Characterization of alpha-gliadin genes from diploid wheats and the comparative analysis with those from polyploid wheats

To carry out the comparative analysis of alpha-gliadin genes on A genomes of diploid and polyploid wheats, 8 full-length alpha-gliadin genes, including 3 functional genes and 5 pseudogenes, were obtained from diploid wheats, among which 2, 2 and 4 alpha-gliadin genes were isolated from T. urartu, T. monococcum and T. boeoticum, respectively. The results indicated that higher number of alpha-gliadin pseudogenes have been present in diploid wheats before the formation of polyploid wheats. Amino acid sequence comparative analysis among 26 alpha-gliadin genes, including 16 functional genes and 10 pseudogenes, from diploid and polyploid wheats was conducted. The results indicated that all alpha-gliadins contained four coeliac toxic peptide sequences (i.e., PSQQ, QQQP, QQPY and QPYP). The polyglutamine domains are highly variable, and the second polyglutamine stretch is usually disrupted by the lysine or arginine residue at the fourth position. The unique domain I is the most conserved domain. There are 4 and 2 conserved cysteine residues in the unique domains I and II, respectively. Comparative analysis indicated that the functional alpha-gliadin genes from A genome are highly conserved, whereas the identity of pseudogenes in diploid wheats are higher than those in hexaploid wheats. Phylogenetic analysis indicated that all the analyzed functional alpha-gliadin genes could be clustered into two major groups, among which one group could be further divided into 5 subgroups. The origin of alpha-gliadin pseudogene and functional genes were also discussed.     

Identification of a peptide from alpha-gliadin resistant to digestive enzymes: implications for celiac disease

Current knowledge indicates that both innate and adaptive immune responses are involved in Celiac disease (CD) driven by different gliadin peptides. By studying a representative recombinant alpha-gliadin form, a further 25-mer peptide resistant to gastric, pancreatic, and human intestinal brush-border membrane enzymes was detected. This peptide latter encompasses the sequence 31-43 known to elicit the innate immune response in CD. The resistance of 25-mer, as well as that of the already described 33-mer related to the CD adaptive immune response, was confirmed on a standard flour wheat sample representative of the most widespread European varieties.     

Proteolytic activity and reduction of gliadin-like fractions by sourdough lactobacilli

AIMS: To characterize the peptide hydrolase system of Lactobacillus plantarum CRL 759 and CRL 778 and evaluate their proteolytic activity in reducing gliadin-like fractions. METHODS AND RESULTS: The intracellular peptide hydrolase system of Lact. plantarum CRL 759 and CRL 778 involves amino-, di- (DP), tri- (TP) and endopeptidase activities. These peptidases are metalloenzymes inhibited by EDTA and 1,10-phenanthroline and stimulated by Co2+. DP and TP activities of Lact. plantarum CRL 759 and CRL 778, respectively, were completely inhibited by Cu2+. Lactobacillus plantarum CRL 778 showed the highest proteolytic activity and amino acids release in fermented dough. The synthetic 31-43 alpha-gliadin fragment was hydrolysed to 36% and 73% by Lact. plantarum CRL 778 and CRL 759 respectively. CONCLUSIONS: Lactobacillus plantarum CRL 759 and CRL 778 have an active proteolytic system, which is responsible for the high amino acid release during sourdough fermentation and the hydrolysis of the 31-43 alpha-gliadin-like fragment. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new information of use when obtaining sourdough starters for bread making. Moreover, knowledge regarding lactobacilli capable of reducing the level of gliadin-like fractions, a toxic peptide for coeliac patients, has a beneficial health impact.     

Interaction of alpha-gliadin with poly(HEMA-co-SS): structural characterization and biological implication

The wheat gluten protein alpha-gliadin, a well known trigger of celiac disease, can be complexed by random copolymers of hydroxyethyl methacrylate (HEMA) and sodium 4-styrene sulfonate (SS). In this work, influence of alpha-gliadin and poly(HEMA-co-SS) concentrations on alpha-gliadin structure was studied using spectroscopic techniques and dynamic light scattering. In 70% ethanol or 0.06M HCl (pH 1.2), alpha-gliadin was found to self-associate upon increasing its concentrations and displayed decreased alpha-helical content and increased beta-turn and beta-sheet contents. At pH 1.2, alpha-gliadin interacted with poly(HEMA-co-SS) to form supra-molecular complex particles. Poly(HEMA-co-SS) induced alpha-gliadin structural changes that mimicked those obtained by varying the protein concentration in pure solution. At pH 6.8, alpha-gliadin was poorly soluble and formed large particles but alpha-helix is still main secondary structure. The influence of the polymer on protein structure was weaker at neutral than acidic pH. Interaction with poly(HEMA-co-SS) disrupted alpha-gliadin conformation and self-association to form new complex particles at neutral pH. This study provides insight into the mechanism of poly(HEMA-co-SS)/alpha-gliadin interaction and the polymer as alpha-gliadin sequestering agents in the supportive treatment of celiac disease.     

Epitopes of peptide 43-88 of guinea pig myelin basic protein: localization with monoclonal antibodies

Two monoclonal antibodies, one raised by immunization with mouse myelin basic protein (MBP) and the second raised by immunization with peptide 68-88 of guinea pig MBP, were compared with respect to specificity. The former antibody (15.32) cross-reacted completely with rat, guinea pig, human, and bovine MBP. It also reacted with peptide 43-88 from each MBP. The latter antibody (22.17) was nonreactive with MBP, but cross-reacted with peptide 43-88 from rat, human, guinea pig, and bovine MBP. When tested with small peptides derived from peptide 43-88, antibody 22.17 reacted with an epitope in the C-terminal region. Antibody 15.32 reacted with an epitope in the N-terminal half of the peptide. The data show that 22.17 reacted with a unique epitope associated only with free peptide, whereas 15.32 recognized an epitope common to both peptide 43-88 and MBP.     

Anti-chymotrypsin and anti-elastase activities of a synthetic bicyclic fragment containing a chymotrypsin-reactive site of soybean Bowman-Birk inhibitor

A bicyclic hexadecapeptide, which corresponds to the sequence 36-51 and contains the chymotrypsin-reactive Leu-43-Ser-44 bond of soybean Bowman-Birk inhibitor, has been synthesized. This peptide consists of two loops formed by disulfide bridges between Cys-36 and Cys-51 and between Cys-41 and Cys-49. The bicyclic peptide showed a strong anti-chymotryptic activity with a Ki of 7.1.10(-7) M. Comparison of inhibitory activity and digestive stability against chymotrypsin with other hexadecapeptides having the same sequence but lacking one or both disulfide bridges suggested that the compact bicyclic structure increases the activity and protects the Leu-Ser bond from chymotryptic digestion. Interestingly, the bicyclic peptide was found to inhibit porcine pancreatic elastase with a Ki of 4.3.10(-5) M, indicating the broad specificity of this ring system.     

The N-Terminal Sequence of Tyrosine Hydroxylase Is a Conformationally Versatile Motif That Binds 14-3-3 Proteins and Membranes

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the synthesis of catecholamine neurotransmitters, and a reduction in TH activity is associated with several neurological diseases. Human TH is regulated, among other mechanisms, by Ser19-phosphorylation-dependent interaction with 14-3-3 proteins. The N-terminal sequence (residues 1–43), which corresponds to an extension to the TH regulatory domain, also interacts with negatively charged membranes. By using X-ray crystallography together with molecular dynamics simulations and structural bioinformatics analysis, we have probed the conformations of the Ser19-phosphorylated N-terminal peptide [THp-(1-43)] bound to 14-3-3γ, free in solution and bound to a phospholipid bilayer, and of the unphosphorylated peptide TH-(1-43) both free and bilayer bound. As seen in the crystal structure of THp-(1-43) complexed with 14-3-3γ, the region surrounding pSer19 adopts an extended conformation in the bound state, whereas THp-(1-43) adopts a bent conformation when free in solution, with higher content of secondary structure and higher number of internal hydrogen bonds. TH-(1-43) in solution presents the highest mobility and least defined structure of all forms studied, and it shows an energetically more favorable interaction with membranes relative to THp-(1-43). Cationic residues, notably Arg15 and Arg16, which are the recognition sites of the kinases phosphorylating at Ser19, are also contributing to the interaction with the membrane. Our results reveal the structural flexibility of this region of TH, in accordance with the functional versatility and conformational adaptation to different partners. Furthermore, this structural information has potential relevance for the development of therapeutics for neurodegenerative disorders, through modulation of TH–partner interactions.     

Comparative studies of the preparation of alpha-gliadin]

alpha-gliadin was prepared from wheat flour by two different methods. The products were compared electrophoretically and by double radial immuno-diffusion. The alpha-gliadin fraction proved to be identical in the immunological test. Only the alpha-gliadin preparation received by ion exchange chromatography is suitable for further purification by multiple gel filtrations.     

Interaction of a 43-kDa receptor-like protein with a 4-kDa hormone-like peptide in soybean

A 43-kDa soybean protein is a receptor-like protein kinase that is capable of interaction with a 4-kDa hormone-like peptide (leginsulin). The 43-kDa protein consists of alpha and beta subunits; the beta subunit has protein kinase activity that is stimulated by the binding of the 4-kDa peptide. The protein kinase activity is believed to be an early step in a signal transduction cascade, triggered by the peptide. Animal insulin also interacts with the 43-kDa protein and stimulates the protein kinase activity, suggesting that the 4-kDa peptide and insulin bind to the 43-kDa protein with similar mechanisms. To determine the mechanism of interaction between the 4-kDa peptide and 43-kDa protein, we investigated the binding region of the 4-kDa peptide on the 43-kDa protein using surface plasmon resonance (SPR) spectroscopy. We found that the N- (amino acids 1-43) and C-terminal (amino acids 228-251) regions of the alpha subunit of the 43-kDa protein are involved in the binding. The interactions of both insulin and the 4-kDa peptide with the 43-kDa protein were compared using SPR spectroscopy, revealing that insulin binds to the C-terminal regions of the alpha subunit of the 43-kDa protein. These results suggest that the C-terminal region is especially important for the biological function. The N-terminal region is thought to play an important role in stabilizing the complex of the 43-kDa protein and the 4-kDa peptide.     

The antigenic reactivity of small fragments derived from human myelin basic protein peptide 43-88

In an effort to develop immunochemical reagents for detecting small peptides originating from myelin basic protein (BP), the antigenic determinants of fragments from human BP peptide 43-88 were examined. Antisera were produced in nine sheep and forty rabbits immunized with BP, BP peptide 43-88, or a region derived from within or containing a portion of BP peptide 43-88. These included custom synthesized peptides 51-67, 67-80, 74-84, 79-88, and 83-95. Reactivities were assessed by double antibody radioimmunoassay (DA-RIA) using radiolabeled BP or BP peptides. For peptides 74-84, 79-88, and 83-95 it was necessary to synthetically add a terminal tyrosine residue for radioiodination. Antisera to peptides 51-67, 67-80, 74-84, 79-88, and 83-95 showed much greater reactivity with the homologous antigen, with variable, but sometimes no, reactivity against BP or BP peptide 43-88. This was particularly evident in displacement DA-RIA. Of the small peptides, antisera to whole BP reacted best with peptide 83-95, whereas antisera to peptide 43-88 reacted best with peptide 79-88. Placement of the synthetically added tyrosine had a marked influence on the reactivity of BP peptide 79-88: antisera to BP peptide 43-88 reacted much better with radioiodinated tyrosinyl peptide 79-88 than with radioiodinated peptide 79-88-tyrosine. These results indicate that within a region of BP encompassing residues 51 through 95 a number of potential antigenic determinants exist. Some of the determinants on the small peptides represent distinctive conformational determinants or are inaccessible in BP peptide 43-88. The ability to detect small BP peptides by immunoassay necessitates that the identity of the peptide be known and that antibody reagents capable of reacting with the peptide(s) be available.     

Lipid bilayer surface association of lung surfactant protein SP-B, amphipathic segment detected by flow immunofluorescence.

Lung surfactant protein, SP-B, and synthetic amphipathic peptides derived from SP-B were studied in model lung surfactant lipid bilayers by immunofluorescent labeling. Liposomes were formed by hydrating a lipid film on the glass viewing port of a temperature controlled flow chamber. Membrane associated peptides were detected by epifluorescence optical microscopy of the binding of anti-peptide polyclonal monospecific antibodies and FITC-conjugated secondary antibodies added to buffer contained in the flow chamber. Liposomes were bound by antibody to residues 1-25 of SP-B if formed from lipid films containing the 1-25 peptide, (SP-B(1-25)), or if SP-B(1-25) was added to already formed liposomes in buffer solution. The distribution of antigen-antibody complex was temperature dependent with aggregation occurring at greater than or equal to 30 degrees C. Surface association was not detected in liposomes formed from lipid films containing the 49-66 peptides (SP-B(49-66)), using an antibody to the 49-66 peptide, or to a synthetic version of the SP-B protein, (SP-B(1-78)), using both antibodies to the 49-66 peptide and the 1-25 peptide. The detection of SP-B(1-78) with antibody to the 49-66 sequence was only possible after reducing SP-B(1-78) with dithiothreitol, suggesting that the COOH-terminus of the full monomer protein is accessible to the bulk aqueous environment unlike the COOH-terminal peptide. The size, number of layers, and fluidity of the liposomes were not altered by protein or peptides, although they were affected by lipid composition and temperature.     

CD69 expression on alpha-gliadin-specific T cells in coeliac disease

Coeliac disease (CD) is a T-cell mediated immunological disease of the small intestine which is triggered in susceptible individuals by ingestion of gluten. The pathogenic mechanism of coeliac disease, and the role that alpha-gliadin specific T cells play in mucosal lesions and their involvement in peripheral blood is not yet explained at all. Previous studies have reported proliferative response to alpha-gliadin measured with the classic assay of 3HTdR incorporation. We analysed the activation antigen CD69 on T cells from CD patients and normal individuals following stimulation with alpha-gliadin and different antigens (tetanus toxoid, peptides unrelated to gliadin and PHA). CD69 coexpression with T cell CD3+ and proliferation marker Ki67 was evaluated with time. CD69 coexpression with T cell CD3+, CD4+ and CD8+ was also evaluated. It was found that peripheral blood mononuclear cells (PBMC) of coeliac patients increased their percentage of CD69 positive T cells when stimulated with alpha-gliadin, in comparison with cells from controls. Significant T cell activation was found only in subjects not treated with the gluten free diet; a positive response was found also in two coeliac patients with selective IgA deficiency, anti-endomisium negative, without circulating IgA anti alpha-gliadin or anti-tissue transglutaminase antibodies. The CD69 expression after stimulation was compared with the standard method of 3HTdR incorporation. Our data show that CD69 expression is useful to asses a specific T cell response to alpha-gliadin in coeliac disease. in a very short time. Moreover, the method allows to investigate T cell response at the lymphocyte subsets level, which represents a useful tool in the diagnosis of coeliac disease.     

Celiac disease association with CD8+ T cell responses: identification of a novel gliadin-derived HLA-A2-restricted epitope

One of the diagnostic hallmarks of the histological lesions associated with celiac disease is the extensive infiltration of the small intestinal epithelium by CD8(+) T cells of unknown Ag specificity. In this study, we report recognition of the gliadin-derived peptide (A-gliadin 123-132) by CD8(+) T lymphocytes from celiac patients. A-gliadin 123-132-specific IFN-gamma production and cytotoxic activity were detected in PBMCs derived from patients on gluten-free diet, but not from either celiac patients on gluten-containing diet or healthy controls. In contrast, A-gliadin 123-132-specific cells were isolated from small intestine biopsies of patients on either gluten-free or gluten-containing diets. Short-term T cell lines derived from the small intestinal mucosa and specific for the 123-132 epitope recognized human APC pulsed with either whole recombinant alpha-gliadin or a partial pepsin-trypsin gliadin digest. Finally, we speculate on a possible mechanism leading to processing and presentation of class I-restricted gliadin-derived epitopes in celiac disease patients.     

Platelet fragmentation requires a specific structural conformation of human monoclonal antibody against beta3 integrin

We have described an autoantibody against beta3 (GPIIIa49-66), a region of platelet integrin alphaIIbbeta3 that is unique. It induces platelet fragmentation in the absence of complement via antibody activation of platelet NADPH oxidase and 12-lipoxygenase to release reactive oxygen species, which destroy platelets. To study the mechanism of anti-GPIIIa antibody-induced platelet fragmentation, we screened a human single chain Fv antibody library with the GPIIIa49-66 peptide. Nine monoclonal antibodies were identified that were capable of binding to GPIIIa49-66. Surprisingly, binding avidity for GPIIIa49-66 did not correlate with activity of induction of platelet fragmentation. We therefore investigated the requirements for platelet fragmentation. Mutations were introduced into the heavy chain complementary-determining region-3 of clones 11, 43, and 54 by site-directed mutagenesis. The capability of these clones to induce platelet fragmentation or bind to GPIIIa49-66 subsequently changed. Molecular modeling of these clones with their mutants revealed that the ability to induce platelet fragmentation is affected by the side chain orientation of positively charged amino acids in the heavy chain of residues 99-102. Thus, a structural change in the conformation of anti-GPIIIa49-66 antibody contributes to its binding to the beta3 integrin and subsequent antibody-induced platelet fragmentation and aggregate dissolution.     

Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain

Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca(2+)-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca(2+) influx reduced Cx43 gap junction conductance (G(j)) by 95%, while increasing cytosolic Ca(2+) concentration threefold. By contrast, Cx40 G(j) declined by <20%. The Ca(2+)-induced decline in Cx43 G(j) was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca(2+)-free extracellular solution, if Ca(2+) chelation was commenced before complete uncoupling, after which g(j) was only 60% recoverable. The Cx43 CL(136-158) mimetic peptide, but not the scrambled control peptide, or Ca(2+)/CaM-dependent kinase II 290-309 inhibitory peptide also prevented the Ca(2+)/CaM-dependent decline of Cx43 G(j). Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca(2+)/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca(2+)/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca(2+) regulatory properties of Cx43 and Cx40.