The kiwi fruit peptide kissper displays anti-inflammatory and anti-oxidant effects in in-vitro and ex-vivo human intestinal models.

Literature reports describe kiwi fruit as a food with significant effects on human health, including anti-oxidant and anti-inflammatory activity. Fresh fruit or raw kiwi fruit extracts have been used so far to investigate these effects, but the molecule(s) responsible for these health-promoting activities have not yet been identified. Kissper is a kiwi fruit peptide displaying pore-forming activity in synthetic lipid bilayers, the composition of which is similar to that found in intestinal cells. The objective of this study was to investigate the kissper influence on intestinal inflammation using cultured cells and ex-vivo tissues from healthy subjects and Crohn's disease (CD) patients. The anti-oxidant and anti-inflammatory properties of kissper were tested on Caco-2 cells and on the colonic mucosa from 23 patients with CD, by challenging with the lipopolysaccharide from Escherichia coli (EC-LPS) and monitoring the appropriate markers by Western blot and immunofluorescence. EC-LPS challenge determined an increase in the intracellular concentration of calcium and reactive oxygen species (ROS). The peptide kissper was highly effective in preventing the increase of LPS-induced ROS levels in both the Caco-2 cells and CD colonic mucosa. Moreover, it controls the calcium increase, p65-nuclear factor (NF)-kB induction and transglutaminase 2 (TG2) activation inflammatory response in Caco-2 cells and CD colonic mucosa. Kissper efficiently counteracts the oxidative stress and inflammatory response in valuable model systems consisting of intestinal cells and CD colonic mucosa. This study reports the first evidence supporting a possible correlation between some beneficial effects of kiwi fruit and a specific protein molecule rather than generic nutrients.

Peamaclein--a new peach allergenic protein: similarities, differences and misleading features compared to Pru p 3.

BACKGROUND: Among the peach-derived allergens which are already known, the lipid transfer protein (Pru p 3) seems to be the one to exert severe allergic reactions. OBJECTIVE: To identify and characterize a new peach allergen causing a clinical picture similar to that of Pru p 3. METHODS: Patients were selected on the basis of their severe clinical reactivity and negative results to a panel of peach allergens available on the ISAC103 microarray. Several in-house and commercial preparations were compared. Several methods were used to characterize the newly identified molecule. Specific IgE and inhibition assays were performed using the Allergen micro-Beads Array (ABA) assay. RESULTS: Negative ISAC results to Pru p 3 were confirmed by additional testing in contrast with the positive results obtained by commercial Pru p 3-enriched peach peel extracts. The analyses of one of these preparations led to the identification of Peamaclein, a new allergenic protein. It is a small, basic, cysteine-rich, heat-stable, digestion-resistant protein, homologous to a potato antimicrobial peptide. Peamaclein was able to trigger positive skin test reactions and to bind IgE in the ABA assay. It displays an electrophoretic mobility and chromatographic behaviour similar to that of Pru p 3; therefore, it can be hidden in Pru p 3 preparations. In fact, Pru p 3-enriched peach peel extracts were found to contain both Pru p 3 and Peamaclein by means of comparative in vivo testing, and by biochemical and immunochemical assays. Commercially available anti-Pru p 3 polyclonal antibodies were found to have a double specificity for the two molecules. CONCLUSIONS AND CLINICAL RELEVANCE: A new allergen from peach belonging to a new family of allergenic proteins has been identified and characterized. This knowledge on Peamaclein will improve our understanding on the clinical aspects of the peach allergy and the quality of diagnostic reagents.

Pru p 3, the nonspecific lipid transfer protein from peach, dominates the immune response to its homolog in hazelnut.

BACKGROUND: Nonspecific lipid transfer proteins (nsLTPs) are important food allergens. Often, patients allergic to the nsLTP in peach suffer from allergy to hazelnuts. We aimed to analyse the T-cell response to Cor a 8, the nsLTP in hazelnut and its immunological cross-reactivity with the nsLTP in peach, Pru p 3. METHODS: Cor a 8-reactive T-cell lines (TCL) established from patients allergic to hazelnut and peach were stimulated with 12-mer peptides representing the complete amino acid sequence of Cor a 8 to identify its T-cell-activating regions and with Pru p 3 to investigate cellular cross-reactivity. T-cell clones specific for different major T-cell-activating regions of Pru p 3 were stimulated with Cor a 8. Both nsLTPs were subjected to endolysosomal degradation assays. Immunoglobulin E (IgE) cross-reactivity between Cor a 8 and Pru p 3 was assessed in inhibition enzyme-linked immunosorbent assay. RESULTS: No major T-cell-activating region was found among 26 T-cell-reactive peptides identified in Cor a 8. Although generated with Cor a 8, 62% of the TCL responded more strongly to Pru p 3. This cross-reactivity was mediated by T cells specific for the immunodominant region Pru p 3(61-75) . Peptide clusters encompassing this region were generated during lysosomal degradation of both nsLTPs. Cor a 8 was more rapidly degraded by lysosomal proteases than Pru p 3. Pre-incubation of sera with Pru p 3 completely abolished IgE binding to Cor a 8, which was not the case vice versa. CONCLUSIONS: T-cell reactivity to Cor a 8 is predominantly based on cross-reactivity with Pru p 3, indicating that the latter initiates sensitisation to its homolog in hazelnut. The limited allergenic potential of Cor a 8 seems to be associated with rapid lysosomal degradation during allergen processing and the lack of major T-cell-activating regions.

Physico-chemical features of the environment affect the protein conformation and the immunoglobulin E reactivity of kiwellin (Act d 5).

BACKGROUND: Allergy diagnostic systems sometimes give false positive or negative results. In this respect, the influence of protein conformational changes on the allergen-IgE interaction sites is worthy to be investigated. OBJECTIVE: To investigate the influence of different experimental conditions on the structural properties and IgE reactivity of kiwellin (Act d 5) as a model system. METHODS: Act d 5 was purified from the natural source. To study its conformational features, experiments of circular dichroism (CD) in different media were performed. The IgE reactivity was investigated by skin testing, immunoblotting and ISAC microarray system, in a population of kiwifruit allergic subjects. RESULTS: CD experiments indicated that Act d 5 has a mainly helical structure and the conformation is strongly affected by the experimental conditions. The protein is more structured in low polarity media and at acidic pH values, similar to those of the natural source. Eleven subjects of 29 (38%) allergic to kiwifruit were positive to purified natural Act d 5 by skin test. Among them, three patients (10%) showed a reaction only to Act d 5 at pH 4.5, and three (10%) showed a reaction only to the allergen in standard neutral conditions. No one of the 11 subjects with positive skin test recognized Act d 5 immobilized on the ISAC system. Eight of nine subjects detected Act d 5 by IgE immunoblotting. One subject did not recognize the sequence epitopes of Act d 5 in IgE immunoblotting experiments and reacted to the skin test only when the allergen was in acidic conditions. CONCLUSIONS AND CLINICAL RELEVANCE: The conformation and IgE reactivity of Act d 5 are affected by the physico-chemical characteristics of the solvent. These findings suggest that the assay conditions influence the results of the diagnostic systems by modulating the pattern of exposed antigenic epitopes.

Biochemical, immunological and clinical characterization of a cross-reactive nonspecific lipid transfer protein 1 from mulberry.

BACKGROUND: Mulberry (Morus spp.) is a genus comprising several species of deciduous trees whose fruits are commonly eaten in southern Europe. Subjects with severe systemic reaction have been described. The aim of this study was to isolate the allergens of this species. METHODS: A nonspecific lipid transfer protein 1 (ns-LTP1) was purified from black mulberry by ion exchange and reverse phase high-performance liquid chromatography, and the primary structure was elucidated by direct protein sequencing. Its allergenic activity was evaluated in vivo by skin prick test and in vitro by Western Blot, CD203c basophil activation assay and high throughput multiplex inhibition method on immunosolid-phase allergen chip (ISAC). RESULTS: Mulberry ns-LTP (Mor n 3) comprises 91 amino acids producing a molecular mass of 9246 Da. This protein shows high sequence identity with several allergenic ns-LTP1. Immunoblot analysis and CD203c activation assay demonstrated its allergenic activity in symptomatic subjects and in ns-LTP allergic patients who are not mulberry consumers. Immunological co-recognition was studied in vivo on a selected group of well-characterized ns-LTP allergic patients showing a high percentage of nMor n 3(+) subjects (88.46%) even in patients who have never eaten mulberry before. IgE inhibition on ISAC micro-array demonstrated an almost complete cross-reactivity to nArt v 3, rCor a 8 and a very high percentage of inhibition to nPru p 3. CONCLUSIONS: Mor n 3 is the first allergen isolated in black mulberry and immunologically characterized. It displayed allergenic activity among symptomatic and nonconsumer patients and a pattern of cross-reactivity to other plant-derived LTPs.

Diagnosing allergic sensitizations in the third millennium: why clinicians should know allergen molecule structures.

Diagnostic tests to detect allergic sensitization were introduced at the end of the nineteenth century but only in the late 1990s did the advent of molecular allergology revolutionize the approach to the allergic patient. Personalized Medicine, a medical procedure that separates patients into different groups with different medical decisions, practices and interventions has sanctioned this change. In fact, in the last few years molecular allergology and the observation that not every patient has the same allergic profile, even when allergic to the same allergenic source, has originated the concept "one size does not fit all". This new approach requires the identification of still unknown allergens, but also the more detailed investigation of those already known. In depth studies of the structure-function relationships in allergenic molecules can reveal the structural determinants involved in the IgE-binding. Then, the knowledge of the epitope profile of each allergen and of the environmental/experimental conditions affecting the exposure of IgE-binding epitopes can provide important contributions to the understanding of cross-reaction processes and to the improvement of diagnosis, immunotherapy and the overall patient treatment. The evolution of diagnostic systems cannot ignore these new needs in this field.

Glucose-6-phosphate dehydrogenase from the blood cells of two antarctic teleosts: correlation with cold adaptation.

Glucose-6-phosphate dehydrogenase (G6PD) has been purified from the blood of two Antarctic teleost species, i.e., from the erythrocytes of Dissostichus mawsoni (family Nototheniidae), and from the plasma and cells of haemoglobinless Chionodraco hamatus (family Channichthyidae). The specific activities in haemolysates of Antarctic blood cells appear higher than that of a lysate of human erythrocytes. The two Antarctic enzymes have an apparent subunit molecular mass slightly higher than that of human G6PD; the electrophoretic behaviour on cellulose acetate is similar. Both Antarctic enzymes are irreversibly heat inactivated through a biphasic process. Km for glucose-6-phosphate (G6P) does not vary significantly with temperature, whereas Km for NADP increases at increasing temperature, kcat increases with temperature, with a break point at 35 degrees C (in human G6PD, the break point is at 15 degrees C). Thermodynamic and kinetic characterisation indicate that the catalytic performance of the enzyme of cold-adapted fish, at temperatures typical of their habitat, is more efficient than that displayed by G6PD from a temperature organism.

L-Glutamate dehydrogenase from the antarctic fish Chaenocephalus aceratus. Primary structure, function and thermodynamic characterisation: relationship with cold adaptation.

In order to study the molecular mechanisms of enzyme cold adaptation, direct amino acid sequence, catalytic features, thermal stability and thermodynamics of the reaction and of heat inactivation of L-glutamate dehydrogenase (GDH) from the liver of the Antarctic fish Chaenocephalus aceratus (suborder Notothenioidei, family Channichthyidae) were investigated. The enzyme shows dual coenzyme specificity, is inhibited by GTP and the forward reaction is activated by ADP and ATP. The complete primary structure of C. aceratus GDH has been established; it is the first amino acid sequence of a fish GDH to be described. In comparison with homologous mesophilic enzymes, the amino acid substitutions suggest a less compact molecular structure with a reduced number of salt bridges. Functional characterisation indicates efficient compensation of Q(10), achieved by increased k(cat) and modulation of S(0.5), which produce a catalytic efficiency at low temperature very similar to that of bovine GDH at its physiological temperature. The structural and functional characteristics are indicative of a high extent of protein flexibility. This property seems to find correspondence in the heat inactivation of Antarctic and bovine enzymes, which are inactivated at very similar temperature, but with different thermodynamics.

Pectin methylesterase inhibitor.

Pectin methylesterase (PME) is the first enzyme acting on pectin, a major component of plant cell wall. PME action produces pectin with different structural and functional properties, having an important role in plant physiology. Regulation of plant PME activity is obtained by the differential expression of several isoforms in different tissues and developmental stages and by subtle modifications of cell wall local pH. Inhibitory activities from various plant sources have also been reported. A proteinaceous inhibitor of PME (PMEI) has been purified from kiwi fruit. The kiwi PMEI is active against plant PMEs, forming a 1:1 non-covalent complex. The polypeptide chain comprises 152 amino acid residues and contains five Cys residues, four of which are connected by disulfide bridges, first to second and third to fourth. The sequence shows significant similarity with the N-terminal pro-peptides of plant PME, and with plant invertase inhibitors. In particular, the four Cys residues involved in disulfide bridges are conserved. On the basis of amino acid sequence similarity and Cys residues conservation, a large protein family including PMEI, invertase inhibitors and related proteins of unknown function has been identified. The presence of at least two sequences in the Arabidopsis genome having high similarity with kiwi PMEI suggests the ubiquitous presence of this inhibitor. PMEI has an interest in food industry as inhibitor of endogenous PME, responsible for phase separation and cloud loss in fruit juice manufacturing. Affinity chromatography on resin-bound PMEI can also be used to concentrate and detect residual PME activity in fruit and vegetable products.

Kiwi protein inhibitor of pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges.

A protein acting as a powerful inhibitor of plant pectin methylesterase was isolated from kiwi (Actinidia chinensis) fruit. The complete amino-acid sequence of the pectin methylesterase inhibitor (PMEI) was determined by direct protein analysis. The sequence comprises 152 amino-acid residues, accounting for a molecular mass of 16 277 Da. The far-UV CD spectrum indicated a predominant alpha-helix conformation in the secondary structure. The protein has five cysteine residues but neither tryptophan nor methionine. Analysis of fragments obtained after digestion of the protein alkylated without previous reduction identified two disulfide bridges connecting Cys9 with Cys18, and Cys74 with Cys114; Cys140 bears a free thiol group. A database search pointed out a similarity between PMEI and plant invertase inhibitors. In particular, the four Cys residues, which in PMEI are involved in the disulfide bridges, are conserved. This allows us to infer that also in the homologous proteins, whose primary structure was deduced only by cDNA sequencing, those cysteine residues are engaged in two disulfide bridges, and constitute a common structural motif. The comparison of the sequence of these inhibitors confirms the existence of a novel class of proteins with moderate but significant sequence conservation, comprising plant proteins acting as inhibitors of sugar metabolism enzymes, and probably involved in various steps of plant development.

Enzymes in antarctic fish: glucose-6-phosphate dehydrogenase and glutamate dehydrogenase.

Glucose-6-phosphate dehydrogenase (G6PD) and L-glutamate dehydrogenase (GDH) from Antarctic fish were isolated and characterized. G6PD was purified from the erythrocytes of red-blooded Dissostichus mawsoni and from the colorless blood of the icefish Chionodraco hamatus. Structural and functional characterization showed that the two enzymes do not differ significantly from each other. GDH was purified from the liver of the icefish Chaenocephalus aceratus. As in other fish ODHs, it showed a marked preference for NAD-. The amino acid sequence of the active-site peptide is virtually identical to that of other fish and vertebrate counterparts. Although the basic structural features of the Antarctic enzymes are similar to those of mesophilic organisms, some catalytic and thermodynamic properties make the Antarctic enzymes more suited to cold-adapted organisms.

Primary structure of human erythrocyte nicotinamide adenine dinucleotide phosphate (NADP[H])-binding protein FX: identification with the mouse tum- transplantation antigen P35B.

Human erythrocytes contain a nicotinamide adenine dinucleotide phosphate (NADP[H])-binding protein, FX, whose levels are significantly increased in erythrocytes from glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals bearing the mediterranean variant of G6PD. Elucidation of the still unknown biologic functions of FX was approached by means of amino acid sequencing of its 25 tryptic peptides. Searching in the EMBL data bank allowed identification of extensive homology between these tryptic peptides and all sequence-aligned regions encompassing the complete structure of a putative protein encoded by the P35B gene in the mouse. This gene, which differs from the normal allele by a point mutation, has been previously cloned from a tum- variant of the murine tumor cell line P815, so defined because it is associated with low tumorigenicity compared with the progenitor P815. The reported P35B cDNA contains an open reading frame (ORF) of 813 bp and encodes a putative protein of 271 amino acids (30 kD), whereas FX protein is 320 amino acids in length (35.81 kD, in good agreement with previous studies). However, a single base shift at position 4,752 of the P35B gene suppresses the stop codon after Phe 271 and allows continuation of the ORF for up to 320 amino acids to reach the same length as FX. The remarkably high extent (92%) of homology indicates that erythrocyte FX protein is the human homolog of the P35B gene product.

Selective deamidation of ribonuclease A. Isolation and characterization of the resulting isoaspartyl and aspartyl derivatives.

Selective deamidation of proteins and peptides is a reaction of great interest, whether it has physiological significance as in protein aging, or occurs as a disturbing event in the preparation of natural or recombinant proteins. Deamidation of bovine pancreatic ribonuclease A, RNase A, a classical model protein, has been reported to occur only after denaturation of the protein, or under harsh conditions. In this paper convenient procedures are described for selective deamidation of Asn67 in native RNase A under mild conditions. Furthermore, for the first time, both products of deamidation were isolated: the aspartyl and the isoaspartyl containing protein derivatives. Replacement of Asn67 with either residue lowers the catalytic activity of the enzyme, on RNA and on model substrates, except when a dinucleotide with a purine on the 5' side is the substrate. In the latter case an intriguing increase in the specificity constant is observed. The Asp67 derivative was found to refold, after full denaturation and reduction, at the same rate as the fully amidated protein, whereas the iso-Asp67 derivative refolded at half that rate. It is hypothesized that this effect is due to a delayed formation of disulfide 65-72 for the presence of the abnormal isopeptide bond between residues 67 and 68.

NADP+-dependent glutamate dehydrogenase in the Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1. Characterization, protein and DNA sequence, and relationship to other glutamate dehydrogenases.

The Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1 contains two distinct glutamate dehydrogenases (GDH), each specific for either NADP+ or NAD+. This feature is quite unusual in bacteria, which generally have a single GDH. NADP+-dependent GDH has been purified to homogeneity and the gene encoding GDH has been cloned and expressed. The enzyme has a hexameric structure. The amino acid sequence determined by peptide and gene analyses comprises 447 residues, yielding a protein with a molecular mass of 49 285 Da. The sequence shows homology with hexameric GDHs, with identity levels of 52% and 49% with Escherichia coli and Clostridium symbiosum GDH, respectively. The coenzyme-binding fingerprint motif GXGXXG/A (common to all GDHs) has Ser at the last position in this enzyme. The overall hydrophilic character is increased and a five-residue insertion in a loop between two alpha-helices may contribute to the increase in protein flexibility. Psychrobacter sp. TAD1 GDH apparent temperature optimum is shifted towards low temperatures, whereas irreversible heat inactivation occurs at temperatures similar to those of E. coli GDH. The catalytic efficiency in the temperature range 10-30 degrees C is similar or lower than that of E. coli GDH. Unlike E. coli GDH the enzyme exhibits marked positive cooperativity towards 2-oxoglutarate and NADPH. This feature is generally absent in prokaryotic GDHs. These observations suggest a regulatory role for this GDH, the most crucial feature being the structural/functional properties required for fine regulation of activity, rather than the high catalytic efficiency and thermolability encountered in several cold-active enzymes.