Enrique Méndez

Reversion of prion protein conformational changes by synthetic b-sheet breaker peptides

BACKGROUND Transmissible spongiform encephalopathies are associated with a structural transition in the prion protein that results in the conversion of the physiological PrPc to pathological PrP(Sc). We investigated whether this conformational transition can be inhibited and reversed by peptides homologous to the PrP fragments implicated in the abnormal folding, which contain specific residues acting as beta-sheet blockers (beta-sheet breaker peptides). METHODS We studied the effect of a 13-residue beta-sheet breaker peptide (iPrP13) on the reversion of the abnormal structure and properties of PrP(Sc) purified from the brains of mice with experimental scrapie and from human beings affected by sporadic and variant Creutzfeldt-Jakob disease. In a cellular model of familial prion disease, we studied the effect of the peptide in the production of the abnormal form of PrP in intact cells. The influence of the peptide on prion infectivity was studied in vivo by incubation time assays in mice with experimental scrapie. FINDINGS The beta-sheet breaker peptide partly reversed in-vitro PrP(Sc) to a biochemical and structural state similar to that of PrPc. The effect of the peptide was also detected in intact cells. Treatment of prion infectious material with iPrP13 delayed the appearance of clinical symptoms and decreased infectivity by 90-95% in mice with experimental scrapie. INTERPRETATION Beta-sheet breaker peptides reverse PrP conformational changes implicated in the pathogenesis of spongiform encephalopathies. These peptides or their derivatives provide a useful tool to study the role of PrP conformation and might represent a novel therapeutic approach for prion-related disorders.

Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol.

Objectives There is currently much call for a reliable enzyme‐linked immunosorbent assay (ELISA) protocol for determining gluten in foods to serve as a basis for further Codex Alimentarius regulations. Given its ability to recognize the potential coeliac‐toxic epitope QQPFP, which occurs repeatedly in &agr;‐, &ggr;‐ and &ohgr;‐gliadins, hordeins and secalins, the monoclonal antibody R5 raised against a secalin extract may prove to be an essential tool for gluten analysis. This study was designed to develop a highly sensitive and specific sandwich ELISA to quantify low levels of wheat, barley and rye prolamins in foods for coeliacs. Methods Simple sandwich ELISA based on the use of a single monoclonal antibody (R5) as both the coating and detection was developed. A quantitative cocktail glutenextraction procedure for heat‐processed foods was also tested. Results R5‐ELISA was able to identify gliadins, hordeins and secalins with assay sensitivities of 0.78, 0.39 and 0.39 ng/ml, respectively. The assays detection limit was 1.5 ng gliadins/ml (1.56 ppm gliadins, 3.2 ppm gluten). The system proved insensitive to the non‐coeliac‐toxic cereals maize, rice and oats, and was non‐cultivar‐dependent. It was also able to detect gliadins and hordeins in unprocessed and heat‐processed wheat‐ and barleybased products, and to estimate the gluten content of hydrolysed foods. Conclusion We present a new generation of a robust sandwich R5‐ELISA with good reproducibility (8.7%) and repeatability (7.7%). Its gluten‐detection limit of 3.2 ppm is lower than the existing threshold of 20‐200 ppm. The ELISA, which is equally sensitive to barley, wheat and rye prolamins, is compatible with the quantitative cocktail extraction procedure for heat‐processed foods. Along with the cocktail procedure, the Working Group on Prolamin Analysis and Toxicity is currently evaluating an R5‐ELISA system as proposed by the Codex Alimentarius Commission.

γ-Purothionins: amino acid sequence of two polypeptides of a new family of thionins from wheat endosperm

Two homologous sulfur‐rich basic polypeptides form wheat endosperm, so‐called γ1‐purothionin and γ2‐purothionin, are described. Purification involves extraction with volatile solvents and ammonium bicarbonate fractionation followed by reversed‐phase high‐performance liquid chromatography. The complete primary structure of these two polypeptides has been determined by automatic degradation of the intact, S‐carboxymethylated γ‐purothionins and peptides obtained by enzymatic cleavage. γ1‐Purothionin and γ2‐purothionin consist of 47 amino acids with an assessed molecular weight of 5239 and 5151 Da, respectively and 8 cysteines organized in 4 disulfide bridges. They present a high degree of homology among themselves (89% of identity) and are the first two thionin‐like polypeptides, so‐called y‐thionins, described from wheat endosperm.

Solution structure of gamma 1-H and gamma 1-P thionins from barley and wheat endosperm determined by 1H-NMR: a structural motif common to toxic arthropod proteins.

The complete assignment of the proton NMR spectra of the homologous gamma 1-hordothionin and gamma 1-purothionin (47 amino acids, 4 disulfide bridges) from barley and wheat, respectively, has been performed by two-dimensional sequence-specific methods. A total of 299 proton-proton distance constraints for gamma 1-H and 285 for gamma 1-P derived from NOESY spectra have been used to calculate the three-dimensional solution structures. Initial structures have been generated by distance geometry methods and further refined by dynamical simulated annealing calculations. Both proteins show identical secondary and tertiary structure with a well-defined triple-stranded antiparallel beta-sheet (residues 1-6, 31-34, and 39-47), an alpha-helix (residues 16-28), and the corresponding connecting loops. Three disulfide bridges are located in the hydrophobic core holding together the alpha-helix and the beta-sheet and forming a cysteine-stabilized alpha-helical (CSH) motif. Moreover, a clustering of positive charges is observed on the face of the beta-sheet opposite to the helix. The three-dimensional structures of the gamma-thionins differ remarkably from plant alpha- and beta-thionins and crambin. However, they show a higher structural analogy with scorpion toxins and insect defensins which also present the CSH motif.

A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins

Objectives Antibodies that detect coeliac-toxic prolamins from wheat, barley and rye are important tools for controlling the diet of coeliac disease patients. Recently, a monoclonal antibody R5 that recognizes wheat gliadin, barley hordein and rye secalin equally was described. In this study, the epitope recognized by R5 was investigated. Methods Both a phage-displayed heptapeptide library and overlapping peptides spanning the sequence of α- and γ-type gliadins (pepscan) were screened for binding of R5. Results Both techniques yielded comparable pentapeptide consensus sequences (phage display QXPW/FP; pepscan QQPFP). According to recent observations, this peptide stretch may be of key importance in the pathogenicity of coeliac disease. This sequence occurs repetitively in prolamins (in γ- and ω-type prolamins more frequently than in α-type prolamins) together with several homologous peptide stretches, which are recognized less strongly. Conclusions R5 seems to be a good candidate for the specific detection of putative coeliac disease-active sequences in prolamins and thus represents a valuable tool for the quality control of gluten-free food.

Analysis and clinical effects of gluten in coeliac disease.

The prolamin working group coordinates research on laboratory gluten analysis in food and on clinical evaluation of patient sensitivity to prolamins. As an observer organization to the Codex Alimentarius Commission, the group summarizes current data on analysis and effects of gluten in coeliac disease. All types of gliadin, the ethanol-soluble fraction of gluten, contain the coeliac-active factor. However, coeliac toxicity and immunogenicity (humoral and cellular) of various prolamins are not identical in coeliac patients. There are no conclusive data on the threshold of gluten sensitivity of coeliac patients. Information as to the long-term risk to coeliac patients exposed to small doses of gliadin is lacking. Therefore, every effort should be made to keep the diet of coeliac patients as gluten-free as possible. The prolamin group is currently evaluating a new enzyme-linked immunosorbent assay (ELISA) protocol for gluten analysis that could serve as a basis for further Codex regulations. The group recommends adherence to a single Codex limit for gluten-free foods. The current limit of 200 ppm gluten is questionable and requires reconsideration based on new information that will be available soon.

Imidazole‐SDS‐Zn reverse staining of proteins in gels containing or not SDS and microsequence of individual unmodified electroblotted proteins

A reverse staining procedure is described for the detection of proteins in acrylamide and agarose gels with and without SDS. Protein detection occurs a few minutes after electrophoresis. The sensitivity on acrylamide gels is higher than that of Coomassie blue staining either on acrylamide gels or on electrotransferred membranes. Sequencing of protein bands only detected by reverse staining on the gel and not by Coomassie blue is demonstrated.

Development of a general procedure for complete extraction of gliadins for heat processed and unheated foods.

Objectives In the past, one of the major problems in gluten analysis has been the unavailability of an efficient, universal, extraction procedure of gliadins – the alcohol-soluble proteins of gluten – from both heat processed and unprocessed products. This study was designed to develop a universal, extraction procedure capable of extracting the totality of gliadins from both unprocessed and heat processed foods for coeliac patients. Methods A simple quantitative extraction solution containing 250 mM 2-mercaptoethanol and 2 M guanidine hydrochloride (‘cocktail’), was developed to extract gliadins from heated foods. Results The diluted reducing and disaggregating agents reaching the micro plate at low concentration do not affect the ELISA system based on the R5 monoclonal antibody. The recovery of gliadins extracted by the cocktail from spiked samples was nearly complete, with an average mean value of 95.5%, which is clearly superior to 44.4% obtained with conventional 60% aqueous ethanol. The cocktail always yielded either slightly similar or higher values than 60% aqueous ethanol depending on the type of foods: 1.1-fold in unheated foods, 1.4-fold in wheat starches and 3.0-fold in heated foods. False positives or negatives were never observed using the cocktail solution. Conclusion We present a general complete gliadin extraction procedure based on reducing and disaggregating agents for both heated and unheated foods as a crucial tool for gliadin analysis. The new extraction solution is used for corresponding proteins from rye (secalins) and barley (hordeins). The cocktail was employed as the extraction method in the international ring trial evaluation of sandwich R5-ELISA as proposed by the Codex Alimentarius and organized by the Working Group on Prolamin Analysis and Toxicity.

Distribution and properties of major ribosome-inactivating proteins (28 S rRNA N-glycosidases) of the plant Saponaria officinalis L. (Caryophyllaceae).

We have studied the distribution of the protein synthesis inhibitory activity in the tissues of Saponaria officinalis L. (Caryophyllaceae). Seven major saporins, ribosome-inactivating proteins, were purified to apparent homogeneity from leaves, roots and seeds using a new procedure of RIPs isolation including ion-exchange and hydrophobic chromatography. They all catalysed the depurination of rat liver ribosomes, which generate the Endos diagnostic rRNA fragment upon treatment with acid aniline, thus indicating that A4324 from the 28S rRNA has been released (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). The molecular mass of saporins by SDS-PAGE ranged between 30.2 and 31.6 kDa and by gel-filtration between 27.5 and 30.1 kDa. Amino acid composition and amino-terminal amino acid sequence indicate that all saporins may be considered isoforms. Only two saporins present in roots were glycosylated (SO-R1 and SO-R3). All saporins are very active on cell-free translation systems derived from rabbit reticulocyte lysates, rat liver, Triticum aestivum L., Cucumis sativus L. and Vicia sativa L. However, they are poor inhibitors of an Escherichia coli translation system. They inhibit protein synthesis in HeLa, BeWo and NB 100 cells, HeLa cells being the most resistant. The enzymatic activity of at least one saporin isoform was dependent on magnesium concentration in the standard rat liver cell-free system.

Report of a collaborative trial to investigate the performance of the R5 enzyme linked immunoassay to determine gliadin in gluten-free food.

Objective Analytical methods for measurements of gluten in the low level range of 20–200 ppm of gluten, as required for gluten-free food, have never been endorsed by the Codex Alimentarius. With the aim of investigating standardized and reliable methods for the detection of gliadin in food with detection limits lower than 200 ppm, as proposed by the Codex Alimentarius, the Working Group on Prolamin Analysis and Toxicity (WGPAT) coordinated a large collaborative study to validate a monoclonal antibody based ELISA, which uses an antibody (R5) against rye secalins. Methods Twelve food samples in which gliadin was present in the range of 0–168 ppm were analysed with two different commercially available R5 ELISA tests and a special extraction solvent, based on a reducing and a dissociating agent designed to extract heat denatured proteins. Twenty laboratories participated in this study. Results Recovery values ranged from 65 to 110% in general. The repeatability (RSDr) and reproducibility (RSDR) figures ranged between 13 and 25, resp. 23 and 47 for one test, and between 11 and 22, resp. 25 and 33 for the other. Conclusion Both assays are comparable and robust. The repeatability and reproducibility data are in a range that is acceptable for ELISAs. Kits from both suppliers fulfilled performance criteria of regular ELISA methods, and it is shown that both ELISA kits guarantee a sensitivity of 1.5 ppm gliadin for gluten-free food.