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Summary Background Allergy to kiwifruit is increasingly reported across Europe. Currently, the reliability of its diagnosis by the measurement of allergen-specific IgE with extracts or by skin testing with fresh fruits is unsatisfying. Objective To evaluate the usefulness of a component-based allergen microarray for the diagnosis of kiwifruit allergy in a large group of patients. Methods With an allergen microarray, we measured specific IgE and IgG4 levels to a panel of nine kiwifruit allergens in sera of 237 individuals with kiwifruit allergy. Sera from 198 allergic patients without kiwifruit allergy served as controls. Furthermore, we determined the extent of sensitization to latex. Results The panel of kiwifruit allergens showed a diagnostic sensitivity of 66%, a specificity of 56% and a positive predictive value of 73%. Sera from kiwifruit-allergic patients contained significantly more frequently Act d 1-specific IgE than sera from control patients. Furthermore, 51% of the positive sera contained IgE directed to a single allergen, namely Act d 1 (45%), Act d 9 (27%) or Act d 7 (13%). Within the control group, 36% sera recognized a single allergen. Out of those, 48% were positive to the cross-reactive glycoallergen Act d 7, 43% to the profilin Act d 9 and only 5% to Act d 1. Allergen-specific IgG4 levels did not differ between kiwifruit-allergic and -tolerant patients. Kiwifruit- and latex-allergic patients contained Hev b 11-specific IgE significantly more frequently than latex-allergic patients without kiwifruit allergy. Conclusions Act d 1 can be considered a marker allergen for genuine sensitization to kiwifruit. We demonstrated that a component-based kiwifruit allergen microarray would improve the prognostic value of in vitro diagnostic tests. Cite this as: M. Bublin, S. Dennstedt, M. Buchegger, M. Antonietta Ciardiello, M. L. Bernardi, L. Tuppo, C. Harwanegg, C. Hafner, C. Ebner, B. K. Ballmer-Weber, A. Knulst, K. Hoffmann-Sommergruber, C. Radauer, A. Mari and H. Breiteneder, Clinical & Experimental Allergy, 2011 (41) 129–136.

Kiwellin is a novel protein of 28 kDa isolated from kiwi (Actinidia chinensis) fruit. It is one of the three most abundant proteins present in the edible part of this fruit. Kiwellin has been purified by ion exchange chromatography. Its N-terminal amino acid sequence revealed high identity with that previously reported for a 28 kDa protein described as one of the most important kiwi allergens. This observation prompted us to fully characterize this protein. The complete primary structure, elucidated by direct sequencing, indicated that kiwellin is a cysteine-rich protein. Serological tests and Western Blotting analysis showed that kiwellin is specifically recognized by IgE of patients allergic to kiwi fruit.

Psychrobacter sp. TAD1 is a psychrotolerant bacterium from Antarctic frozen continental water that grows from 2 to 25 degrees C with optimal growth rate at 20 degrees C. The new isolate contains two glutamate dehydrogenases (GDH), differing in their cofactor specificities, subunit sizes and arrangements, and thermal properties. NADP+-dependent GDH is a hexamer of 47 kDa subunits and it is comparable to other hexameric GDHs of family-I from bacteria and lower eukaria. The NAD+-dependent enzyme, described in this communication, has a subunit weight of 160 kDa and belongs to the novel class of GDHs with large size subunits. The enzyme is a dimer; this oligomeric arrangement has not been reported previously for GDH. Both enzymes have an apparent optimum temperature for activity of approximately 20 degrees C, but their cold activities and thermal labilities are different. The NAD+-dependent enzyme is more cold active: at 10 C it retains 50% of its maximal activity, compared with 10% for the NADP+-dependent enzyme. The NADP+-dependent enzyme is more heat stable, losing only 10% activity after heating for 30 min, compared with 95% for the NAD+-dependent enzyme. It is concluded that in Psychrobacter sp. TAD1 not only does NAD+-dependent GDH have a novel subunit molecular weight and arrangement, but that its polypeptide chains are folded differently from those of NADP+-dependent GDH, providing different cold-active properties to the two enzymes.

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.

Glutamate dehydrogenase (GDH) was purified from the liver of the teleost Chaenocephalus aceratus (Notothenioidei: Channichthyidae) and the microorganism Psychrobacter sp. TAD1, from Antarctic marine and terrestrial environments, respectively. GDH isolated from C. aceratus liver had a hexameric molecular structure very similar to that of other vertebrates and displayed preference for NAD+, a feature shared with other fish enzymes. The bovine and fish GDH activity and stability were differently affected by temperature and hydrostatic pressure. At low temperatures, the specific activity of fish GDH was higher than that measured with the homologous bovine enzyme. Psychrobacter sp. TAD1 showed a feature quite unusual in bacteria, i.e. the presence of two distinct GDHs specific either for NADP+ or for NAD+. NADP+ -dependent GDH was purified and characterised. It has a hexameric structure with a subunit molecular weight similar to that described for this class of GDHs and a specific activity at low and moderate temperatures similar to that measured with the homologous enzyme from Escherichia coli. The kinetic properties of NADP+ -dependent GDH of Psychrobacter sp. TAD1 and the presence of another NAD+-dependent GDH suggest that, during the cold-adaptation process, this enzymatic function acquired a pattern of changes different from that of C. aceratus.

Gill cytoplasmic carbonic anhydrase of the haemoglobinless Antarctic icefish Chionodraco hamatus (Ice-CA) was directly sequenced and consists in 259 residues with an acetylated N-terminus. The molecular mass, deduced from the sequence, was 28.45 kDa, while mass spectrometry analysis of the native protein gave higher values. Treatment with dithiothreitol abolished this difference, indicating possible post-translational modifications. Isoelectric focusing analysis of Ice-CA suggested S-thiolation, which was identified as S-glutathionylation by immunostaining. Deglutathionylated Ice-CA maintained the anhydrase activity but showed higher susceptibility to hydrogen peroxide, suggesting that glutathione binding to Cys residues may have a role in the defence against oxidative damage. Ice-CA is characterized by lower thermal stability, higher activity and lower activation energy than its homologue gill CA of the temperate European eel, confirming the adaptation of the catalytic capacity to low temperatures. Alignment of Ice-CA with homologous enzymes from other fish shows high identity; the enzyme is grouped with a previously described fish CA monophyletic clade although Ice-CA shows several characteristics that can increase protein-solvent interaction and structural flexibility.

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.

In 2012, we received 683 submissions and published 20 editorials, 38 reviews, 11 letters and 128 original articles. This represents an acceptance rate for original papers in the range of 20%. About 30% of original papers were triaged not to go out to review, either because the editors did not feel they had sufficient priority for publication or because the topic did not feel right for the readers of the journal. We place great emphasis on obtaining sufficient high-quality reviews to make our decisions on publication fair and consistent. Inevitably, however, there is a degree of luck about what gets published and which papers miss out, and we are always happy to receive an appeal on our decisions either at the triage stage or after review. This gives us the opportunity to revisit the decision and revise it or explain in more detail to the authors the basis for the decision. Once again in 2012, we were delighted by the quality of the papers submitted and the breadth and depth of research into allergic disease that it revealed. The pattern of papers submitted was similar in previous years with considerable emphasis on all aspects of asthma and rhinitis. We were particularly pleased with our special issue on severe asthma. Elucidating mechanisms using either animal models or patients has always been a major theme of the journal, and the excellent work in these areas has been summarized by Harissios Vliagoftis with a particularly interesting section on early-life events guiding the development of allergic disease, which understandably continue to be a major theme of research. Magnus Wickman summarized the papers looking at the epidemiology of allergic disease including work from birth cohorts, which are an increasingly rich source of data on risk factors for allergic disease, and two papers on the epidemiology of anaphylaxis. Giovanni Passalacqua discussed the papers in the clinical allergy section of the journal, and Adriano Mari who runs the excellent Allergome website discussed the papers looking at allergens including characterization and the relative usefulness of allergen arrays versus single extracts in diagnosis and management.

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.

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

Kiwellin, an allergenic protein formerly isolated from green kiwi fruit, has been identified as the most abundant component of the gold kiwi species. A protein named KiTH, showing a 20 kDa band on reducing SDS-PAGE and 100% identity with the C-terminal region of kiwellin, has been identified in the extract of the ripe green species. In vitro treatment of purified kiwellin with the protease actinidin from green kiwi fruit originated KiTH and kissper, a recently described pore-forming peptide. Primary structure analysis and experimental evidence suggest that kiwellin is a modular protein with two domains. It may undergo in vivo proteolytic processing by actinidin, thus producing KiTH and kissper. When probed with sera recognizing kiwellin from green kiwi fruit, KiTH showed IgE binding, with reactivity levels sometimes different from those of kiwellin. The IgE-binding capacity of kiwellin from gold kiwi fruit appears to be similar to that of the green species.

Pectin methylesterase was purified from kiwi (Actinidia chinensis) and kaki fruit ( Diospyros kaki). The pH values of the fruit homogenates were 3.5 and 6.2, respectively. The kiwi enzyme is localized in the cell wall and has a neutral-alkaline pI, whereas the kaki enzyme is localized in the soluble fraction and has a neutral-acidic pI. The molecular weights of the kiwi and kaki enzymes were 50 and 37 kDa, respectively. The two enzymes showed a similar salt and pH dependence of activity, and a different pH dependence of the inhibition by the kiwi proteinaceous inhibitor.

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.

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.

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.