Current concepts of the structure and nature of mammalian salivary mucous glycoproteins

Summary The visco-elastic properties of salivary secretions are due to high molecular-weight glyco-proteins, known as mucins. Mucins are composed of numerous oligosaccharide side-chains O-glycosidically linked through 2-acetamido-2deoxy--d-galactose to the hydroxyl groups of seryl and threonyl residues of the protein core; on the average, every fourth amino acid residue is involved in such a bond. This work conveys their isolation and purification, compiles the compositional analysis of several mammalian submaxillary and sublingual mucins; defines the conditions of the alkaline -elimination reaction, its mechanism and importance in structural studies of glycoproteins, and briefly discusses the influence of stimuli on mucous secretions, as well as biosynthesis, structural diversity, and physiological role of salivary mucous glycoproteins.     

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     

Synthesis of sulfated oligosaccharides by cystic fibrosis trachea epithelial cells

The mucin glycoproteins in tracheal mucus of patients with cystic fibrosis is more highly sulfated than the corresponding secretions from healthy individuals [16]. In order to further characterize these differences in sulfation and possibly also glycosylation patterns, we compared the structures of sulfated mucin oligosaccharides synthesized by continuously cultured human tracheal cells transformed by siman virus 40. The synthesis of highly sulfated oligosaccharide chains in mucins secreted by normal human epithelial and submucosal cell lines were compared with mucins formed by cystic fibrosis tracheal epithelial and submucosal cell lines.The epithelial cell lines from cystic fibrosis trachea showed a higher rate of sulfate uptake and a significantly higher rate of synthesis and sulfation of high molecular weight chains. Mucins synthesized by each cell line in the presence of ³⁵SO₄ were isolated and oligosaccharide chains were released by beta-elimination and separated by ion exchange chromatography and gel filtration. The sulfated high molecular weight chains synthesized by the cystic fibrosis cell lines were characterized by methylation analysis and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GlcNAc in a ratio of 1:2:2.2 and only one galactosaminitol residue for about every 150-200 sugar residues present. The average molecular size of oligosaccharide chains in these fractions was between 30,000-40,000 daltons.These studies show that increased sulfation of oligosaccharides in mucins synthesized by cells from cystic fibrosis trachea is accompanied by a significant increase in the extension of a basic branched structure present in many of the lower molecular weight oligosaccharides.     

Aggregation of oral bacteria by human salivary mucins in comparison to salivary and gastric mucins of animal origin

Seventeen strains of oral bacteria of the genera Actinomyces (5), Bacteroides (3), and Streptococcus (9) were tested for aggregation by the human whole salivary mucin fraction (HWSM) in comparison to three types of animal mucin preparations from submandibular glands of cow (BSM) and sheep (OSM), and from the stomach of pig (PGM). Considerable variation was seen with respect to the rate and titer of aggregation induced by these mucins. The aggregating activity of HWSM varied widely among the different bacterial strains. The Bacteroides group showed hardly any induced aggregation, whereas the final aggregation titers varied for S. sanguis (3 strains) between 12 and 48, for S. oralis (3 strains) between 6 and 48, for the S. mutans group (3 strains) between 6 and 96, and for the five Actinomyces strains even between 6 and 192. For a particular strain, similar differences in titer were seen between the four mucins. For a human salivary mucin (MG-2) it has been described that sialic acid in the sequence NeuAc (2,3)Gal(1,3)GalNac- was specifically involved in the interaction with S. sanguis strains, in contrast to S. rattus BHT. Our results, however, indicate that this sugar sequence is not a prerequisite for the aggregation of S. sanguis, as animal mucins, devoid of this structure, were equally well or even better capable of inducing aggregation. On the other hand, desialization of BSM and OSM largely abolished their aggregating capability towards S. rattus BHT. Moreover, it was found that BSM and OSM, which are comparable with respect to their major oligosaccharide structure, show considerable differences in aggregating activity towards the same bacterial strain. The results indicate that the interaction and aggregation of oral bacteria with mucins is not necessarily dictated by specific oligosaccharide structures of the mucins, but may be caused instead by common physico-chemical features of the mucins as well.     

Structures of sulfated oligosaccharides in human trachea mucin glycoproteins

The structures of high molecular weight sulfated oligosaccharide chains in mucins purified from the sputum of a patient with cystic fibrosis and blood group H determinant were established. Reduced oligosaccharides released by treatment with alkaline borohydride were separated by ion exchange chromatography on DEAE-Agarose and a fraction containing multisulfated chains was further purified by lectin affinity chromatography to completely remove small amounts of sialylated chains. A major sulfated oligosaccharide fraction containing chains with an average of 160 to 200 sugar residues was isolated by gel filtration on BioGel P-10 columns and individual subfractions were characterized by methylation analysis, periodate oxidation and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GldNAc in a ratio of 1:2:2.1 and only one galactosaminitol residue for every 160-to 200 sugar residues. The average molecular weight of oligosaccharide chains in these fractions was between 27,000 and 40,000 daltons. Structural analysis showed that these high molecular weight chains contained varying amounts of the repeating unit shown in the following oligosaccharide. Only one in about every 10 repeating units contained sulfate esters.Several shorter chains which contain 2 to 3 sulfate esters were also isolated from this multisulfated oligosaccharide fraction. The structures proposed for these oligosaccharides indicate that they are lower molecular weight chains with the same general structure as those found in the high molecular weight sulfated oligosaccharides. Taken collectively, the results of these studies show that a major sulfated oligosaccharide fraction in resporatory mucin purified from the mucus of patients with cystic fibrosis contains high molecular weight branched chains that consist of a repeating oligosaccharide sequence with sulfate linked to the 6 positions of galactose and possibly GlcNAc residues in the side chains.     

Primary structure of the majorO-glycosidically linked carbohydrate unit of human von Willebrand factor

A reduced tetrasaccharide chain was obtained from human von Willebrand factor (vWF) by mild alkaline borohydride treatment. The purification of thisO-glycosidically-linked oligosaccharide was achieved by serial affinity chromatography on immobilized concanavalin A andLens culinaris agglutinin and finally gel filtration. Its structure was determined by a combination of methylation studies and 500 MHz¹H-NMR spectroscopy to be: NeuAc(2-3)Gal(1-3)[NeuAc(2-6)]GalNAc-ol.Abbreviations ConA concanavalin A - LCA Lens culinaris agglutinin - vWF von Willebrand factor - NeuAc N-acetylneuraminic acid - Gal d-galactose - GalNAc-ol N-acetyl-d-galactosaminitol - HMW high molecular weight - LMW low molecular weight     

Sulfated glycans on oral mucin as receptors for Helicobacter pylori

Helicobacter pylori is able to colonize gastric epithelia, causingchronic active gastritis, gastric and duodenal ulcers and presumablygastric malignancies. Attempts to identify the natural reservoirfor this microorganism other than the stomach have been unsuccessful.It is suspected that H.pylori can be transmitted orally, sincethe microorganism has been detected at various sites of theoral cavity. The aim of the present study was to determine whetherH.pylori can bind to salivary mucins, which in vivo coat theoral epithelia, and characterize further the interaction. Bindingof salivary mucins and of synthetic oligosaccharides was studiedIn ELISA and immunoblotting, using specific mono-and polyclonalantibodies, and synthetic neoglycoconjugates. H.pylori boundmost avidly to a highly sulfated sub-population of high molecularweight salivary mucins, secreted from the palatine salivaryglands, and with less avidity to mucin species secreted by thesublingual and submandibular salivary glands, which are lesssulfated. Binding was strongly enhanced upon decreasing pH from6.0 to 5.0. Using synthetic polyacrylamide coupled oligosaccharidesit was found that SO₃-3-Gal and the SO₃-3-Lewis^a blood groupantigen bound to H.pylori. In contrast, binding of sialylatedLewis^a and Lewis^b antigens was much weaker. This study indicatesthat sulfated oligosaccharides on salivary mucins may providereceptor structures for adhesion of H.pylori to oral surfaces. H.pylori saliva sulfomucin nickel oligosaccharide     

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.     

Human salivary gland glycoconjugates: a lectin histochemical study

Summary The glycoconjugate content of normal salivary glands has been extensively investigated in humans by biochemical means and in non-human mammals by histochemical methods. However, there have been few histochemical studies of human tissues. This paper describes the findings obtained in parotid, submandibular and minor salivary glands by applying a panel of 13 biotinylated lectins, directed against a range ofn-linked, fucosylated and galactosylated sequences, using an avidin-peroxidase technique, with appropriate enzymatic and inhibitory sugar controls. The results were generally in accord with those observed in biochemical assays but the use of lectin histochemistry permitted the localizationin situ of small amounts of oligosaccharide and, therefore, allowed the recognition of subtle tissue differences. This study expands the current knowledge on the glycoconjugate composition of salivary glands and their lectin histochemistry and serves as a baseline for further studies, particularly in the field of neoplasia.     

Glycomic mapping of <Emphasis Type="Italic">O-</Emphasis> and <Emphasis Type="Italic">N-</Emphasis>linked glycans from major rat sublingual mucin

Carbohydrate moieties of salivary mucins play various roles in life processes, especially as a microbial trapping agent. While structural details of the salivary O-glycans from several mammalian sources are well studied, very little information is currently available for the corresponding N-glycans. The existence of N-glycans alongside O-glycans on mucin isolated from rat sublingual gland has previously been implicated by total glycosyl compositional analysis but the respective structural data are both lacking. The advent of facile glycomic mapping and sequencing methods by mass spectrometry (MS) has enabled a structural reinvestigation into many previously unsolved issues. For the first time, high energy collision induced dissociation (CID) MALDI-MS/MS as implemented on a TOF/TOF instrument was applied to permethyl derivatives of mucin type O-glycans and N-glycans, from which the linkage specific fragmentation pattern could be established. The predominant O-glycans carried on the rat sublingual mucin were defined as sialylated core 3 and 4 types whereas the N-glycans were determined to be non-bisected hybrid types similarly carrying a sialylated type II chain. The masking effect of terminal sialylation on the tight binding of rat sublingual mucin to Galβ1→4GlcNAc specific lectins and three oligomannose specific lectins were clearly demonstrated in this study.     

Occurrence of two l-threonine (l-serine) dehydratases in the thermophile Chloroflexus aurantiacus

The thermophilic phototrophic prokaryote, Chloroflexus aurantiacus was shown to contain high constitutive l-threonine (l-serine) deaminating activity. Separation of cellular proteins by DE 52-cellulose chromatography and by polyacrylamide gel electrophoresis with subsequent activity staining of the gels yielded two bands, one representing an isoleucine-sensitive, the other one an isoleucine-insensitive form of l-threonine dehydratase. Both enzymes had a molecular weight of 120,000 but were distinguished by their different affinities to the two substrates, l-threonine and l-serine.Abbreviations SDH l-serine dehydratase - TDH l-threonine dehydratase     

Binding and precipitating activities ofErythrina lectins with complex type carbohydrates and synthetic cluster glycosides. A comparative study of the lectins fromE. corallodendron,E. cristagalli,E. flabelliformis,andE. indica

Erythrina lectins possess similar structural and carbohydrate binding properties. Recently, tri- and tetra-antennary complex type carbohydrates with non-reducing terminal galactose residues have been shown to be precipitated as tri- and tetravalent ligands, respectively, with certainErythrina lectins [Bhattacharyya L, Haraldsson M, Brewer CF (1988) Biochemistry 271034-41]. The present work describes a comparative study of the binding and precipitating activities of fourErythrina lectins,viz. E. corallodendron, E. cristagalli, E. flabelliformis, andE. indica, with multi-antennary complex type carbohydrates and synthetic cluster glycosides. The results show that though their binding affinities are very similar, theErythrina lectins show large differences in their precipitating activities with the carbohydrates. The results also indicate significant dependence of the precipitating activities of the lectins on the core structure of the carbohydrates. These findings provide a new dimension to the structure-activity relationship of the lectins and their interactions with asparagine-linked carbohydrates.Abbreviations EAL, ECorL, ECL, EFL, and EIL represent the lectins from the seeds ofErythrina arborescens, - E. corallodendron, E. cristagalli, E. flabelliformis, andE. indica respectively - AFOS thetri-antennary complex type oligosaccharide from asialofetuin - AFGP the tri-antennary glycopeptide from asialofetuin - MeGal methyl -d-galactopyranoside Unless stated otherwise all sugars are in thed-configuration.     

A lipid linked oligosaccharide which contains hexosamine,mannose and glucose

Summary Previous work showed that liver microsomes catalyze the transfer of glucose from dolichol monophosphate glucose to an endogenous acceptor believed to be a dolichol pyrophosphate derivative of an oligosaccharide. This oligosaccharide has now been prepared on a larger scale so as to permit the determination of its sugars. The purification procedure includes, as a last step, a thin layer chromatography on kieselguhr-silica gel to obviate glucose-containing contaminants. After complete hydrolysis mannose, glucose and a small amount of hexosamine were detected.Abbreviations DolMp dolichyl monophosphate - DolDP dolichyl diphosphate - GEA glucosylated endogenous acceptor solvent 1103, chloroform-methanol-water (1:1:0.3) This paper is dedicated to Dr.Luis Federico Leloir on the occasion of his 70th birthday.     

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.

     

Isolation and characterization of acetamidocinnamate acylase,a new enzyme suitable for production of l-phenylalanine

Summary A new acylase catalyzing the deacetylation of acetamidocinnamic acid (ACA) was found in strains of Brevibacterium sp. Such strains could be isolated from soil samples by their ability to grow on ACA as well as on l-phenylalanine. A 110-fold enrichment of the enzyme with an over-all yield of 48% was obtained in 4 steps resulting in an electrophoretically pure preparation of 28.6 U·mg^-1. Important enzymological data concerning the application of the enzyme are: K _M (ACA) 0.45 mM, pH-optimum 7.5, heat stability up to 52°C, molecular weight of 50.000 Dalton, two subunits. Deacetylation of ACA resulted in phenylpyruvate via the unstable enamine-imine derivative. Coupling the acylase with l-phenylalanine dehydrogenase proved to be an alternative route for l-phenylalanine production avoiding substrate inhibition by phenylpyruvate and its instability. The substrate specifity of ACA-acylase revealed that the enzyme probably acts as a dipeptidase in its biological function.Abbreviations ACA acetamidocinnamate, acetamidocinnamic acid - FPLC fast protein liquid chromatography - pheDH l-Phenylalanine dehydrogenase - HicDH Hydroxyisocaproate dehydrogenase - OD optical density - BSA bovine serum albumin - FDH formate dehydrogenase Dedicated to Professor Dr. H. J. Rehm on the occasion of his 60th birthday     

Mutants of the obligate methylothroph Methylobacillus flagellatum KT defective in genes of the ribulose monophosphate cycle of formaldehyde fixation

Pseudomonas cepacia MBA4 able to utilize monobromoacetic acid as a sole source of carbon and energy was isolated from soil by enrichment culture. In batch culture the ability to utilize the substrate was conferred by a single halidohydrolase-type dehalogenase which demonstrated a high activity towards the enrichment substrate. The purified enzyme, designated as dehalogenase IVa by activity-stain polyacrylamide gel electrophoresis, had a relative molecular weight of 45,000 and was comprised of two electrophoretically identical subunits with relative molecular weights of 23,000. Dehalogenase IVa demonstrated isomer specificity, being active towards the L-isomer of 2-monochloropropionic acid only. The significance of activity-stain polyacrylamide gel electrophoresis in characterizing dehalogenases and their ubiquitous distribution among bacterial genera are discussed.Abbreviations MCA Monochloroacetic acid - DCA dichloroacetic acid - MBA monobromoacetic acid - 2MPCA 2-monochloropropionic acid - 2MBPA 2-monobromopropionic acid     

Chromosomal structure of populations of drosophila flavopilosa studied in larvae collected in their natural breeding sites

Summary D. flavopilosa Frey, is a neotropical species of Drosophila living in Chile that, together with 13 other species, has been included by Wheeler, Takada und Brncic (1962) in the flavopilosa species group. Despite the fact that it has not been possible to breed this species in the laboratory, the circumstance that its habitat is well known, has allowed the study of the chromosomal structure of some natural populations of this species, analyzing larvae directly taken to the laboratory from their natural breeding sites: the flowers of the solanacean Cestrum parqui L'Héritier.The present paper describes the mitetic and salivary gland chromosomes of D. flavopilosa, and gives a composite map of the Standard gene arrangement. In central Chile, natural populations of this species are polymorphic with respect to the gene orders in their chromosomes, due to the presence of four independent inversions, all located in one of the six chromosomes (the right arm of the V-chromosome). Quantitative data on the distribution of the inversions shows that there is an altitudinal gradient in the frequencies of two of the four inversions. Heterozygotes for Inversion A, are more frequent at high altitudes than at sea level. On the contrary, heterozygotes for inversion B, are abundant at sea level, but practically disappear at high altitudes. These altitudinal clines have been observed in two valleys near Santiago, Chile, which run from the Andes mountains to the Pacific coast.The work reported in this article has been carried out under Contract AT (30-1) 2465 US Atomic Energy Commission, and partially supported by Grants from the University of Chile, Faculty of Medicine and the Rockefeller Foundation under a joint program.     

Glutamate-mediated synaptic transmission between neuron B4 and salivary cells ofHelisoma trivolvis

In this study, we have further characterized the morphology and physiology of the neuroglandular synapse between the identified buccal neuron, B4, and the salivary gland ofHelisoma. We demonstrate that the coupling coefficient between salivary cells within an individual acinus is approximately 1.0. We also demonstrate that synapses within the salivary gland are located near a superficial muscle layer. We examine the effects of glutamate on the salivary gland and on the B4-salivary gland EPSP.l-glutamate produces a transient, rapid onset depolarization of salivary gland cells. The response is mimicked by high concentrations ofl-homocysteic acid, but not by NMDA,l-aspartate,d-glutamate or kainate. The response is blocked by the presence ofl- ord-glutamate in the bath, but not by CNQX, DNQX, DGG,d-AP5, orl-AP3. The depolarization is primarily dependent on the presence of calcium in the bathing solution. When eitherl- ord-glutamate is present in the bathing solution, the amplitude of the B4-salivary gland EPSP is reversibly reduced. The similar pharmacological properties of the response of the salivary gland to glutamate and the B4 epsp indicate thatl-glutamate is a strong candidate for the fast excitatory neurotransmitter at theHelisoma neuroglandular synapse.     

Structural analysis of a polysaccharide from<Emphasis Type="Italic">Chlorella kessleri</Emphasis> by means of gas chromatography—mass spectrometry of its saccharide alditols

Interpretation of gas chromatographic-mass spectrometric data of oligosaccharide alditols was used to determine their structures and to derive the structure of a water soluble polysaccharide isolated fromChlorella kessleri.¹H- and¹³C-NMR was employed to assess the configuration of glycosidic bonds and individual monosaccharides were assigned to thel ord series by means of gas chromatography of the acetylated (S)-2-butyl glycosides.