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 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 Low-Methoxyl Pectin Stimulates Small Intestinal Mucin Secretion Irrespective of Goblet Cell Proliferation and Is Characterized by Jejunum Muc2 Upregulation in Rats

2012 ◽  
Vol 143 (1) ◽  
pp. 34-40 ◽  
Author(s):  
Shingo Hino ◽  
Kei Sonoyama ◽  
Hiroyuki Bito ◽  
Hirokazu Kawagishi ◽  
Seiichiro Aoe ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Goblet Cell ◽  
Mucin Secretion ◽  
Intestinal Mucin ◽  
Small Intestinal

The molecular basis for epitopes on the free β-subunit of human chorionic gonadotrophin (hCG), its carboxyl-terminal peptide and the hCGβ-core fragment

1994 ◽  
Vol 141 (1) ◽  
pp. 153-162 ◽  
Author(s):  
S Dirnhofer ◽  
S Madersbacher ◽  
J-M Bidart ◽  
P B W Ten Kortenaar ◽  
G Spöttl ◽  
...  
Keyword(s):  
Amino Acid ◽  
Molecular Basis ◽  
Tertiary Structure ◽  
Solid Phase ◽  
Critical Role ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Antigenic Determinants ◽  
Β Subunit ◽  
Carboxyl Terminal

Abstract The molecular basis for antigenic determinants on the free β-subunit of human chorionic gonadotrophin (hCGβ), its carboxyl-terminal peptide (hCGβCTP) and the hCGβcore fragment (hCGβcf) was elucidated by means of monoclonal antibodies (MCAs). The objective of the present study was to resolve the antigenic topography of these three molecules in terms of epitope identification at different levels of structural organization as well as analysis of their spatial arrangement. An hCGβcf preparation, a synthetic peptide corresponding to the hCGβCTP (β109–145), overlapping synthetic peptides spanning the entire amino acid sequence of hCGβ, and a reduced and alkylated hCGβ preparation were assayed in a solid-phase one-site enzyme-linked immunoassay and in a solublephase direct-binding radioimmunoassay (RIA) or competitive RIA. The antigenic topography was mapped by incorporating the MCAs into two-site binding assays. On the surface of free hCGβ, nine different epitopes (β1–β9), arranged in three spatially distinct domains, could be distinguished. Epitopes β1–β7 were located in a single large domain on both hCGβ and the hCGβcf whereas hCGβCTP contained two topographically distant determinants, designated β8 and β9 respectively. All but the two epitopes located on hCGβCTP (β8 and β9) were destroyed by reducing and alkylating hCGβ, suggesting that most antigenic determinants are predominantly non-contiguous and require an intact tertiary structure whereas the molecular structure of hCGβCTP is linear. At a molecular level, amino acid residues spanning hCGβ 45–52, hCGβ 137–144 and hCGβ 113–116 contributed to the formation of epitopes β5, β8 and β9 respectively. We have also shown that the hCGβcf represents the immunodominant part of the free β-subunit of hCG, containing seven mainly conformationally determined epitopes, one of which has a share of the sequence β45–52. The hCGβCTP does not play a critical role in the immunologically important tertiary structure of hCGβ and was itself found to be a predominantly continuous sequence also within the native hormone, expressing two spatially distant antigenic determinants located within residues β113–116 and β137–144 respectively. Journal of Endocrinology (1994) 141, 153–162

scholarly journals Characterization and localization of the putative ‘link’ component in rat small-intestinal mucin

1987 ◽  
Vol 243 (3) ◽  
pp. 631-640 ◽  
Author(s):  
R E F Fahim ◽  
R D Specian ◽  
G G Forstner ◽  
J F Forstner
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Intestinal Lumen ◽  
Western Blots ◽  
Peptide Bonds ◽  
Disulphide Bonds ◽  
Purification Technique ◽  
Intestinal Mucin ◽  
Small Intestinal ◽  
Monospecific Antibodies ◽  
Link Component

Rat intestinal mucin is polymerized by a putative ‘link’ component of Mr 118,000 that can be released from the native mucin by thiol reduction [Fahim, Forstner & Forstner (1983) Biochem. J. 209, 117-124]. To confirm that this component is an integral part of the mucin and independent of the mucin purification technique, rat mucin was purified in the present study by three independent techniques. In all cases, the 118,000-Mr component was released after reduction. The 118 kDa band was electroeluted from SDS/polyacrylamide gels and its composition shown to resemble closely that of the link component of human intestinal mucin [Mantle, Forstner & Forstner (1984) Biochem. J. 224, 345-354]. Carbohydrates were present, including significant (10 mol/100 mol) amounts of mannose, suggesting the presence of N-linked oligosaccharides. Monospecific antibodies prepared against the rat 118,000-Mr component established its tissue localization in intestinal goblet cells. Mucins subjected to SDS/polyacrylamide-gel electrophoresis and Western blots using the same antibody, established that the link components of rat and human intestinal mucin are similar antigenically. Brief exposure (10 min) of native rat mucin to trypsin or Pronase (enzyme/mucin protein, 1:500, w/w) also released a 118,000-Mr component that reacted with the monospecific antibody. Thus the 118,000-Mr component is an integral part of the mucin and, although linked to large glycopeptides by disulphide bonds, this component also has proteinase-sensitive peptide bonds, presumably at terminal locations such that brief treatment with proteinases releases the molecule in a reasonably intact form. Under physiological conditions, therefore, one might expect that, after mucin is secreted into the intestinal lumen, luminal proteinases would rapidly remove the link component, thereby causing the mucin to depolymerize.

scholarly journals Recognition Signal for C-Mannosylation of Trp-7 in RNase 2 Consists of Sequence Trp-x-x-Trp

1998 ◽  
Vol 9 (2) ◽  
pp. 301-309 ◽  
Author(s):  
Joachim Krieg ◽  
Steffen Hartmann ◽  
Anna Vicentini ◽  
Wolfgang Gläsner ◽  
Daniel Hess ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Three Dimensional ◽  
Structural Features ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Dimensional Structure ◽  
Interleukin 12 ◽  
Sequence Motif ◽  
Protein Databases ◽  
Human Rnase

C2-α-Mannosyltryptophan was discovered in human RNase 2, an enzyme that occurs in eosinophils and is involved in host defense. It represents a novel way of attaching carbohydrate to a protein in addition to the well-known N- andO-glycosylations. The reaction is specific, as in RNase 2 Trp-7, but never Trp-10, which is modified. In this article, we address which structural features provide the specificity of the reaction. Expression of chimeras of RNase 2 and nonglycosylated RNase 4 and deletion mutants in HEK293 cells identified residues 1–13 to be sufficient for C-mannosylation. Site-directed mutagenesis revealed the sequence Trp-x-x-Trp, in which the first Trp becomes mannosylated, as the specificity determinant. The Trp residue at position +3 can be replaced by Phe, which reduces the efficiency of the reaction threefold. Interpretation of the data in the context of the three-dimensional structure of RNase 2 strongly suggests that the primary, rather than the tertiary, structure forms the determinant. The sequence motif occurs in 336 mammalian proteins currently present in protein databases. Two of these proteins were analyzed protein chemically, which showed partial C-glycosylation of recombinant human interleukin 12. The frequent occurrence of the protein recognition motif suggests that C-glycosides could be part of the structure of more proteins than assumed so far.

Methods for making stereoslides and -prints, with freeze-etched olfactory epithelium as example

1984 ◽  
Vol 42 ◽  
pp. 704-705
Author(s):  
Bert Ph. M. Menco ◽  
Ido F. Menco ◽  
Frans L.T. Verdonk
Keyword(s):  
Electron Microscope ◽  
Olfactory Epithelium ◽  
Three Dimensional ◽  
Supporting Cell ◽  
Structural Features ◽  
Extensive Study ◽  
Sprague Dawley ◽  
Freezing Method ◽  
Respiratory Epithelia ◽  
Three Dimensional Presentation

Previously we presented an extensive study of the distributions of intramembranous particles of structures in apical surfaces of nasal olfactory and respiratory epithelia of the Sprague-Dawley rat. For the same structures these distributions were compared in samples which were i) chemically fixed and cryo-protected with glycerol before cryo-fixation, after excision, and ii)ultra-rapidly frozen by means of the slam-freezing method. Since a three-dimensional presentation markedly improves visualization of structural features micrographs were presented as stereopairs. Two exposures were made by tiling the sample stage of the electron microscope 6° in either direction with an eucentric goniometer. The negatives (Agfa Pan 25 Professional) were reversed with Kodak Technical Pan Film 2415 developed in D76 1:1. The prints were made from these reversed negatives. As an example tight-junctional features of an olfactory supporting cell in a region where this cell conjoined with two other cells are presented (Fig. 1).

Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain

1998 ◽  
Vol 79 (01) ◽  
pp. 104-109 ◽  
Author(s):  
Osamu Takamiya
Keyword(s):  
Monoclonal Antibodies ◽  
Tissue Factor ◽  
Human Factor ◽  
Solid Phase ◽  
Three Dimensional ◽  
Coagulation Factors ◽  
Factor Vii ◽  
Dimensional Structure ◽  
Epidermal Growth

SummaryMurine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

Insights into the Structural Features, Conformational Stability and Functional Activity of the Olneya tesota PF2 Lectin

2020 ◽  
Vol 27 ◽  
Author(s):  
Edgar Acedo-Espinoza ◽  
Irlanda Lagarda-Diaz ◽  
Rosina Cabrera ◽  
Ana M. Guzman-Partida ◽  
Amir Maldonado-Arce ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Proteolytic Enzymes ◽  
Conformational Stability ◽  
Structural Features ◽  
Beta Sheets ◽  
Hemagglutinating Activity ◽  
Effect Of Temperature ◽  
Hydrodynamic Diameter ◽  
Bioinformatic Tools ◽  
Structural Levels

Background: The O. tesota lectin PF2 is a tetrameric protein with subunits of 33 kDa that recognizes only complex carbohydrates, resistant to proteolytic enzymes and has insecticidal activity against Phaseolus beans pest. Objective: To explore PF2 lectin features at different protein structural levels and to evaluate the effect of temperature and pH on its functionality and conformational stability. Methods: PF2 lectin was purified by affinity chromatography. Its primary structure was resolved by mass spectrometry and analyzed by bioinformatic tools, including its tertiary structure homology modeling. The effect of temperature and pH on its conformational traits and stability was addressed by dynamic light scattering, circular dichroism, and intrinsic fluorescence. The hemagglutinating activity was evaluated using a suspension of peripheral blood erythrocytes. Results: The proposed PF2 folding comprises a high content of beta sheets. At pH 7 and 25 °C, the hydrodynamic diameter (Dh) was found to be 12.3 nm which corresponds to the oligomeric native state of PF2 lectin. Dh increased under the other evaluated pH and temperature conditions, suggesting protein aggregation. At basic pH, PF2 exhibited low conformational stability. The native PF2 (pH 7) retained its full hemagglutinating activity up to 45 °C and exhibited one transition state with a melting temperature of 76.8 °C. Conclusion: PF2 showed distinctive characteristics found in legume lectins. The pH influences the functionality and conformational stability of the protein. PF2 lectin displayed a relatively narrow thermostability to the loss of secondary structure and hemagglutinating activity.

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