Snooker Structure-Based Pharmacophore Model Explains Differences in Agonist and Blocker Binding to Bitter Receptor hTAS2R39
Roland, W.S.U. ; Sanders, M.P.A. ; Buren, L. van; Gouka, R.J. ; Gruppen, H. ; Vincken, J.P. ; Ritschel, T. - \ 2015
PLoS ONE 10 (2015)3. - ISSN 1932-6203
protein-coupled receptors - class-a gpcrs - taste receptors - activation - identification - requirements - peptides - family - assay - t2r1
The human bitter taste receptor hTAS2R39 can be activated by many dietary (iso)flavonoids. Furthermore, hTAS2R39 activity can be blocked by 6-methoxyflavanones, 4’-fluoro-6-methoxyflavanone in particular. A structure-based pharmacophore model of the hTAS2R39 binding pocket was built using Snooker software, which has been used successfully before for drug design of GPCRs of the rhodopsin subfamily. For the validation of the model, two sets of compounds, both of which contained actives and inactives, were used: (i) an (iso)flavonoid-dedicated set, and (ii) a more generic, structurally diverse set. Agonists were characterized by their linear binding geometry and the fact that they bound deeply in the hTAS2R39 pocket, mapping the hydrogen donor feature based on T5.45 and N3.36, analogues of which have been proposed to play a key role in activation of GPCRs. Blockers lack hydrogen-bond donors enabling contact to the receptor. Furthermore, they had a crooked geometry, which could sterically hinder movement of the TM domains upon receptor activation. Our results reveal characteristics of hTAS2R39 agonist and bitter blocker binding, which might facilitate the development of blockers suitable to counter the bitterness of dietary hTAS2R39 agonists in food applications.
Determination of the influence of the pH of hydrolysis on enzyme selectivity of Bacillus licheniformis protease towards whey protein isolate
Butre, C.I. ; Sforza, S. ; Wierenga, P.A. ; Gruppen, H. - \ 2015
International Dairy Journal 44 (2015). - ISSN 0958-6946 - p. 44 - 53.
differential scanning calorimetry - beta-lactoglobulin - peptides - endopeptidases - kinetics
Enzymatic protein hydrolysis is typically described by the degree of hydrolysis and by the enzyme specificity. While the specificity describes which cleavage sites can potentially be cleaved, it does not describe which are preferred. To identify the relative rate at which each individual cleavage site is hydrolysed, enzyme selectivity has recently been introduced. To test the effect of pH on selectivity, whey protein isolate (WPI) was hydrolysed with Bacillus licheniformis protease (BLP) at pH 7.0, 8.0 or 9.0. At all pH values, large differences in the enzyme selectivity (from
Protein redesign by learning from data
Berg, B.A. van den; Reinders, M.J.T. ; Laan, J.M. van der; Roubos, J.A. ; Ridder, D. de - \ 2014
Protein Engineering, Design & Selection 27 (2014)9. - ISSN 1741-0126 - p. 281 - 288.
computational enzyme design - aspergillus - stabilization - optimization - generation - prediction - secretion - hydrolase - peptides - tools
Protein redesign methods aim to improve a desired property by carefully selecting mutations in relevant regions guided by protein structure. However, often protein structural requirements underlying biological characteristics are not well understood. Here, we introduce a methodology that learns relevant mutations from a set of proteins that have the desired property and demonstrate it by successfully improving production levels of two enzymes by Aspergillus niger, a relevant host organism for industrial enzyme production. We validated our method on two enzymes, an esterase and an inulinase, creating four redesigns with 5-45 mutations. Up to 10-fold increase in production was obtained with preserved enzyme activity for small numbers of mutations, whereas production levels and activities dropped for too aggressive redesigns. Our results demonstrate the feasibility of protein redesign by learning. Such an approach has great potential for improving production levels of many industrial enzymes and could potentially be employed for other design goals.
Dilute self-healing hydrogels of silk-collagen-like block copolypeptides at neutral pH
Golinska, M.D. ; Wlodarczyk-Biegun, M.K. ; Werten, M.W.T. ; Cohen Stuart, M.A. ; Wolf, F.A. de; Vries, R.J. de - \ 2014
Biomacromolecules 15 (2014)3. - ISSN 1525-7797 - p. 699 - 706.
spider dragline silk - drug-delivery - fibrous biomaterials - actin solutions - contact-lenses - scaffolds - gels - peptides - polymers - elasticity
We report on self-healing, pH-responsive hydrogels that are entirely protein-based. The protein is a denovo designed recombinant triblock polypeptide of 66 kg/mol consisting of a silk-like middle block (GAGAGAGH)48, flanked by two long collagen-inspired hydrophilic random coil side blocks. The pH-dependent charge on the histidines in the silk block controls folding and stacking of the silk block. At low pH the protein exists as monomers, but above pH 6 it readily self-assembles into long fibers. At higher concentrations the fibers form self-healing physical gels. Optimal gel strength and self-healing are found at a pH of around 7. The modulus of a 2 wt % gel at pH 7 is G' = 1700 Pa. Being protein-based, and amenable to further sequence engineering, we expect that these proteins are promising scaffold materials to be developed for a broad range of biomedical applications.
Introducing enzyme selectivity: a quantitative parameter to describe enzymatic protein hydrolysis
Butré, C.I. ; Sforza, S. ; Gruppen, H. ; Wierenga, P.A. - \ 2014
Analytical and Bioanalytical Chemistry 406 (2014)24. - ISSN 1618-2642 - p. 5827 - 5841.
bovine beta-lactoglobulin - tryptic hydrolysis - substrate-specificity - mass-spectrometry - release kinetics - casein - peptides - identification - ph - proteases
Enzyme selectivity is introduced as a quantitative parameter to describe the rate at which individual cleavage sites in a protein substrate are hydrolyzed relative to other cleavage sites. Whey protein isolate was hydrolyzed by Bacillus licheniformis protease, which is highly specific for Glu and Asp residues. The molar concentration of all peptides (58) from ß-lactoglobulin formed during hydrolysis was determined from the UV214 signal. The quality of identification and quantification of the peptides were described by newly defined parameters: the peptide sequence coverage (on average 94 %) and the molar sequence coverage (on average 75 %). The selectivity was calculated from the rate of hydrolysis of each cleavage site, and showed differences of up to a factor of 5,000. The ability to quantitatively discriminate the enzyme preference towards individual cleavage sites is considered essential to the understanding of enzymatic protein hydrolysis.
6-Methoxyflavanones as Bitter Taste Receptor Blockers
Roland, W.S.U. ; Gouka, R.J. ; Gruppen, H. ; Driesse, M. ; Buren, L. van; Smit, G. ; Vincken, J.P. - \ 2014
PLoS ONE 9 (2014)4. - ISSN 1932-6203
structural requirements - activate - peptides - subunits
Many (dietary) bitter compounds, e.g. flavonoids, activate bitter receptor hTAS2R39 in cell-based assays. Several flavonoids, amongst which some flavanones, are known not to activate this receptor. As certain flavanones are known to mask bitter taste sensorially, flavanones might act as bitter receptor antagonists. Fourteen flavanones were investigated for their potential to reduce activation of hTAS2R39 by epicatechin gallate (ECG), one of the main bitter compounds occurring in green tea. Three flavanones showed inhibitory behavior towards the activation of hTAS2R39 by ECG: 49-fluoro-6-methoxyflavanone, 6,39-dimethoxyflavanone, and 6-methoxyflavanone (in order of decreasing potency). The 6-methoxyflavanones also inhibited activation of hTAS2R14 (another bitter receptor activated by ECG), though to a lesser extent. Dose-response curves of ECG at various concentrations of the full antagonist 49-fluoro-6-methoxyflavanone and wash-out experiments indicated reversible insurmountable antagonism. The same effect was observed for the structurally different agonist denatonium benzoate
Nonlinear Amplification of a Supramolecular Complex at a Multivalent Interface
Hsu, S.H. ; Yilmaz, M.D. ; Reinhoudt, D.N. ; Velders, A.H. ; Huskens, J. - \ 2013
Angewandte Chemie-International Edition 52 (2013)2. - ISSN 1433-7851 - p. 714 - 719.
self-assembled monolayers - molecular printboards - chemistry - binding - membrane - nanostructures - organization - selection - discrete - peptides
Competition with a monovalent cyclodextrin host (blue cones) in solution drives the multivalent binding of a Eu3+ complex and a sensitizer molecule to cyclodextrin monolayers through a nonlinear self-assembly process. Adamantyl groups (light-blue spheres) are attached to the EDTA ligand (black) and the antenna molecule (orange), which has a carboxylate group for coordination to the Eu3+ ion (yellow or red in free or complexed form, respectively).
Influence of high solid concentrations on enzymatic wheat gluten hydrolysis and resulting functional properties
Hardt, N.A. ; Goot, A.J. van der; Boom, R.M. - \ 2013
Journal of Cereal Science 57 (2013)3. - ISSN 0733-5210 - p. 531 - 536.
ultrafiltration - protein - starch - improvement - fractions - peptides - systems - flour
Enzymatic hydrolysis at increased solid concentrations is beneficial with regard to energy and water consumption. This study examines the influence of the solid concentration on the enzymatic hydrolysis of wheat gluten and the resulting functional properties of the hydrolysate. Wheat gluten was mildly hydrolyzed at a solid concentration varying from 10% to 60% to degrees of hydrolysis (DH%) ranging from 3.2% to 10.2%. The gluten was susceptible to hydrolysis at all solid concentrations but the hydrolysis rate was influenced by increasing solid concentrations. Size-exclusion high-performance liquid chromatography revealed an increase in the ratio of peptides with a molecular mass >25 kDa for solid concentrations of 40% and 60%. The water solubility increased on hydrolysis and was independent of the solid concentration during proteolysis. The foam stability was not influenced by the solid concentration at low DH%. At DH% higher than 8%, high solid concentrations increased the foam stability, which might be related to the presence of more peptides with a molecular mass >25 kDa. In addition, we found increased reactor productivity. The results show the potential of hydrolyzing wheat gluten at high solid concentrations, which could lead to large savings for water and energy when applied industrially.
Computational protein design with electrostatic focusing: experimental characterization of a conditionally folded helical domain with a reduced amino acid alphabet
Suarez Diez, M. ; Pujol, M. ; Matzapetakis, M. ; Jaramillo, A. ; Iranzo, O. - \ 2013
Biotechnology Journal 8 (2013)7. - ISSN 1860-6768 - p. 855 - 864.
solution nmr structure - structural basis - peptides - recognition - prediction - sequences - dynamics - energy - trifluoroethanol - optimization
Automated methodologies to design synthetic proteins from first principles use energy computations to estimate the ability of the sequences to adopt a targeted structure. This approach is still far from systematically producing native-like sequences, due, most likely, to inaccuracies when modeling the interactions between the protein and its aqueous environment. This is particularly challenging when engineering small protein domains (with less polar pair interactions than with the solvent). We have re-designed a three-helix bundle, domain B, using a fixed backbone and a four amino acid alphabet. We have enlarged the rotamer library with conformers that increase the weight of electrostatic interactions within the design process without altering the energy function used to compute the folding free energy. Our synthetic sequences show less than 15% similarity to any Swissprot sequence. We have characterized our sequences in different solvents using circular dichroism and nuclear magnetic resonance. The targeted structure achieved is dependent on the solvent used. This method can be readily extended to larger domains. Our method will be useful for the engineering of proteins that become active only in a given solvent and for designing proteins in the context of hydrophobic solvents, an important fraction of the situations in the cell
16 kDa Heat Shock Protein from Heat-Inactivated Mycobacterium tuberculosis Is a Homodimer – Suitability for Diagnostic Applications with Specific Llama VHH Monoclonals
Srivastava, S.K. ; Ruigrok, V.J.B. ; Thompson, N.J. ; Trilling, A.K. ; Heck, A.J.R. ; Rijn, C.J.M. van; Beekwilder, M.J. ; Jongsma, M.A. - \ 2013
PLoS ONE 8 (2013)5. - ISSN 1932-6203
hiv-associated tuberculosis - immunological diagnosis - antibody fragments - mass-spectrometry - skin-test - antigen - complexes - responses - peptides - epitopes
Background: The 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). Its use in serum-based diagnostics is limited, but for the direct identification of M.tb. bacteria in sputum or cultures it may represent a useful tool. Recently, a broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored. Methodology/Principal Findings: To identify the epitopes recognized by the nine (randomly selected from a set of twelve 16 kDa specific VHH antibodies) distinct VHH antibodies, 14 overlapping linear epitopes (each 20 amino acid long) were characterized using direct and sandwich ELISA techniques. Seven out of 14 epitopes were recognized by 8 out of 9 VHH antibodies. The two highest affinity binders B-F10 and A-23 were found to bind distinct epitopes. Sandwich ELISA and SPR experiments showed that only B-F10 was suitable as secondary antibody with both B-F10 and A-23 as anchoring antibodies. To explain this behavior, the epitopes were matched to the putative 3D structure model. Electrospray ionization time-of-flight mass spectrometry and size exclusion chromatography were used to determine the higher order conformation. A homodimer model best explained the differential immunological reactivity of A-23 and B-F10 against heat-treated M.tb. lysates. Conclusions/Significance: The concentrations of secreted antigens of M.tb. in sputum are too low for immunological detection and existing kits are only used for identifying M.tb. in cultures. Here we describe how specific combinations of VHH domains could be used to detect the intracellular HSP antigen. Linked to methods of pre-concentrating M.tb. cells prior to lysis, HSP detection may enable the development of protein-based diagnostics of sputum samples and earlier diagnosis of diseases.
Polymerisation of Beta-alanine through catalytic ester-amide exchange
Steunenberg, P. ; Könst, P.M. ; Scott, E.L. ; Franssen, M.C.R. ; Zuilhof, H. ; Sanders, J.P.M. - \ 2013
European Polymer Journal 49 (2013)7. - ISSN 0014-3057 - p. 1773 - 1781.
nitrogen-containing chemicals - ring-opening polymerization - amino-acids - biobased production - cyanophycin - peptides - esterification - acrylamide - polymers - mild
Herein we present the use of group (IV) metal alkoxides as catalysts for the polymerisation of esters of p-alanine and its derivatives. The influence of different group (IV) metal alkoxides, different esters, temperature and solvents on the polymerisation are investigated. The order in which the group (IV) metal alkoxides catalyse the polymerisation is: Hf(Ot-Bu)(4) - >= Zr(Ot-Bu)(4) > Ti(Oi-Pr) 4 > Ti(On-Bu)(4). Polymers with the highest degree of polymerisation are obtained performing a neat polymerisation of p-alanine methyl ester in the presence of Hf(Ot-Bu)(4) as a catalyst at 50 degrees C. The polymerisation can also be carried out efficiently in solution, in different solvents and at higher reaction temperatures. This method could also be applied for the formation of other polypeptides. (c) 2013 Elsevier Ltd. All rights reserved.
Enzyme-Catalyzed Polymerization of Beta-alanine Esters, A Sustainable Route Towards the Formation of Poly-Beta-alanine
Steunenberg, P. ; Uiterweerd, M. ; Sijm, M. ; Scott, E.L. ; Zuilhof, H. ; Sanders, J.P.M. ; Franssen, M.C.R. - \ 2013
Current Organic Chemistry 17 (2013)7. - ISSN 1385-2728 - p. 682 - 690.
nitrogen-containing chemicals - ring-opening polymerization - amino-acids - organic-solvents - biobased production - peptides - lactams - lipases - biotransformations - esterification
The synthesis of poly-ß-alanine by a lipase catalyzed polycondensation reaction between ß-alanine esters is reported. The effect of different solvents, reaction temperatures and substrate/enzyme concentrations on polymer yield and degree of polymerization (DP) was determined. Also the effect of methyl substituents at the a and ß position of ß-alanine was studied. Cross-Linked Enzyme Aggregates (CLEA) of Candida antarctica lipase B converted ß-alanine esters into polymers in high yield (up to 90%) and with high (DP) (up to 53 units). Optimum results were obtained when methyl esters are used in methyl tert-butyl ether (MTBE) as the solvent, at 50°C for 16 h.
Discovery of T cell epitopes implementing HLA-peptidomics into a reverse immunology approach
Hombrink, P. ; Hassan, C. ; Kester, M.G.D. ; Ru, A.H. ; Bergen, C.A.M. ; Nijveen, H. ; Drijfhout, J.W. ; Falkenburg, J.H.F. ; Heemskerk, M.H.M. ; Veelen, P.A. van - \ 2013
The Journal of Immunology 190 (2013)8. - ISSN 0022-1767 - p. 3869 - 3877.
minor histocompatibility antigens - versus-host-disease - restricted tissue distribution - identification - peptides - leukemia - gene - immunotherapy - lymphocytes - complexes
T cell recognition of minor histocompatibility Ags (MiHA) plays an important role in the graft-versus-tumor effect of allogeneic stem cell transplantation. Selective infusion of T cells reactive for hematopoiesis-restricted MiHA presented in the context of HLA class I or II molecules may help to separate the graft-versus-tumor effects from graft-versus-host disease effects after allogeneic stem cell transplantation. Over the years, increasing numbers of MiHA have been identified by forward immunology approaches, and the relevance of these MiHA has been illustrated by correlation with clinical outcome. As the tissue distribution of MiHA affects the clinical outcome of T cell responses against these Ags, it would be beneficial to identify additional predefined MiHA that are exclusively expressed on hematopoietic cells. Therefore, several reverse immunology approaches have been explored for the prediction of MiHA. Thus far, these approaches frequently resulted in the identification of T cells directed against epitopes that are not naturally processed and presented. In this study we established a method for the identification of biologically relevant MiHA, implementing mass spectrometry–based HLA-peptidomics into a reverse immunology approach. For this purpose, HLA class I binding peptides were eluted from transformed B cells, analyzed by mass spectrometry, and matched with a database dedicated to identifying polymorphic peptides. This process resulted in a set of 40 MiHA candidates that were evaluated in multiple selection steps. The identification of LB-NISCH-1A demonstrated the technical feasibility of our approach. On the basis of these results, we present an approach that can be of value for the efficient identification of MiHA or other T cell epitopes.
The lipid droplet coat protein perilipin 5 also localizes to muscle mitochondria
Bosma, M. ; Minnaard, R. ; Sparks, L.M. ; Schaart, G. ; Losen, M. ; Baets, M.H. de; Duimel, H. ; Kersten, A.H. ; Bickel, P.E. ; Schrauwen, P. ; Hesselink, M.K.C. - \ 2012
Histochemistry and Cell Biology 137 (2012)2. - ISSN 0948-6143 - p. 205 - 216.
carnitine-palmitoyltransferase-i - human skeletal-muscle - substrate metabolism - lipolysis - adipocytes - peptides - promotes - tissues - family - signal
Perilipin 5 (PLIN5/OXPAT) is a lipid droplet (LD) coat protein mainly present in tissues with a high fat-oxidative capacity, suggesting a role for PLIN5 in facilitating fatty acid oxidation. Here, we investigated the role of PLIN5 in fat oxidation in skeletal muscle. In human skeletal muscle, we observed that PLIN5 (but not PLIN2) protein content correlated tightly with OXPHOS content and in rat muscle PLIN5 content correlated with mitochondrial respiration rates on a lipid-derived substrate. This prompted us to examine PLIN5 protein expression in skeletal muscle mitochondria by means of immunogold electron microscopy and Western blots in isolated mitochondria. These data show that PLIN5, in contrast to PLIN2, not only localizes to LD but also to mitochondria, possibly facilitating fatty acid oxidation. Unilateral overexpression of PLIN5 in rat anterior tibialis muscle augmented myocellular fat storage without increasing mitochondrial density as indicated by the lack of change in protein content of five components of the OXPHOS system. Mitochondria isolated from PLIN5 overexpressing muscles did not possess increased fatty acid respiration. Interestingly though, (14)C-palmitate oxidation assays in muscle homogenates from PLIN5 overexpressing muscles revealed a 44.8% (P = 0.05) increase in complete fatty acid oxidation. Thus, in mitochondrial isolations devoid of LD, PLIN5 does not augment fat oxidation, while in homogenates containing PLIN5-coated LD, fat oxidation is higher upon PLIN5 overexpression. The presence of PLIN5 in mitochondria helps to understand why PLIN5, in contrast to PLIN2, is of specific importance in fat oxidative tissues. Our data suggests involvement of PLIN5 in directing fatty acids from the LD to mitochondrial fatty acid oxidation.
Controlling the aggregation and gelation of ß-lactoglobulin by the addition of its peptides
Kosters, H.A. - \ 2012
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Peter Wierenga. - S.l. : s.n. - ISBN 9789461732040 - 171
bèta-lactoglobuline - aggregatie - gelering - peptiden - eiwithydrolysaten - beta-lactoglobulin - aggregation - gelation - peptides - protein hydrolysates
In this thesis the effects of peptides, or protein hydrolysates on the heat-induced aggregation and gelation of (concentrated) protein systems were studied. First, it was investigated if specific peptides could influence the heat-induced denaturation and aggregation of intact proteins solutions, and which peptide properties dominated the different interactions. Next, the effects of the peptides on the heat-induced gelation of intact proteins as a model for a potential high protein food system were studied.
It was found that certain peptides in the hydrolysate show binding to native proteins, and some additional peptides bind to unfolded proteins. Since the same peptides were shown to bind to not only β-lactoglobulin, but also other to proteins, it is concluded that the binding does not depend on specific molecular details of the protein. The hydrophobicity and charge were found to be important in determining the binding and the effect on aggregation. With the hydrolysates, as well as with two synthesized peptides (modelled on those found in the hydrolysate) it was confirmed that the addition of these binding peptides has significant effects on the heat-induced aggregation. In the gelation experiments performed in this study a dominant effect was found for peptides containing free SH groups. While it is expected that the changes in aggregation behaviour, induced by the binding of non-cysteine-containing peptides also affects the gel properties, this was not found with the techniques used. Finally, disulfide-containing peptides were found to reduce the presence of sulfurous volatiles formed after heating of β-lactoglobulin, WPI and lysozyme.
Since only certain peptides exhibit binding to intact proteins, it is expected that control over the hydrolysis process and, thereby the concentrations of such specific peptides, can be used to produce hydrolysates with specific functionalities in this respect.
The influence of casein and urea as nitrogen sources on in vitro equine caecal fermentation
Santos, A.S. ; Ferreira, L.M.M. ; Martin-Rosset, W. ; Cotovio, M. ; Silva, F. ; Bennett, R.N. ; Cone, J.W. ; Bessa, R.J.B. ; Rodrigues, M.A.M. - \ 2012
Animal 6 (2012)7. - ISSN 1751-7311 - p. 1096 - 1102.
rumen microbial-growth - gas-production - amino-acids - protein - ponies - digestibility - metabolism - peptides - kinetics - invitro
To access the fermentative response of equine caecal microbial population to nitrogen availability, an in vitro study was conducted using caecal contents provided with adequate energy sources and nitrogen as limiting nutrient. Two nitrogen (N) sources were provided, protein (casein) and non-protein (urea). Caecal fluid, taken from three cannulated horses receiving a hay–concentrate diet, was mixed with a N-free buffer–mineral solution. The influence of four N levels (3.7, 6.3, 12.5 or 25 mg of N in casein or urea) was studied using the gas production technique. Total volatile fatty acids (VFA), NH3-N and gas production were measured after a 24-h incubation period. Microbial biomass was estimated using adenine and guanine bases as internal markers, and ATP production was estimated stoichiometrically. Microbial growth efficiency (YATP) and gas efficiency (Egas) were estimated. Fermentation with casein as the sole N source was generally characterized by lower total VFA, NH3-N, total gas production and higher acetate : propionate (A : P) ratio and YATP than with urea. Results herein presented indicate that, under these in vitro conditions, caecal microbial population does in fact use urea N, but less efficiently than casein in terms of microbial growth
Identification and localization of the structural proteins of anguillid herpesvirus 1
Beurden, S.J. van; Leroy, B. ; Wattiez, R. ; Haenen, O.L.M. ; Boeren, S. ; Vervoort, J.J.M. ; Peeters, B.P.H. ; Rottier, P.J.M. ; Engelsma, M.Y. ; Vanderplasschen, A.F. - \ 2011
Veterinary Research 42 (2011). - ISSN 0928-4249 - 15 p.
herpes-simplex-virus - channel catfish virus - koi herpesvirus - proteomic analysis - genome sequences - european eel - virions - peptides
Many of the known fish herpesviruses have important aquaculture species as their natural host, and may cause serious disease and mortality. Anguillid herpesvirus 1 (AngHV-1) causes a hemorrhagic disease in European eel, Anguilla anguilla. Despite their importance, fundamental molecular knowledge on fish herpesviruses is still limited. In this study we describe the identification and localization of the structural proteins of AngHV-1. Purified virions were fractionated into a capsid-tegument and an envelope fraction, and premature capsids were isolated from infected cells. Proteins were extracted by different methods and identified by mass spectrometry. A total of 40 structural proteins were identified, of which 7 could be assigned to the capsid, 11 to the envelope, and 22 to the tegument. The identification and localization of these proteins allowed functional predictions. Our findings include the identification of the putative capsid triplex protein 1, the predominant tegument protein, and the major antigenic envelope proteins. Eighteen of the 40 AngHV-1 structural proteins had sequence homologues in related Cyprinid herpesvirus 3 (CyHV-3). Conservation of fish herpesvirus structural genes seemed to be high for the capsid proteins, limited for the tegument proteins, and low for the envelope proteins. The identification and localization of the structural proteins of AngHV-1 in this study adds to the fundamental knowledge of members of the Alloherpesviridae family, especially of the Cyprinivirus genus.
Natural variation in avenin epitopes among oat varieties: implications for Celiac
Mujico, J.R. ; Mitea, C. ; Gilissen, L.J.W.J. ; Ru, A. ; Veelen, P. van; Smulders, M.J.M. ; Koning, F. de - \ 2011
Journal of Cereal Science 54 (2011)1. - ISSN 0733-5210 - p. 8 - 12.
t-cells - gliadin - gluten - wheat - peptides - prevalence - antibodies - toxicity - children - hla-dq2
Celiac disease (CD) is a chronic inflammatory disease affecting the small intestinal mucosa. The causative agents have been identified as gluten proteins from wheat, barley and rye, and the only available treatment for CD patients is a lifelong gluten-free diet. Non-gluten containing cereals would be a valuable contribution to the gluten-free diet. In this respect, oats are a good choice. However, commercial lots of oat flakes and flour frequently are contaminated with wheat, barley and rye, and two studies have reported that some peptides derived from the gluten-like avenin storage proteins of oat can trigger an immune response in some CD patients. In the present study we have initiated the investigation whether all oat varieties contain similar amounts of potentially harmful sequences by biochemical and immunological methods. We confirm that commercial oat preparations are contaminated with other cereals that contain gluten or gluten-like proteins. Moreover, our results demonstrate that contamination-free oat varieties differ in their capacity to stimulate an avenin-sensitive gamma-gliadin specific T cell line derived from a patient with CD, indicative for differences in the two known avenin epitopes among oat varieties, implying that selection and breeding of completely safe oat varieties for all CD patients may be a realistic possibility.
Soya bean tempe extracts show antibacterial activity against Bacillus cereus cells and spores
Roubos-van den Hil, P.J. ; Dalmas, E. ; Nout, M.J.R. ; Abee, T. - \ 2010
Journal of Applied Microbiology 109 (2010)1. - ISSN 1364-5072 - p. 137 - 145.
rhizopus-oligosporus - food biopreservation - bacteriocins - peptides
Aims: Tempe, a Rhizopus ssp.-fermented soya bean food product, was investigated for bacteriostatic and/or bactericidal effects against cells and spores of the food-borne pathogen Bacillus cereus. Methods and results: Tempe extract showed a high antibacterial activity against B. cereus ATCC 14579 based on optical density and viable count measurements. This growth inhibition was manifested by a 4 log CFU ml-1 reduction, within the first 15 min of exposure. Tempe extracts also rapidly inactivated B. cereus spores upon germination. Viability and membrane permeability assessments using fluorescence probes showed rapid inactivation and permeabilization of the cytoplasmic membrane confirming the bactericidal mode of action. Cooked beans and Rhizopus grown on different media did not show antibacterial activity, indicating the unique association of the antibacterial activity with tempe. Subsequent characterization of the antibacterial activity revealed that heat treatment and protease addition nullified the bactericidal effect, indicating the proteinaceous nature of the bioactive compound. Conclusions: During fermentation of soya beans with Rhizopus, compounds are released with extensive antibacterial activity against B. cereus cells and spores. Significance and Impact of Study: The results show the potential of producing natural antibacterial compounds that could be used as ingredients in food preservation and pathogen control
In search of tetraploid wheat accessions reduced in celiac disease-related gluten epitopes
Broeck, H.C. van den; Hongbing, C. ; Lacaze, X. ; Dusautoir, J.C. ; Gilissen, L.J.W.J. ; Smulders, J.M. ; Meer, I.M. van der - \ 2010
Molecular BioSystems 6 (2010)11. - ISSN 1742-206X - p. 2206 - 2213.
durum-wheat - storage proteins - polyploid wheat - bread wheat - subunit genes - domestication - gliadin - peptides - triticum - complex
Tetraploid wheat (durum wheat) is mainly used for the preparation of pasta. As a result of breeding, thousands of tetraploid wheat varieties exist, but also tetraploid landraces are still maintained and used for local food preparations. Gluten proteins present in wheat can induce celiac disease, a T-cell mediated auto-immune disorder, in genetically predisposed individuals after ingestion. Compared to hexaploid wheat, tetraploid wheat might be reduced in T-cell stimulatory epitopes that cause celiac disease because of the absence of the D-genome. We tested gluten protein extracts from 103 tetraploid wheat accessions (obtained from the Dutch CGN genebank and from the French INRA collection) including landraces, old, modern, and domesticated accessions of various tetraploid species and subspecies from many geographic origins. Those accessions were typed for their level of T-cell stimulatory epitopes by immunoblotting with monoclonal antibodies against the a-gliadin epitopes Glia-a9 and Glia-a20. In the first selection, we found 8 CGN and 6 INRA accessions with reduced epitope staining. Fourteen of the 57 CGN accessions turned out to be mixed with hexaploid wheat, and 5 out of the 8 selected CGN accessions were mixtures of two or more different gluten protein chemotypes. Based on single seed analysis, lines from two CGN accessions and one INRA accession were obtained with significantly reduced levels of Glia-a9 and Glia-a20 epitopes. These lines will be further tested for industrial quality and may contribute to the development of safer foods for celiac patients.