ATPase from the uncD412 mutant of Escherichia coli.

Properties of purified F1-ATPase from Escherichia coli mutant strain AN484 (uncD412) have been studied in an attempt to understand why the amino acid substitution in the beta-subunit of this enzyme causes a tenfold reduction from normal MgATP hydrolysis rate. In most properties that were studied, uncD412 F1-ATPase resembled normal E. coli F1-ATPase. Both enzymes were found to contain a total of six adenine-nucleotide-binding sites, of which three were found to be non-exchangeable and three were exchangeable (catalytic) sites. Binding of the non-hydrolysable substrate analogue adenosine 5'-[beta gamma-imido]triphosphate (p[NH]ppA) to the three exchangeable sites showed apparent negative co-operativity. The binding affinities for p[NH]ppA, and also ADP, at the exchangeable sites were similar in the two enzymes. Both enzymes were inhibited by efrapeptin, aurovertin and p[NH]ppA, and were inactivated by dicyclohexylcarbodi-imide, 4-chloro-7-nitrobenzofurazan and p-fluorosulphonyl-benzoyl-5'-adenosine. Km values for CaATP and MgATP were similar in the two enzymes. uncD412 F1-ATPase was abnormally unstable at high pH, and dissociated into subunits readily with consequent loss of activity. The reason for the impairment of catalysis in uncD412 F1-ATPase cannot be stated with certainty from these studies. However we discuss the possibility that the mutation interrupts subunit interaction, thereby causing a partial impairment in the site-site co-operativity which is required for 'promotion' of catalysis in this enzyme.

Trypsin cleavage of the alpha-subunit of beef heart F1-ATPase abolishes ATP synthesis and ATP-driven energy-transduction capabilities.

Previous work has shown that mild trypsin treatment eliminates energy-transduction capability and tight (non-exchangeable)nucleotide binding in beef heart mitochondrial F1-ATPase (Leimgruber, R.M. and Senior, A.E. (1976) J. Biol. Chem. 251, 7103-7109). The structural change brought about by trypsin was, however, too subtle to be identified by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, and was not defined. In this work we have applied two-dimensional electrophoresis (isoelectric focussing then sodium dodecyl sulfate polyacrylamide gradient electrophoresis) to the problem, and have determined that the alpha-subunit of F1 is altered by the mild trypsin treatment, whereas no change was detected in beta-, gamma-, delta- or epsilon-subunits. Binding of ADP to the trypsin-treated F1 was compared to binding to control enzyme over a range of 0-40 muM ADP in a 30 min incubation period. There was no difference between the two enzymes, KADPd in Mg2+ -containing buffer was about 2 muM in each. Since the tight (nonexchangeable)sites are abolished in trypsin-treated F1, this shows that tight exchangeable ADP-binding sites are different from the tight nonexchangeable ADP-binding sites. There was no effect of trypsin cleavage of the alpha-subunit on beta-subunit conformation as judged by aurovertin fluorescence studies. The cleavage of the alpha-subunit which occurred was judged to occur very close to the C- or N-terminus of the subunit and constitutes therefore a small and specific chemical modification which abolishes overall function in F1 but leaves partial functions intact.