FAB-MS characterization of sialyl Lewis x determinants on polylactosamine chains of human airway mucins secreted by patients suffering from cystic fibrosis or chronic bronchitis.

Although a large body of structural data exists for bronchial mucins from cystic fibrosis (CF) and chronic bronchitis (CB) patients, little is known about terminal structures carried on poly-N-acetyllactosamine antennae. Such structures are of interest because they are potential ligands for bacterial adhesins and other lectins. In this study, we have used fast atom bombardment mass spectrometry (FAB-MS) to examine terminal sequences released by endo-beta-galactosidase from O-glycans obtained by reductive elimination of bronchial mucins purified from the sputum of 8 CF and 8 CB patients. Our data show that, although the polylactosamine antennae of CF and CB mucins have several terminal sequences in common, they differ significantly in their sialyl Lewis(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAcbeta1-) content. Thus all examined mucins from CF patients carry sialyl Lewis(x) on their polylactosamine antennae, whereas this type of epitope is present on only three out of the eight CB mucins examined, notably in the airways of one CB patient which were heavily infected by Pseudomonas aeruginosa as are the airways of all the CF patients. This suggests that, in airway mucins, the expression of sialyl Lewis(x) on polylactosamine antennae is probably more related to inflammation and infection than to a direct effect of the CF defect.

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 TNFalpha. 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.

O-glycan variability of egg-jelly mucins from Xenopus laevis: characterization of four phenotypes that differ by the terminal glycosylation of their mucins.

Eggs from Xenopus laevis are surrounded by several layers of jelly that are needed for proper fertilization. Jelly coat is composed of high-molecular-mass glycoconjugates to which are bound many globular proteins. O-glycans released from the jelly coat of X. laevis have been partially described in previous studies. In this study, we compared the glycosylation pattern of the egg jelly coat isolated from six specimens of X. laevis. The O-glycans were released from jelly coats by alkali/borohydride treatment. Structural characterization was performed through a combination of one- and two-dimensional (1)H-NMR and methylation analysis. This allowed the description of a new family of sulphated O-glycans present in jelly coats of all X. laevis. However, the jelly O-glycans showed a low extent of polymorphism between specimens. This intra-specific variability was restricted to the terminal substitution of O-linked oligosaccharides. The differential expression of two glycosyltransferase [an alpha-(1-->4) galactosyltransferase and an alpha-(1-->3) fucosyltransferase] activities resulted in the characterization of four phenotypes of X. laevis. Furthermore, electrophoretic analysis suggested that the high-molecular-mass fraction of jelly coat was mostly composed of mucin-type glycoproteins. Blot analysis with lectins confirmed that the glycan variability was borne by these mucin-type components. However, fertilization assays suggested that the glycan polymorphism had no repercussion on egg fertilizability.

The sialylation of bronchial mucins secreted by patients suffering from cystic fibrosis or from chronic bronchitis is related to the severity of airway infection.

Bronchial mucins were purified from the sputum of 14 patients suffering from cystic fibrosis and 24 patients suffering from chronic bronchitis, using two CsBr density-gradient centrifugations. The presence of DNA in each secretion was used as an index to estimate the severity of infection and allowed to subdivide the mucins into four groups corresponding to infected or noninfected patients with cystic fibrosis, and to infected or noninfected patients with chronic bronchitis. All infected patients suffering from cystic fibrosis were colonized by Pseudomonas aeruginosa. As already observed, the mucins from the patients with cystic fibrosis had a higher sulfate content than the mucins from the patients with chronic bronchitis. However, there was a striking increase in the sialic acid content of the mucins secreted by severely infected patients as compared to noninfected patients. Thirty-six bronchial mucins out of 38 contained the sialyl-Lewis x epitope which was even expressed by subjects phenotyped as Lewis negative, indicating that at least one alpha1,3 fucosyltransferase different from the Lewis enzyme was involved in the biosynthesis of this epitope. Finally, the sialyl-Lewis x determinant was also overexpressed in the mucins from severely infected patients. Altogether these differences in the glycosylation process of mucins from infected and noninfected patients suggest that bacterial infection influences the expression of sialyltransferases and alpha1,3 fucosyltransferases in the human bronchial mucosa.

Elastase derived elastin peptides: putative autoimmune targets in giant cell arteritis.

OBJECTIVE: Histological analysis of giant cell arteritis (GCA) reveals a granulomatous reaction around the internal elastic lamina. Elastolysis by multinucleated giant cells has also been reported. We investigated elastin derived peptides as putative recall antigens for peripheral blood mononuclear cells (PBMC) from patients with GCA. METHODS: PBMC were collected from 17 patients with GCA (Group 1), 17 patients with vascular diseases, connective tissue diseases, or polymyalgia rheumatica without GCA (Group 2), and 17 healthy controls (Group 3). Cultures of PBMC with different elastin derived peptides or elastase were analyzed. RESULTS: A proliferative response was obtained only with elastate derived elastin peptides in 12/13 untreated patients with GCA. Steroid treatment was believed to abolish this proliferative response in 4 patients with GCA. PBMC from only 3/34 non-GCA subjects responded to these antigens. No proliferative response was obtained for other elastin derived peptides or elastase in any subject. CONCLUSION: Degradation of native elastin by leukocyte elastase can provide elastin derived peptides that act as autoimmune targets for T cells in GCA.

The binding of surface proteins from Staphylococcus aureus to human bronchial mucins.

Colonization by Staphylococcus aureus is frequently observed in obstructive lung diseases, particularly in cystic fibrosis. It has been shown that the bacteria bind to mucins, the main constituent of bronchial secretions. The binding mechanism, however, remains unclear. We have investigated the interactions of two strains of S. aureus, one mucoid and one nonmucoid, with human bronchial mucins. Using a solution phase assay, the binding capacity of the two strains to radiolabelled bronchial mucins was assessed. The bacterial constituents were released by lysostaphin lysis and the surface components of the nonmucoid strain were extracted with the use of a detergent (3-([3-cholamidopropyl] dimethylammonio)-1-propane sulphonate (CHAPS)). All were analysed for mucin-binding using an overlay assay. The amount of mucins bound to the nonmucoid strain was threefold greater than that of the mucoid strain. In the lysostaphin extract from the mucoid strain, only a 57 kDa protein faintly bound 125I-labelled mucins, whereas three mucin-binding proteins (52, 57 and 71 kDa) were identified from the nonmucoid strain. Two surface proteins, one major at 60 kDa and one minor at 71 kDa, bound radiolabelled bronchial mucins and their binding was almost completely inhibited by ovine submaxillary mucin. These results indicate: 1) differences in the mucin-binding capacity from one strain of S. aureus to another; and 2) the presence of external and internal adhesins binding to human respiratory mucins in the nonmucoid strain.

Expression of human mucous proteinase inhibitor in respiratory tract: a study by in situ hybridization.

Human mucous proteinase inhibitor (MPI) is present in bronchial secretions, where it participates in the protection of lung structures against degradation by leukocyte elastase. The protein has been localized by immunohistochemical studies and immunogold labeling essentially in the serous cells of the submucosal glands and also in the surface epithelial cells of central and peripheral airways. However, until now no gene expression study has been performed at the tissue level. In this study, in situ hybridization was used to precisely study MPI mRNA expression in bronchial tissue sections with a specific radiolabeled oligonucleotide probe. By light microscopy, MPI gene expression was localized exclusively in the serous and seromucinous acini of the submucosal glands of large airways. The MPI gene was expressed in submucosal glands of normal and carcinomatous tissue sections, whereas it was not expressed in bronchial and bronchiolar surface epithelia.

Influence of iron depletion on growth kinetics, siderophore production, and protein expression of Staphylococcus aureus.

Growth rates, siderophore secretion, and bacterial proteins of two clinical isolates of Staphylococcus aureus were studied over 72 h of growth in iron-supplemented and iron-restricted chemically defined media. Under iron restriction the growth rates were decreased to different extents depending on the strain. Production of siderophore was detected in the mid-exponential and stationary phases of growth. The expression of iron-regulated proteins of 81, 23, and 17 kDa was time-dependent, associated with the same stage of growth, and might be involved in siderophore efficiency.

Western blotting of basic proteins after nondenaturing electrophoresis in acid conditions using the PhastSystem.

Electroblotting of basic proteins was performed from minigels after electrophoresis, under nondenaturing acidic conditions, by using the automated PhastSystem. Depending on the molecular masses of the proteins to be studied, various precast gel media were chosen. The transfer membranes with various types (nitrocellulose and polyvinylidene difluoride) and pore sizes (0.45 and 0.2 micron) were chosen accordingly. For the semidry electric transfer, a simple, discontinuous two-buffer system was used. The anode solution contained 0.3 M Tris, pH 10.4, and the cathode solution, 40 mM 6-amino-n-hexanoic acid, pH 7.6, with 20% v/v methanol each. The addition of 0.1% sodium dodecyl sulfate (SDS) in the cathode solution facilitated the elution of proteins from the gels and directed the migration of the negative SDS-protein complexes towards the anode membranes. The transfer conditions following native polyacrylamide gel electrophoresis allowed the visualization of basic proteins, with molecular weights ranging from 29,000 to 5,000, for which isoforms could be resolved and which retained their biological properties.

Analysis of the conformation and stability of human bronchial lysozyme by circular dichroism.

1. Human bronchial lysozyme was isolated from nonpurulent secretions and studied by circular dichroism (CD) spectroscopy for its conformational properties. 2. The two negative bands at 208 and 222 nm indicated that the peptide chain adopted an alpha-helical structure in physiological conditions. 3. The molecule was stable at pH 1.0 but not at pH 12.0. 4. Increasing ionic strength by adding NaCl up to 1 M did not change the CD spectra. 5. Complete unfolding of the molecule by guanidinium chloride was obtained only at the concentration of 6 M. 6. Bronchial lysozyme was also denatured by sodium dodecyl sulphate. 7. The molecule was stable when mild reduction was performed at 37 degrees C for 30 min but was completely unfolded after heating at 100 degrees C for 3 min.

Strong ionic interactions between mucins and two basic proteins, mucus proteinase inhibitor and lysozyme, in human bronchial secretions.

1. Mucins were isolated from sputum from a patient with chronic bronchitis and subjected to two different preparation procedures. 2. In the first procedure, CsBr density-gradient centrifugation gave rise to two well-separated fractions. Mucins recovered in the high-density fraction still contained mucus proteinase inhibitor (MPI) and lysozyme (LSZ). 3. Mucins were purified after a second step of CsBr density-gradient centrifugation or after gel-filtration chromatography with a buffer of high ionic strength, containing 0.5 M NaCl. 4. In the second procedure, trichloroacetic acid treatment of whole sputum followed by cation-exchange chromatography allowed the obtention of a non-retained fraction composed of mucins. 5. Gel-filtration in buffer containing 0.5 M NaCl, allowed the release of MPI and LSZ from mucins, thus confirming that interactions still occurred between those components. 6. The chemical compositions of the mucins isolated by the two above procedures were quite similar. 7. These data support the hypothesis of the existence of ionic interactions between basic amino acid residues of MPI and LSZ and acid residues of the carbohydrate chains of mucins in the secretions of the large airways. 8. These interactions could play a role in the protection of mucins against proteolysis and consequently in the maintenance of rheological properties of the mucus gel in disease.

Interaction between secretory leucocyte proteinase inhibitor and bronchial mucins or glycopeptides. Physiopathological implications for the protection of mucins against proteolysis by human leucocyte elastase.

The interaction of secretory leucocyte proteinase inhibitor with bronchial mucins and glycopeptides was studied by means of c.d. spectroscopy. The interaction with mucins was characterized by an increase in organized structure of alpha-helical type, as evidenced by the appearance in the difference spectra of two positive bands at 208 and 218 nm. This phenomenon was correlated with the amount of inhibitor present in the mixtures, suggesting that the change was inherent to the inhibitor. Surprisingly, when the inhibitor was mixed with acid glycopeptides, difference c.d. spectra showed a decrease in organized structure, characterized by a negative minimum at 196 nm. Glycopeptides treated with neuraminidase gave similar profiles of difference spectra in three different mixtures, indicating that the interaction was smaller. The interaction between the inhibitor and mucins was also studied for its ability to modify in vitro the proteolytic activity of human leucocyte elastase. Mucins alone were degraded by that proteinase into glycopeptides of Mr 400,000-500,000, whereas mucins mixed with inhibitor before adding elastase were proteolysed to a lesser extent. These data demonstrate that the secretory leucocyte proteinase inhibitor interacts with mucins and consequently is capable of protecting the mucins against proteolysis by elastase.

A rapid periodic acid-Schiff staining procedure for the detection of glycoproteins using the PhastSystem.

A procedure for the analysis of glycoproteins and glycopeptides using the PhastSystem with detection by the periodic acid-Schiff stain is described. Following sodium dodecyl sulfate or nondenaturing polyacrylamide gel electrophoresis and also isoelectric focusing, samples are stained directly for the presence of carbohydrates. By using the PhastSystem, the method is rapid, sensitive, reliable and allows storage of the gels without a change in the stain. As little as 0.1 micrograms of protein-associated carbohydrates can be detected. The staining procedure is also used following isoelectric focusing of mucin-derived glycopeptides to visualize their charge differences and the increase of their isoelectric point after neuraminidase treatment.

Separation of the two domains of human mucus proteinase inhibitor: inhibitory activity is only located in the carboxy-terminal domain.

Human mucus proteinase inhibitor is a two-domain protein which inactivates bovine trypsin and chymotrypsin, leukocyte elastase and cathepsin G. In order to localize the site(s) responsible for these inhibitory activities, the two domains were isolated after specific cleavage of the Asp49-Pro50 bond following mild acid treatment of the bronchial inhibitor. The carboxy-terminal domain was active against leukocyte elastase, trypsin and chymotrypsin whereas the amino-terminal domain, which contained a putative antitryptic active site, was devoid of activity. This implicates that, in the whole molecule, the inhibitory activity region is localized only in the carboxy-terminal domain.

Comparison of the effects of purified human alpha 1-antichymotrypsin and alpha 1-proteinase inhibitor on NK cytotoxicity: only alpha 1-proteinase inhibitor inhibits natural killing.

While an inhibitory effect on natural killer (NK) cell activity was demonstrated with partially purified alpha 1 Achy, neither highly purified alpha 1 Achy from two healthy donors nor from one patient with giant-cell arteritis, which carries more highly branched glycans, inhibited the NK cytotoxicity. Our purification procedure, based on immunoaffinity chromatography and gel filtration, was not in question since the pure alpha 1-proteinase inhibitor (alpha 1PI) prepared in our laboratory by using a similar procedure continued to inhibit the NK cytotoxicity. If an inhibitory effect not related to antiprotease activity occurs with alpha 1PI, it is surprising that it is not shared by alpha 1 Achy which, like alpha 1PI, belongs to the serpin family and which possesses a strong structural homology with alpha 1PI. Our finding that alpha 1PI is able to affect human NK cytotoxicity while alpha 1 Achy (even with more highly branched glycans) is unable to suggests that events controlling NK activity may involve other enzymes than chymotrypsin-like enzymes.

Electrotransfer of basic proteins from nondenaturing polyacrylamide acid gels to nitrocellulose: detection of enzymatic and inhibitory activities and retention of protein antigenicity.

We have developed a method for electrotransfer of strongly basic proteins (lysozyme, pI 11; mucus proteinase inhibitor, pI greater than 10; bovine pancreas trypsin inhibitor; pI 10.5; human leukocyte elastase, pI greater than 9) from nondenaturing acid gels (pH 4.5) to nitrocellulose sheets. Buffers were those used in a discontinuous system for transfer from sodium dodecyl sulfate (SDS)-containing polyacrylamide gels with one modification in the cathode buffer which contained 0.1% SDS. This method was compared to electrotransfer performed in 0.7% acetic acid. The basic proteins studied, which were positively charged in the gel, formed with SDS negative complexes which migrated toward the anode and were efficiently transferred to the nitrocellulose. Moreover, their biological properties were preserved: inhibitory activity, enzyme activity, and antigenicity. This method is advantageous because it is simple, is sensitive, and can be applied to various biological fluids to detect inhibitors, enzymes, and other proteins which have a basic character, after electrophoretic separation under their native forms.

Special neutrophil elastase inhibitory activity in BAL fluid from patients with silicosis and asbestosis.

Pneumoconiosis is defined as the disease resulting from a chronic exposure to different inorganic dusts. In order to assess the lung defence against the effects of dust exposure, we studied the bronchoalveolar lavage (BAL) fluids from 30 silicotic patients (9 of them having a diagnosis of progressive massive fibrosis (PMF)) and 8 subjects with a diagnosis of asbestosis. Total protein content, N-acetyl-beta-D-glucosaminidase activity, free elastase-like activity, immunoreactive alpha 1-proteinase inhibitor (alpha 1PI) and neutrophil elastase inhibitory capacity (NEIC) were determined, and the values obtained were compared to those of 14 control BAL fluids. In all of the patients, our data showed a significant increase of total protein content and free elastase-like activity. In contrast, N-acetyl-beta-D-glucosaminidase activities did not reach statistical significance. Values concerning immunoreactive alpha 1PI and NEIC were significantly raised only in patients with PMF and with asbestosis. When the ratio NEIC/immunoreactive alpha 1PI was calculated, a significant difference was noticed in the asbestosis group; on the other hand, this ratio was significantly reduced in the group of PMF patients. After neutrophil elastase addition, an electrophoretic study by SDS-PAGE and immunoblotting was carried out; it showed more proteolysed alpha 1PI in the BAL fluids having a lowered NEIC/alpha 1PI ratio. These facts could be explained by the presence of inhibitors of neutrophil elastase different from alpha 1PI.

Evidence for the tight binding of human mucus proteinase inhibitor to highly glycosylated macromolecules in sputum.

Two extraction procedures of non-purulent sputum for the isolation of human mucus proteinase inhibitor (MPI) in its free and bound forms have been assayed. The dissociating procedure involved sputum homogenization in 1M NaCl and 4% (w/v) trichloroacetic treatment. When the soluble material was applied to a CM-Trisacryl column, a non-negligible, MPI-related inhibitory activity was recovered with the highly glycosylated constituents not retained on the column; the amount of MPI released in a free form was retained and eluted from the column according to the basic character of this inhibitor. The non-dissociating procedure consisted in a high water dilution (1:12) of sputum, known to bring into solution the macromolecular, fibrillar constituents, which was followed by ultrafiltration on selected Mr cut-off membranes. All the inhibitory activity was recovered with the high Mr (greater than 100,000) fraction which was shown on SDS-PAGE to be essentially composed of strongly glycosylated material; on electrophoretic analysis under non-reducing conditions, the MPI activity was visualized as three bands which corresponded to the inhibitor released from this high Mr fraction in the presence of SDS. As mucin-type molecules are the major, highly glycosylated constituents of bronchial secretions, it is suggested that they are responsible for the entrapping of MPI within their macromolecular network; it would appear that, as well as for lysozyme, electrostatic interactions occur between the acid charges of mucins and the basic charges of MPI. The possible in vivo consequences of these interactions on MPI activity are discussed.

Studies on the interactions of human pancreatic elastase 2 with human alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin.

The interactions of human pancreatic elastase 2 with alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin were compared by studies in vitro. The equimolar complexes obtained between the enzyme and either inhibitor were relatively stable at 25 degrees C since they could be visualized for up to 5 days by an electrophoretic method. However, in both cases, a slow dissociation occurred with release of active enzyme. As the kass. rate constants are of the same order of magnitude, with a slightly lower value for alpha 1-proteinase inhibitor when compared with alpha 1-antichymotrypsin [(5.6 +/- 1.2) X 10(5) and (8.9 +/- 1.3) X 10(5) M-1.s-1 respectively], partition of human pancreatic elastase 2 between both inhibitors in human plasma is mainly dependent on their respective concentrations. A comparative study by crossed immunoelectrophoresis of the interactions of this enzyme with the two inhibitors contained in normal human plasma and in a mimetic mixture of pure inhibitors was carried out. This allowed the visualization of complexes with either inhibitor. Formation of such a complex with alpha 1-antichymotrypsin had never been demonstrated previously. The patterns obtained are similar when working with normal plasma or with the synthetic mixture, suggesting that, in the conditions used, alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin are the main inhibitors of human pancreatic elastase 2 in the plasma sample. However, it is also shown that part of the enzyme may be taken up by alpha 2-macroglobulin, which is responsible for the remaining enzyme activity on a synthetic substrate. The present work suggests that, according to the delay times of inhibition of human pancreatic elastase 2 calculated from the normal plasma concentrations of alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin, a significant role can be assigned to both inhibitors. Moreover, the role of alpha 1-antichymotrypsin would be enhanced in alpha 1-proteinase-inhibitor deficiency.