Corrigendum to "Large Gliadin Peptides Detected in the Pancreas of NOD and Healthy Mice following Oral Administration".

[This corrects the article DOI: 10.1155/2016/2424306.].

Large Gliadin Peptides Detected in the Pancreas of NOD and Healthy Mice following Oral Administration.

Gluten promotes type 1 diabetes in nonobese diabetic (NOD) mice and likely also in humans. In NOD mice and in non-diabetes-prone mice, it induces inflammation in the pancreatic lymph nodes, suggesting that gluten can initiate inflammation locally. Further, gliadin fragments stimulate insulin secretion from beta cells directly. We hypothesized that gluten fragments may cross the intestinal barrier to be distributed to organs other than the gut. If present in pancreas, gliadin could interact directly with the immune system and the beta cells to initiate diabetes development. We orally and intravenously administered 33-mer and 19-mer gliadin peptide to NOD, BALB/c, and C57BL/6 mice and found that the peptides readily crossed the intestinal barrier in all strains. Several degradation products were found in the pancreas by mass spectroscopy. Notably, the exocrine pancreas incorporated large amounts of radioactive label shortly after administration of the peptides. The study demonstrates that, even in normal animals, large gliadin fragments can reach the pancreas. If applicable to humans, the increased gut permeability in prediabetes and type 1 diabetes patients could expose beta cells directly to gliadin fragments. Here they could initiate inflammation and induce beta cell stress and thus contribute to the development of type 1 diabetes.

The complex interplay between ligand binding and conformational structure of the folate binding protein (folate receptor): Biological perspectives.

This review analyzes how interplay between folate binding and changes in folate binding protein (FBP) conformation/self-association affects the biological function of FBP. Concentration-dependent, reversible self-association of hydrophobic apo-FBP at pI=7.4 is associated with decreased affinity for folate, probably due to shielding of binding sites between interacting hydrophobic patches. Titration with folate removes apo-monomers, favoring dissociation of self-associated apo-FBP into apo-monomers. Folate anchors to FBP through a network of hydrogen bonds and hydrophobic interactions, and the binding induces a conformational change with formation of hydrophilic and stable holo-FBP. Holo-FBP exhibits a ligand-mediated concentration-dependent self-association into multimers of great thermal and chemical stability due to strong intermolecular forces. Both ligand and FBP are thus protected against biological/physicochemical decomposition. In biological fluids with low FBP concentrations, e.g., saliva, semen and plasma, hydrophobic apo-monomers and hydrophilic holo-monomers associate into stable asymmetrical complexes with aberrant binding kinetics unless detergents, e.g., cholesterol or phospholipids are present.

The interrelationship between ligand binding and self-association of the folate binding protein. The role of detergent-tryptophan interaction.

BACKGROUND: The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of folate binding. METHODS: Self-association behavior of apo- and holo-FBP was addressed through size exclusion chromatography, SDS-PAGE, mass spectrometry, surface plasmon resonance and fluorescence spectroscopy. RESULTS: Especially holo-FBP exhibits concentration-dependent self-association at pH 7.4 (pI), and is more prone to associate into stable complexes than apo-FBP. Even more pronounced was the tendency to complexation between apo-FBP and holo-FBP in accord with a model predicting association between apo and holo monomers [19]. This will lead to removal of apo monomers from the reaction scheme resulting in a weak incomplete ligand binding similar to that observed at FBP concentrations <10nM. The presence of synthetic and natural detergents normalized folate binding kinetics and resulted in appearance of monomeric holo-FBP. Fluorescence spectroscopy indicated molecular interactions between detergent and tryptophan residues located in hydrophobic structures of apo-FBP which may participate in protein associations. GENERAL SIGNIFICANCE: Self-association into multimers may protect binding sites, and in case of holo-FBP even folate from biological degradation. High-affinity folate binding in body secretions, typically containing 1-10nM FBP, requires the presence of natural detergents, i.e. cholesterol and phospholipids, to avoid complexation between apo- and holo-FBP.

Cooperative formation of native-like tertiary contacts in the ensemble of unfolded states of a four-helix protein.

In studies of the ensembles of unfolded structures of a four-helix bundle protein, we have detected the presence of potential precursors of native tertiary structures. These observations were based on the perturbation of NMR chemical shifts of the protein backbone atoms by single site mutations. Some mutations change the chemical shifts of residues remote from the site of mutation indicating the presence of an interaction between the mutated and the remote residues, suggesting that the formation of helix segments and helix-helix interactions is cooperative. We can begin to track down the folding mechanism of this protein using only experimental data by combining the information available for the rate limiting structure formation during the folding process with measurements of the site specific hydrogen bond formation in the burst phase, and with the existence prior to the folding reaction of tertiary structures in the ensemble of otherwise unfolded structures observed in the present study.

A chemometric analysis of ligand-induced changes in intrinsic fluorescence of folate binding protein indicates a link between altered conformational structure and physico-chemical characteristics.

Ligand binding alters the conformational structure and physico-chemical characteristics of bovine folate binding protein (FBP). For the purpose of achieving further information we analyzed ligand (folate and methotrexate)-induced changes in the fluorescence landscape of FBP. Fluorescence excitation and emission two-dimensional (2D) spectra were recorded over a wide range of wavelengths on a Perkin-Elmer LS 55 spectrofluorometer at varying pH in different buffers, and the resulting three-dimensional data were subjected to a chemometric analysis, parallel factor analysis (PARAFAC). The most important finding was the occurrence of two maximum intensity emission wavelengths of tryptophan, 350 nm (component one) and 330 nm (component two). In contrast to the first component, the score of the short wavelength component increased with increasing ligation of FBP. Since the emission wavelengths of indole groups in tryptophan shorten with increasing distance from the solvent surface of proteins, an increasing number of the 11 tryptophan residues seem to reorientate from the solvent surface to the interior of FBP with increasing ligation. The sharp decrease in hydrophobicity at pI=7-8 following binding of folate accords fairly well with the disappearance of strongly hydrophobic tryptophan residues from the solvent-exposed surface of FBP. The PARAFAC has thus proven useful to establish a hitherto unexplained link between parallel changes in conformational structure and physico-chemical characteristics of FBP induced by folate binding. Parameters for ligand binding derived from PARAFAC analysis of the fluorescence data were qualitatively and quantitatively similar to those obtained from binding of radiofolate to FBP. Herein, methotrexate exhibited a higher affinity for FBP than in competition with radiofolate. This could suggest a rapid and firm complexation of folate to FBP, blocking access of competing ligands.

Peptide binding specificity of the chaperone calreticulin.

Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composition. A large set of available synthetic peptides (n=127) was tested for binding to calreticulin and the results analysed by multivariate data analysis. The parameter that correlated best with binding was hydrophobicity while beta-turn potential disfavoured binding. Only hydrophobic peptides longer than 5 amino acids showed binding and a clear correlation with hydrophobicity was demonstrated for oligomers of different hydrophobic amino acids. Insertion of hydrophilic amino acids in a hydrophobic sequence diminished or abolished binding. In conclusion our results show that calreticulin has a peptide-binding specificity for hydrophobic sequences and delineate the fine specificity of calreticulin for hydrophobic amino acid residues.

Correcting attenuated total reflection-Fourier transform infrared spectra for water vapor and carbon dioxide.

Fourier transform infrared (FT-IR) spectroscopy is a valuable technique for characterization of biological samples, providing a detailed fingerprint of the major chemical constituents. However, water vapor and CO(2) in the beam path often cause interferences in the spectra, which can hamper the data analysis and interpretation of results. In this paper we present a new method for removal of the spectral contributions due to atmospheric water and CO(2) from attenuated total reflection (ATR)-FT-IR spectra. In the IR spectrum, four separate wavenumber regions were defined, each containing an absorption band from either water vapor or CO(2). From two calibration data sets, gas model spectra were estimated in each of the four spectral regions, and these model spectra were applied for correction of gas absorptions in two independent test sets (spectra of aqueous solutions and a yeast biofilm (C. albicans) growing on an ATR crystal, respectively). The amounts of the atmospheric gases as expressed by the model spectra were estimated by regression, using second-derivative transformed spectra, and the estimated gas spectra could subsequently be subtracted from the sample spectra. For spectra of the growing yeast biofilm, the gas correction revealed otherwise hidden variations of relevance for modeling the growth dynamics. As the presented method improved the interpretation of the principle component analysis (PCA) models, it has proven to be a valuable tool for filtering atmospheric variation in ATR-FT-IR spectra.

Application of near-infrared and Fourier transform infrared spectroscopy in the characterization of ligand-induced conformation changes in folate binding protein purified from bovine milk: influence of buffer type and pH.

Fourier transform infrared (FT-IR) and near-infrared (NIR) spectroscopy have been applied to detect structural alterations in folate binding protein (FBP) induced by ligation in different buffer types. The amide I region pointed to a beta-sheet to alpha-helix transition upon ligation in acetate and phosphate buffers, and the formation of intermolecular beta-sheet was indicated at pH 5.0, in agreement with a dimerization of FBP taking place at this pH. The ligand-induced changes in the 2100-2300 nm NIR region were significant for FBP in acetate and phosphate buffers of pH 5.0, and the variations were interpreted as secondary structure changes, based on previous assignments of secondary structures to the combination bands in the NIR region. In the case of acetate buffer, variations in the amide combination bands agreed with the amide I analysis, but for the other buffer types some discrepancies were found and explained by side-chain contributions to the NIR, which could reflect the tertiary and quaternary structure differences. NIR spectra of FBP at pH 7.4 and 5.0 revealed contradictory effects on the side chains, reflecting different polymerization events at the two pH values, whereas the amide I region indicated similar changes at the two pH values. Therefore, we suggest that FT-IR and NIR spectroscopy may complement each other, such that the two techniques in combination may give information on all three types of protein conformational changes. While the secondary structure changes are revealed by FT-IR, the tertiary and quaternary structure changes are reflected in the NIR spectra, although the general influence of the latter changes on the NIR spectra remains to be confirmed.

Multivariate approaches in plant science.

The objective of proteomics is to get an overview of the proteins expressed at a given point in time in a given tissue and to identify the connection to the biochemical status of that tissue. Therefore sample throughput and analysis time are important issues in proteomics. The concept of proteomics is to encircle the identity of proteins of interest. However, the overall relation between proteins must also be explained. Classical proteomics consist of separation and characterization, based on two-dimensional electrophoresis, trypsin digestion, mass spectrometry and database searching. Characterization includes labor intensive work in order to manage, handle and analyze data. The field of classical proteomics should therefore be extended to also include handling of large datasets in an objective way. The separation obtained by two-dimensional electrophoresis and mass spectrometry gives rise to huge amount of data. We present a multivariate approach to the handling of data in proteomics with the advantage that protein patterns can be spotted at an early stage and consequently the proteins selected for sequencing can be selected intelligently. These methods can also be applied to other data generating protein analysis methods like mass spectrometry and near infrared spectroscopy and examples of application to these techniques are also presented. Multivariate data analysis can unravel complicated data structures and may thereby relieve the characterization phase in classical proteomics. Traditionally statistical methods are not suitable for analysis of the huge amounts of data, where the number of variables exceed the number of objects. Multivariate data analysis, on the other hand, may uncover the hidden structures present in these data. This study takes its starting point in the field of classical proteomics and shows how multivariate data analysis can lead to faster ways of finding interesting proteins. Multivariate analysis has shown interesting results as a supplement to classical proteomics and added a new dimension to the field of proteomics.

Antigenic specificity of serum antibodies in mice fed soy protein.

BACKGROUND: Soybean protein is used in a number of food products but unfortunately is also a common cause of food allergy. Upon ingestion of soy protein, healthy mice like other animals and humans generate a soy-specific antibody response in the absence of signs of illness. Not much is known about the relationship between the immunogenic proteins involved in this nondeleterious antibody response and the pathological response associated with food allergy. The objective of the present study was to characterize the antigenic specificity of the soy protein-specific antibody response generated in healthy mice ingesting soy protein. METHODS: Blood from mice fed a soy-containing diet was analyzed using ELISA and immunoblot for antibody reactivity towards various soy protein fractions and pure soy proteins/subunits. Mice bred on a soy-free diet were used as controls. RESULTS: The detectable antigenic specificity of the serum antibodies of soy-consuming mice comprised glycinin and beta-conglycinin. Immunoblots with soy protein extract demonstrated antibody reactivity towards both the basic and the acidic chains of glycinin and the beta-conglycinin subunits with an individual response pattern among mice. Moreover, antibody reactivity was found towards the native quaternary structure of glycinin. CONCLUSIONS: Mice ingesting soy protein generate an antibody response with reactivity towards glycinin and beta-conglycinin. Antibody reactivity found towards the native quaternary structure of glycinin indicates an oral immunogenicity of the highly processing-resistant oligomerized glycinin.