scholarly journals Intestinal mucins from normal subjects and patients with cystic fibrosis. Variable contents of the disulphide-bound 118 kDa glycoprotein and different reactivities with an anti-(118 kDa glycoprotein) antibody

1989 ◽  
Vol 259 (1) ◽  
pp. 243-253 ◽  
Author(s):  
M Mantle ◽  
G Stewart
Keyword(s):  
Cystic Fibrosis ◽  
Concanavalin A ◽  
Specific Antibody ◽  
Small Proportion ◽  
Three Dimensional ◽  
Normal Subjects ◽  
Antigenic Determinants ◽  
Western Blots ◽  
Coomassie Blue ◽  
Intestinal Mucin

1. A specific antibody was developed against the disulphide-bound 118 kDa glycoprotein of human intestinal mucin and used to establish an e.l.i.s.a. Fourteen purified mucins [eight normal (N) and six cystic fibrosis (CF)] had the same affinity for the antibody in the e.l.i.s.a., but their relative immunoreactivities varied widely (approx. 100,000-fold). In general, CF mucins were more antigenic than N mucins. 2. Variations (approx. 10-fold) were detected in the 118 kDa glycoprotein content of both N and CF mucins (assessed from Coomassie Blue-stained polyacrylamide gels), but these did not appear to be responsible for the differences in mucin immunoreactivity. 3. Variations (approx. 6-fold) were also observed in the size of the 118 kDa peak produced by N and CF mucins on Western blots. These were mostly due to differences in the 118 kDa glycoprotein content of mucins, although a small proportion resulted from changes in the number of antigenic determinants within individual 118 kDa glycoproteins. 4. After concanavalin A affinity chromatography of four reduced mucins (two N and two CF), purified 118 kDa glycoprotein was recovered in the bound fractions from the column, specifically eluted by methyl alpha-mannoside. 5. The amounts of 118 kDa glycoprotein isolated from the four mucins varied as predicted from the size of their 118 kDa bands on Coomassie Blue-stained gels. 6. Three 118 kDa glycoproteins (one N and two CF) showed almost identical reactivity in the e.l.i.s.a.; the fourth had fewer antigenic determinants. 7. Since differences in 118 kDa glycoprotein content and in the number of antigenic determinants within the 118 kDa glycoprotein did not account for variations in the reactivity of native mucins in the e.l.i.s.a., it appeared that accessibility of the 118 kDa glycoprotein to antibody binding may be critical in determining mucin immunoreactivity. This suggests that the three-dimensional conformation of CF mucins may differ from that of N mucins, leading to increased antigenicity.

scholarly journals Antigenic and structural features of goblet-cell mucin of human small intestine

1984 ◽  
Vol 217 (1) ◽  
pp. 159-167 ◽  
Author(s):  
M Mantle ◽  
G G Forstner ◽  
J F Forstner
Keyword(s):  
Goblet Cell ◽  
Tertiary Structure ◽  
Solid Phase ◽  
Three Dimensional ◽  
Structural Features ◽  
Antigenic Determinants ◽  
Western Blots ◽  
Intestinal Mucin ◽  
Small Intestinal ◽  
Three Dimensional Configuration

With the use of a newly developed solid-phase radioimmunoassay method, the major antigenic determinants of human small-intestinal goblet-cell mucin were investigated and related to the overall tertiary structure of the mucin. Preliminary hapten inhibition studies with various oligosaccharides of known sequence and structure suggested that the determinants did not reside in carbohydrate. Exhaustive thiol reduction, however, almost abolished antigenicity, caused breakdown of the mucin into small heterogeneous glycopeptides, and liberated a ‘link’ peptide of Mr 118000. Western ‘blots’ of reduced mucin from polyacrylamide gels on to nitrocellulose sheets showed that a small amount of residual antigenicity remained in large-Mr glycopeptides (Mr greater than 200000). The ‘link’ peptide was not antigenic. Timed Pronase digestion of native mucin resulted in a progressive loss of antigenic determinants. Gel electrophoresis revealed that after 8h of digestion the 118000-Mr peptide had disappeared, whereas antigenicity, which was confined to large-Mr glycopeptides, was destroyed much more slowly with time (70% by 24h, 100% by 72h). Despite the loss of antigenicity, 72h-Pronase-digested glycopeptides retained all of the carbohydrate of the native mucin. Therefore the antibody to human small-intestinal mucin appears to recognize a ‘naked’ (non-glycosylated and Pronase-susceptible) peptide region(s) of mucin glycopeptides. For full antigenicity, however, disulphide bonds are required to stabilize a specific three-dimensional configuration of the ‘naked’ region.

scholarly journals Biochemical characterization of the component parts of intestinal mucin from patients with cystic fibrosis

1984 ◽  
Vol 224 (2) ◽  
pp. 345-354 ◽  
Author(s):  
M Mantle ◽  
G G Forstner ◽  
J F Forstner
Keyword(s):  
Cystic Fibrosis ◽  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Equilibrium Density ◽  
Antigenic Determinants ◽  
Intestinal Mucin ◽  
Sepharose 4B

Previous studies have shown that human small-intestinal mucin consists of high-Mr glycoproteins and a smaller S-S-bonded protein of 118 kDa. The major antigenic determinants of the mucin were associated with the large glycoproteins, but depended for stability on intact disulphide bonds, and were destroyed by digestion with Pronase. In the present study we isolated and analysed the component parts of mucin from patients with cystic fibrosis with special attention being paid to the peptide constituents. After reduction with 0.2 M-beta-mercaptoethanol [5 min, 100 degrees C in 1% SDS (sodium dodecyl sulphate)], the large glycoproteins and smaller peptide with an apparent molecular size of 118 kDa were separated by equilibrium density-gradient centrifugation in CsCl, Sepharose 4B chromatography or preparative SDS/polyacrylamide-gel electrophoresis. The large glycoproteins contained about 70% of the protein of the native mucin. Digestion with Pronase resulted in a further loss of ‘naked’ protein (10% of the native mucin protein) from the C-terminal end of the glycoprotein peptide core, and left behind highly glycosylated proteins comprised mainly (70 mol%) of threonine, serine and proline. The 118 kDa component, which contained about 30% of the native mucin protein, consisted mainly of aspartic acid, serine, glutamic acid and glycine (40 mol%), plus threonine, proline, alanine, valine and leucine (35 mol%). Together with the ‘naked’ protein segment, the 118 kDa component contained most of the cysteine residues of the native mucin. Surprisingly, the peptide also contained carbohydrate (less than or equal to 5% of the native mucin carbohydrate but 50% by weight of the 118 kDa component), which included 9 mol% mannose, suggesting the presence of N-linked oligosaccharides. The peptide exhibited strong non-covalent interactions with the high-Mr glycoproteins and a tendency to self-aggregate in the absence of dissociating agents. Our findings therefore suggest that native mucin consists of large glycoproteins capable of forming disulphide bridges from their C-terminal ‘naked’ (antigenic) regions to a smaller glycopeptide having an Mr of 118 000.

scholarly journals The Membrane Skeleton of Pseudomicrothorax

1991 ◽  
Vol 100 (4) ◽  
pp. 693-706 ◽  
Author(s):  
ROBERT K. PECK ◽  
FRANÇOISE DUBORGEL ◽  
IRM HUTTENLAUCH ◽  
GERARD DE HALLER
Keyword(s):  
Concanavalin A ◽  
Peptide Mapping ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Dimensional Structure ◽  
Western Blots ◽  
Major Band ◽  
Thin Sections ◽  
Dilute Acid Solution ◽  
Β Tubulin

The epiplasm membrane skeleton of the ciliated protozoan Pseudomicrothorax dubius has been isolated and its three-dimensional structure and constituent proteins have been examined. The epiplasm functions as a cytoskeleton to define cell shape and the position of some cortical organelles. Scanning electron microscopy of the isolated epiplasm reveals a rococo cytoarchitecture in which basal bodies and trichocyst attachment sites are arranged in precise geometric arrays. SDS-PAGE reveals 40 bands, one of which is quantitatively the major band of the epiplasm and is composed of at least 3 different proteins and numerous isoelectric variants, as revealed by two-dimensional electrophoresis and peptide mapping. Polyclonal antisera were produced against native (antiserum 15) and SDS-denatured (antiserum 18) epiplasm. On immunoblots, antiserum 15 labels the hydrophilic proteins that are extracted from the epiplasm by treatment with dilute acid solution and that are predominantly glycoproteins, four of which are labeled with Concanavalin A on Western blots. On Lowicryl thin sections, antiserum 15 labels the epiplasm uniformly, except for the terminal plates, indicating that the glycoproteins are integral components of the epiplasm and are not membrane contaminants in the epiplasm fraction. Concanavalin A labeling of Lowicryl sections supports the latter result. On immunoblots, antiserum 18 labels the acid-insoluble epiplasm bands, the major structural elements of the epiplasm. One of the epiplasm bands at 52x1O3Mr is labeled by an anti-β tubulin monoclonal antibody. Evidence is presented that this β tubulin is not due to microtubule contamination of the epiplasm fraction.

scholarly journals Crystal Structure of an Aquabirnavirus Particle: Insights into Antigenic Diversity and Virulence Determinism

2009 ◽  
Vol 84 (4) ◽  
pp. 1792-1799 ◽  
Author(s):  
Fasséli Coulibaly ◽  
Christophe Chevalier ◽  
Bernard Delmas ◽  
Félix A. Rey
Keyword(s):  
Crystal Structure ◽  
Three Dimensional ◽  
Salient Feature ◽  
Infectious Pancreatic Necrosis Virus ◽  
Antigenic Determinants ◽  
Binding Motif ◽  
Icosahedral Symmetry ◽  
Fold Axis ◽  
Cell Internalization ◽  
Pancreatic Necrosis Virus

ABSTRACT Infectious pancreatic necrosis virus (IPNV), a pathogen of salmon and trout, imposes a severe toll on the aquaculture and sea farming industries. IPNV belongs to the Aquabirnavirus genus in the Birnaviridae family of bisegmented double-stranded RNA viruses. The virions are nonenveloped with a T=13l icosahedral capsid made by the coat protein VP2, the three-dimensional (3D) organization of which is known in detail for the family prototype, the infectious bursal disease virus (IBDV) of poultry. A salient feature of the birnavirus architecture is the presence of 260 trimeric spikes formed by VP2, projecting radially from the capsid. The spikes carry the principal antigenic sites as well as virulence and cell adaptation determinants. We report here the 3.4-Å resolution crystal structure of a subviral particle (SVP) of IPNV, containing 20 VP2 trimers organized with icosahedral symmetry. We show that, as expected, the SVPs have a very similar organization to the IBDV counterparts, with VP2 exhibiting the same overall 3D fold. However, the spikes are significantly different, displaying a more compact organization with tighter packing about the molecular 3-fold axis. Amino acids controlling virulence and cell culture adaptation cluster differently at the top of the spike, i.e., in a central bowl in IBDV and at the periphery in IPNV. In contrast, the spike base features an exposed groove, conserved across birnavirus genera, which contains an integrin-binding motif. Thus, in addition to revealing the viral antigenic determinants, the structure suggests that birnaviruses interact with different receptors for attachment and for cell internalization during entry.

scholarly journals Characterization of Wild-Type and ΔF508 Cystic Fibrosis Transmembrane Regulator in Human Respiratory Epithelia

2005 ◽  
Vol 16 (5) ◽  
pp. 2154-2167 ◽  
Author(s):  
Silvia M. Kreda ◽  
Marcus Mall ◽  
April Mengos ◽  
Lori Rochelle ◽  
James Yankaskas ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Normal Subjects ◽  
Cell Types ◽  
Cystic Fibrosis Transmembrane Regulator ◽  
Ciliated Cells ◽  
Metabolic Fate ◽  
Specific Expression ◽  
Δf508 Cftr ◽  
Cftr Protein ◽  
Cystic Fibrosis Transmembrane

Previous studies in native tissues have produced conflicting data on the localization and metabolic fate of WT and ΔF508 cystic fibrosis transmembrane regulator (CFTR) in the lung. Combining immunocytochemical and biochemical studies utilizing new high-affinity CFTR mAbs with ion transport assays, we examined both 1) the cell type and region specific expression of CFTR in normal airways and 2) the metabolic fate of ΔF508 CFTR and associated ERM proteins in the cystic fibrosis lung. Studies of lungs from a large number of normal subjects revealed that WT CFTR protein localized to the apical membrane of ciliated cells within the superficial epithelium and gland ducts. In contrast, other cell types in the superficial, gland acinar, and alveolar epithelia expressed little WT CFTR protein. No ΔF508 CFTR mature protein or function could be detected in airway specimens freshly excised from a large number of ΔF508 homozygous subjects, despite an intact ERM complex. In sum, our data demonstrate that WT CFTR is predominantly expressed in ciliated cells, and ΔF508 CFTR pathogenesis in native tissues, like heterologous cells, reflects loss of normal protein processing.

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