Further characterization, by a combined high-performance liquid chromatography/1H-NMR approach, of the heterogeneity displayed by the neutral carbohydrate chains of human bronchial mucins

From bronchial mucins of cystic fibrosis patients with blood group O, the carbohydrate chains were released in the form of oligosaccharide-alditols by alkaline borohydride treatment. Application of high-performance liquid chromatography directly on the pool of neutral oligosaccharides afforded 23 fractions. Twenty oligosaccharide structures were characterized by employing 500-MHz 1H-NMR spectroscopy in conjunction with sugar analysis. Thirteen among these had been revealed before to occur in human bronchial mucins [Van Halbeek, H., Dorland, L., Vliegenthart, J. F. G., Hull, W. E., Lamblin, G., Lhermitte, M., Boersma, A., and Roussel, P. (1982) Eur. J. Biochem. 127, 7-20], when paper-chromatographic fractionation of this pool of neutral oligosaccharides was employed. High-performance liquid chromatography enabled to obtain another seven pentasaccharide and hexasaccharide alditols; the largest-size representatives are: (Formula see text). Thereby, this approach afforded deeper insight into the structural heterogeneity displayed by the carbohydrate chains of bronchial mucins.     

Structural analysis of 20 oligosaccharide-alditols released from the jelly coat of Rana palustris eggs by reductive beta-elimination characterization of the polymerized sequence [Gal(beta1, 3)GalNAc(alpha1-4)]n.

The eggs of amphibians are surrounded by three to eight layers of jelly coats. This extracellular matrix is mainly composed of hydrated mucin-type glycoproteins. These highly glycosylated molecules are synthesized by oviduct and play an important role in the fertilization process. Recent structural analyses have shown the strict species-specificity of the O-linked oligosaccharides which constitute 60-70% of these oviducal mucins. Consequently, these carbohydrate chains represent new phenotypic markers, and from a biological point of view, can influence parasite tropism or can be involved in species-specific interaction of gametes. The primary structure of 20 oligosaccharide-alditols, released by alkali/borohydride treatment from the mucin of Rana palustris egg jelly coats, was established by 1H and 13C-NMR analysis. Thirteen of these components possess new structures and the polymerization of the sequence Gal(beta1-3)GalNAc(alpha1-4) characterizes the species-specificity of R. palustris.     

Structural studies of the carbohydrate chains of human gamma-interferon

Human gamma-interferon (IFN-gamma) was prepared biotechnologically using Chinese hamster ovary cells. These cells were shown to be able to produce glycosylated IFN-gamma. Sugar analysis revealed the presence of Man, Gal, GlcNAc, NeuAc and Fuc residues in a molar ratio of 3.8:2.0:3.5:0.6:0.4 suggesting the occurrence of N-glycosidically linked N-acetyllactosamine type of carbohydrate chains. For structure determination of these chains, the glycoprotein was subjected to the hydrazinolysis procedure, yielding oligosaccharide-alditols. The latter compounds were analysed by 500-MHz 1H-NMR spectroscopy. The carbohydrate material was found to consist of biantennary structures, exhibiting microheterogeneity as to the terminal sialic acids and the core Fuc residue: (Formula: see text). As similar carbohydrates are present on several human secreted proteins, this glycosyl group is not expected to be immunogenic in man. It remains to be established to what extent the carbohydrate chains of this biotechnologically produced IFN-gamma are identical to those of naturally occurring human IFN-gamma.     

Structural analysis of the oligosaccharide-alditols released by reductive β-elimination from oviducal mucins of Rana temporaria

The carbohydrate chains of the mucins which constitute the jelly coat surrounding the eggs of Rana temporaria were released by alkaline borohydride treatment. Neutral and acidic oligosaccharide-alditols were purified by ion-exchange chromatography and HPLC. From the structural analysis, based upon 1H and 13C-NMR spectroscopy in combination with MALDI-TOF, the following glycan units are proposed. Abbreviations: MALDI-TOF, matrix assisted laser desorption ionization - time of flight; HPLC, high performance liquid chromatography; COSY, correlation spectroscopy; HSQC, heteronuclear single-quantum coherence spectroscopy; HMQC, heteronuclear multiple-quantum coherence spectroscopy; ROESY, rotating-frame overhauser enhancement spectroscopy; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; GalNAc-ol, N-acetylgalactosaminitol; GlcA, glucuronic acid     

The complexity of mucins

Mucins represent the main components of gel-like secretions, or mucus, secreted by mucosae or some exocrine glands. These high-molecular-weight glycoproteins are characterized by the large number of carbohydrate chains O-glycosidically linked to the peptide. The determination of mucin molecular weight and conformation has been controversial for several reasons: 1) the methods used to solubilize mucus and to purify mucins are different and 2) the molecules have a strong tendency to aggregate or to bind to other molecules (peptides or lipids). Recently, electron microscopy has shown the filamentous shape of most mucins and their polydisperse character which, in some secretions, might correspond to a polymorphism of the peptide part of these molecules. The recent development of high pressure liquid chromatography and high-resolution proton NMR spectroscopy has allowed major progress in the structural study of mucin carbohydrate chains. These chains may have from 1 to about 20 sugars and bear different antigenic determinants, such as A, B, H, I, i, X, Y or Cad antigens. In some mucins, such as human respiratory mucins, the carbohydrate chain diversity is remarkable, which raises many questions. Mucins are molecules located at the interface between mucosae and the external environment. The carbohydrate chain diversity might allow many interactions between mucins and microorganisms and play a major role in the colonization or the defense of mucosae.     

Acquisition of species-specific O-linked carbohydrate chains from oviducal mucins in Rana arvalis. A case study.

The extracellular matrix surrounding amphibian eggs is composed of mucin-type glycoproteins, highly O-glycosylated and plays an important role in the fertilization process. Oligosaccharide-alditols were released from the oviducal mucins of the anuran Rana arvalis by alkali-borohydride treatment in reduced conditions. Neutral and acidic oligosaccharides were fractionated by ion-exchange chromatographies and purified by HPLC. Each compound was identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) spectrometry, NMR spectroscopy, electrospray ionization-tandem mass spectroscopy (ESI-MS/MS) and permethylation analyses. This paper reports on the structures of 19 oligosaccharide-alditols, 12 of which have novel structures. These structures range in size from disaccharide to octasaccharide. Some of them are acidic, containing either a glucuronic acid or, more frequently, a sulfate group, located either at the 6 position of GlcNAc or the 3 or 4 positions of Gal. This latter sulfation is novel and has only been characterized in the species R. arvalis. This structural analysis led to the establishment of several novel carbohydrate structures, demonstrating the structural diversity and species-specificity of amphibian glycoconjugates.     

The carbohydrate moiety of human platelet glycocalicin: the structures of the major Asn-linked sugar chains

Glycocalicin, a predominant glycoprotein on the human platelet surface, has been purified from a platelet suspension by means of sonication, ammonium sulfate precipitation and acid treatment followed by chromatography on columns of wheat germ agglutinin-Sepharose and Mono Q. Asparagine-linked (N-linked) oligosaccharides were released by hydrazinolysis, and then N-acetylated and reduced with NaBH4 or NaB3H4. The released carbohydrate chains were found to be of the complex-type from their interaction with immobilized lectin columns. The structures of the two major oligosaccharide-alditols separated by ion-exchange chromatography on a Mono Q column were investigated by means of methylation analysis, glycosidase digestion, and Smith periodate degradation, and they were assigned as typical di- and trisialylated complex-type oligosaccharide-alditols with two and three peripheral chains consisting of Gal-GlcNAc sequences, respectively.     

The structure of a fucose-containingO-glycosidic carbohydrate chain of human platelet glycocalicin

The hydrazinolysis procedure currently used for the release ofN-glycosidic carbohydrate chains was applied to glycocalicin. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis into a neutral (5%) and several acidic fractions. The neutral compounds were passed over Bio-Gel P-4. SomeN-glycosidic oligosaccharide-alditols, of theN-acetyllactosamine type as well as of the oligomannoside type, were found to be present. However, oligosaccharide-alditols derived fromO-glycosidic carbohydrate chains were also found, indicating a partial cleavage of GalNAc1-OSer/Thr linkages under the hydrazinolysis conditions applied. One of the neutralO-glycosidic components was characterized, by 500-MHz¹H-NMR spectroscopy in combination with sugar analysis, as the following pentasaccharidealditol: In addition the afuco analogue of this compound was obtained.     

Primary structure determination of seven novel N-linked carbohydrate chains derived from hemocyanin of Lymnaea stagnalis. 3-O-methyl-D-galactose and N-acetyl-D-galactosamine as constituents of xylose-containing N-linked oligosaccharides in an animal glycoprotein

Hemocyanin from the freshwater snail Lymnaea stagnalis is a high-molecular-mass copper-containing glyco-protein which functions as oxygen carrier in the hemolymph. To release the carbohydrate chains, the protein was digested by pronase followed by hydrazinolysis and reduction. The oligosaccharide-alditols were purified by gel permeation chromatography on Bio-Gel P-4, followed by HPLC on a Lichrosorb-NH2 column. Using 500-MHz 1H-NMR spectroscopy, in conjunction with sugar, methylation and deamination analysis, the following seven novel primary oligosaccharide structures could be unravelled. (Formula: see text).     

Determination of the structure of the carbohydrate chains of prostaglandin endoperoxide synthase from sheep

Prostaglandin endoperoxide synthase was isolated from sheep seminal vesicles. Sugar analysis of the glycoprotein revealed the presence of mannose and N-acetylglucosamine only. The carbohydrate moiety was released from the polypeptide backbone by hydrazinolysis. After re-N-acetylation and reduction, the resulting mixture of oligosaccharide-alditols was fractionated on Bio-Gel P-4 and their structures were investigated by 500-MHz1H-NMR spectroscopy. The carbohydrate chains turned out to be of the oligomannoside type containing six to nine mannose residues. The largest and most abundant compound was established to be: (formula; see text) For the smaller structures heterogeneity occurs with respect to the outer alpha(1----2)-linked mannose residues. Furthermore, a small amount of Man6GlcNAc-ol (artefact of the hydrazinolysis procedure) was detected by 1H-NMR spectroscopy and fast atom bombardment mass spectrometry.     

Confocal evaluation of native and induced lectin binding contributes to discriminate between lingual gland glycocomponents in quail

A confocal analysis was performed on the quail (Coturnix coturnix japonica) lingual salivary glands where the carbohydrate chains were studied by lectin histochemistry. For this purpose, appropriate FITC- and TRITC-conjugates were used for double binding also accomplished with sialidase digestion. The glycosidic components of the quail lingual salivary glands were found to be heterogeneously distributed on the different secretory structures as well as on the single secretory elements of each adenomere. The rostral portion of the anterior lingual gland was found to only secrete neutral glycocomponents, characterized by terminal beta-galactose, N-acetylgalactosamine and fucose residues in contrast to the caudal portion that was shown to be extremely heterogeneous and to produce sialylated glycoconjugates characterized by the terminal sequences sialic acid-beta-galactose-N-acetylgalactosamine, sialic acid-beta-galactose-N-acetylglucosamine, and sialic acid-alpha-N-acetylgalactosamine partly codistributed within secretory adenomeres. The posterior lingual gland was observed to be the major contributor to the secretion of salivary mucins containing sialoglycoconjugates with terminal sialic acid residues linked to beta-galactose-N-acetylgalactosamine or alpha-N-acetylgalactosamine often located in distinct secretory elements.     

Human airway mucin glycosylation: a combinatory of carbohydrate determinants which vary in cystic fibrosis

Human airway mucins represent a very broad family of polydisperse high molecular mass glycoproteins, which are part of the airway innate immunity. Apomucins, which correspond to their peptide part, are encoded by at least 6 different mucin genes (MUC1, MUC2, MUC4, MUC5B, MUC5AC and MUC7). The expression of some of these genes (at least MUC2 and MUC5AC) is induced by bacterial products, tobacco smoke and different cytokines.Human airway mucins are highly glycosylated (70–80% per weight). They contain from one single to several hundred carbohydrate chains. The carbohydrate chains that cover the apomucins are extremely diverse, adding to the complexity of these molecules. Structural information is available for more than 150 different O-glycan chains corresponding to the shortest chains (less than 12 sugars).The biosynthesis of these carbohydrate chains is a stepwise process involving many glycosyl- or sulfo-transferases. The only structural element shared by all mucin O-glycan chains is a GalNAc residue linked to a serine or threonine residue of the apomucin. There is growing evidence that the apomucin sequences influence the first glycosylation reactions. The elongation of the chains leads to various linear or branched extensions. Their non-reducing end, which corresponds to the termination of the chains, may bear different carbohydrate structures, such as histo-blood groups A or B determinants, H and sulfated H determinants, Lewis a, Lewis b, Lewis x or Lewis y epitopes, as well as sialyl- or sulfo- (sometimes sialyl- and sulfo-) Lewis a or Lewis x determinants. The synthesis of these different terminal determinants involves three different pathways with a whole set of glycosyl- and sulfo-transferases.Due to their wide structural diversity forming a combinatory of carbohydrate determinants as well as their location at the surface of the airways, mucins are involved in multiple interactions with microorganisms and are very important in the protection of the underlying airway mucosa.Airway mucins are oversulfated in cystic fibrosis and this feature has been considered as being linked to a primary defect of the disease. However, a similar pattern is observed in mucins from patients suffering from chronic bronchitis when they are severely infected. Airway mucins from severely infected patients suffering either from cystic fibrosis or from chronic bronchitis are also highly sialylated, and highly express sialylated and sulfated Lewis x determinants, a feature which may reflect severe mucosal inflammation or infection.These determinants are potential sites of attachment for Pseudomonas aeruginosa, the pathogen responsible for most of the morbidity and mortality in cystic fibrosis, and the expression of the sulfo- and glycosyl-transferases involved in their biosynthesis is increased by TNF.In summary, airway inflammation may simultaneously induce the expression of mucin genes (MUC2 and MUC5AC) and the expression of several glycosyl- and sulfo-transferases, therefore modifying the combinatory glycosylation of these molecules.     

Sulfated N-linked carbohydrate chains in porcine thyroglobulin

N-linked carbohydrate chains of porcine thyroglobulin were released by the hydrazinolysis procedure. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis, the acidic fractions were further separated by high-performance liquid chromatography on Lichrosorb-NH2, and analyzed by 500-MHz 1H-NMR spectroscopy and, partially, by permethylation analysis. Of the acidic oligosaccharide-alditols, the following sulfated carbohydrate chains could be identified: NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3[(SO3Na----3)Gal beta 1----4GlcNAc beta1----2-Mana alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc-ol and NeuAc alpha 2----6Gal beta 1----4(SO3Na----)0-1 GlcNAc beta 1----2-Man alpha 1----3[NeuAc alpha 2----6Gal beta 1----4(SO3Na----6)1-0GlcNAc beta 1----2Man alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc- ol. The sulfated structural elements for porcine thyroglobulin form novel details of N-linked carbohydrate chains. They contribute to the fine structure of these oligosaccharides and are another type of expression of microheterogeneity.     

Pseudomonas aeruginosa binds to neoglycoconjugates bearing mucin carbohydrate determinants and predominantly to sialyl-Lewis x conjugates.

Pseudomonas aeruginosa plays an important role in the colonization of the airways of patients suffering from cystic fibrosis. It binds to the carbohydrate part of respiratory and salivary mucins and its binding to cystic fibrosis mucins is even higher, suggesting that qualitative or/and quantitative modifications of the carbohydrate chains may be involved in this process. In order to find out the best carbohydrate receptors for P.aeruginosa, a flow cytometry technique using a panel of polyacrylamide based glycoconjugates labeled with fluorescein was developed. The neoglycoconjugates contained neutral, sialylated or sulfated chains analogous to carbohydrate determinants found at the periphery of respiratory mucins (Le(a), Le(y), Le(x), sialyl- and 3'-sulfo-Le(x), and blood group A determinants). We used also neoglycoconjugates containing Gal(alpha1-2)Galbeta and sialyl- N -acetyllactosamine determinants. The interaction of these glycoconjugates with the nonpiliated strain of P.aeruginosa, 1244-NP, was saturable except for the glycoconjugates containing blood group A or sialyl- N -acetyllactosamine epitopes. The measure of Kd indicated that strain 1244-NP had a higher affinity for the glycoconjugate bearing the sialyl-Le(x)determinant than for all the other glycoconjugates studied. The role of sialic acid was confirmed by competition assay using mainly sialylated mucin glycopeptides. In order to find out if this behavior was the same for pathological strains as for the 1244-NP mutant, four mucoid strains of P.aeruginosa isolated from cystic fibrosis patients were analyzed with the Le(x)neoglycoconjugate, its sialylated and its sulfated derivatives. Individual variations in the binding of these strains to the three glycoconjugates were observed. However, three strains out of four had a higher affinity for the sialyl-Le(x)than for the 3'-sulfo-Le(x)derivative.     

Typing of core and backbone domains of mucin-type oligosaccharides from human ovarian-cyst glycoproteins by 500-MHz 1H-NMR spectroscopy

Human blood-group A active glycoproteins from ovarian-cyst fluid were subjected to Smith degradation and subsequent beta-elimination. The resulting oligosaccharide-alditols represent the core and backbone domains of the O-linked carbohydrate chains. Nine of these, ranging in size from disaccharides to hexasaccharides, were investigated by 1H-NMR spectroscopy. Their primary structures could be adequately characterized. In particular, the core types, i.e. the substitution patterns of N-acetylgalactosaminitol (GalNAc-ol) as well as the types of backbone, i.e. the linkage types of alternating Gal-GlcNAc sequences, were unambiguously identified. The core type GlcNAc beta(1-3)GalNAc-ol is described for the first time as occurring in ovarian-cyst glycoprotein.     

Oligosaccharide structures of mucins secreted by the human colonic cancer cell line CL.16E.

Cl.16E, a stably differentiated clonal derivative of the human colonic cancer cell line HT29, was used to investigate the structure of oligosaccharide chains of mucins in colonic cancer. Secretory mucins were purified by equilibrium density gradient centrifugation in CsCl. Oligosaccharide side chains were isolated after beta-elimination. Compositional analysis of oligosaccharide-alditols performed after purification by gel filtration on a Bio-gel P-6 column showed 1) that GalNAc residues were located exclusively at the reducing ends of the chains, and 2) that fucose was absent from the preparation. Oligosaccharide-alditols were separated by high performance liquid chromatography (HPLC) on quaternary amine packings into a minor neutral fraction representing about 6.5% by weight of released oligosaccharides and four acidic fractions. Two acidic fractions, namely FI and FII encompassing mono- and disialylated structures, respectively, and containing 78% of total oligosaccharide alditols, were separated by HPLC. Structural determinations were carried out using methylation analysis, 1H NMR spectroscopy, and fast atom bombardment-mass spectrometry. Twelve oligosaccharide structures were determined which ranged in size from 3 to 8 residues. These oligosaccharides were based on core types 1, 2, and 4. Elongation of oligosaccharide chains was terminated by addition of sialic acid in alpha 2-3 linkage to Gal beta 1-3R and to Gal beta 1-4R residues. The predominant structure was a hexasaccharide (fraction FII-4). This contrasts with normal colonic mucins whose oligosaccharides were previously found to be based on core 3 structures and carry sialic acids in alpha (2-6) linkage to Gal beta 1-3R, to Gal beta 1-4R, and to GalNAc alpha-R (Podolsky, D.K. (1985) J. Biol. Chem. 260, 8262-8271; Podolsky, D.K. (1985) J. Biol. Chem. 260, 15510-15515). Collectively our findings suggest that Cl.16E colon cancer cells are able to synthesize mucin oligosaccharides of gastric type whose elongation is truncated by premature sialylation.     

Diversity of O-linked glycosylation patterns between species. Characterization of 25 carbohydrate chains from oviducal mucins of Rana ridibunda.

Amphibia egg jelly coats are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they pass along the different oviducal portions. Mucin type glycoproteins are the major constituents of the egg jelly coats. In this study, the O-linked carbohydrate chains of the jelly coats surrounding the eggs of Rana ridibunda were released by alkaline borohydride treatment. Fractionation of the mixture of O-linked oligosaccharide-alditols was achieved by a combination of chromatographic techniques including gel-permeation chromatography, ion-exchange chromatography and high-performance liquid chromatography using an amino-bonded silica column. The primary structures of these O-glycans were determined by one-dimensional and two-dimensional 1H-NMR spectroscopy and matrix-assisted laser-desorption-ionization-time-of flight mass spectrometry. 25 oligosaccharide structures, possessing a core consisting of Gal(beta1-3)GalNAc-ol with or without branching through a GlcNAc residue linked (beta1-6) to the GalNAc residue (core type 2 or core type 1, respectively) are described. The most representative antennae are: HSO3(6)[Fuc(alpha1-3)]GlcNAc; Gal(beta1-2)Gal; Gal(beta1-2)Gal(alpha1-3)[Fuc(alpha1-2)]Gal; GlcA(beta1-3)-Gal(beta1-3)[Fuc(alpha1-2)]Gal; GalNAc(alpha1-4)Gal(beta1-4)Gal; Gal(beta1-3)GalNAc(alpha1-4)Gal(beta1-4)Gal and GlcA(beta1-3)Gal(beta1-3)GalNAc. These results confirm the species-specific O-glycosylation of Amphibia oviducal mucins. The significance of this observation should be linked to a symbiotic role of carbohydrates involved in host-parasite interactions.     

Structures of the sugar chains of recombinant human granulocyte-colony-stimulating factor produced by Chinese hamster ovary cells

Recombinant human granulocyte-colony-stimulating factor (G-CSF) was purified from Chinese hamster ovary cells transfected with human G-CSF cDNA. The recombinant human G-CSF was treated with alkaline borohydride and the oligosaccharide-alditols liberated were fractioned by gel filtration on a Bio-Gel P-4 column, followed by high-performance liquid chromatography by use of a strong anion exchanger. Two oligosaccharide-alditols were obtained and their structures were identified by component analysis and 500-MHz 1H-NMR spectroscopy. The structures of the sugar chains were NeuAc alpha 2-3Gal beta 1-3GalNAcol and NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAcol.     

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.