Patrick Gruber

Publications of Patrick Gruber

• Influence of the maillard reaction on the allergenicity of rAra h 2, a recombinant major allergen from peanut (Arachis hypogaea), its major epitopes, and peanut agglutinin.

  Authors: Patrick Gruber, Wolf-Meinhard Becker, Thomas Hofmann

  Journal of agricultural and food chemistry. 04/2005; 53(6):2289-96.

  The influence of thermal processing and nonenzymatic browning reactions on the IgE-binding activity of rAra h 2 was studied and compared to findings recently reported for the allergen's naturalThe influence of thermal processing and nonenzymatic browning reactions on the IgE-binding activity of rAra h 2 was studied and compared to findings recently reported for the allergen's natural counterpart. ELISA experiments as well as inhibition assays revealed that thermal treatment of rAra h 2 in the presence of reactive carbohydrates and carbohydrate breakdown products induces a strong increase of the IgE-binding activity, thus collaborating with the data reported for the natural protein isolated from peanuts. To localize the Ara h 2 sequences responsible for the formation of highly IgE-affine glycation sites, model peptides have been synthesized mimicking sequences which contain possible targets for glycation as well as the immunodominant epitopes. Immunological evaluation of these peptides heated in the absence or presence of reducing sugars and carbonyls, respectively, revealed that neither the two lysine residues of Ara h 2 nor its N-terminus are involved in the formation of IgE-affine structures by Maillard reaction. Also, the cysteine-containing major epitope 3 (aa 27-36) was found to lose its IgE-binding capacity upon heating. By contrast, the overlapping major epitopes 6 and 7, which do not contain any lysine or arginine moieties, showed a distinct higher level of IgE binding when subjected to Maillard reaction, thus giving the first evidence that nonbasic amino acids might be accessible for nonenzymatic glycation reactions and that these posttranslational modifications might induce increased IgE binding of the glycated Ara h 2. Analogous experiments were performed with peanut agglutinin, considered in the literature as a minor allergen. ELISA experiments revealed that the majority of tested sera samples from peanut-sensitive patients showed a high level of IgE binding to the lectin even after heat treatment. In contradiction to published data, nonenzymatic browning reactions seem to deteriorate the IgE affinity of the lectin.

• Development of an epitope-specific analytical tool for the major peanut allergen Ara h 2 using a high-density multiple-antigenic peptide strategy.

  Authors: Patrick Gruber, Martin Suhr, Andreas Frey, Wolf-Meinhard Becker, Thomas Hofmann

  Molecular nutrition & food research. 12/2004; 48(6):449-58.

  Using the major peanut allergen Ara h 2 as an example, an analytical tool enabling the determination of immunoglobulin E (IgE)-epitopes in processed food allergens was developed. We synthesized aUsing the major peanut allergen Ara h 2 as an example, an analytical tool enabling the determination of immunoglobulin E (IgE)-epitopes in processed food allergens was developed. We synthesized a multiple-antigenic peptide (MAP) of the IgE-reactive linear epitope 3 (amino acid positions 27-36) of Ara h 2 and raised a monospecific antiserum against this epitope to obtain a positive control for future epitope resolved diagnostics. First, a MAP of epitope 3, having a molecular mass of 7770 Da, was synthesized, purified, and its structure confirmed by liquid chromatography-mass spectrometry (electrospray ionization) (LC-MS(ESI)), matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), and Edman sequencing. The MAP was then used to raise high titer antibodies in rabbits using the adjuvant Titermax and to characterize the specificity of IgE from allergenic patients sensitized to Ara h 2. The antiserum exclusively detects Ara h 2 in crude peanut extract with a titer of 10(7) by Western blot and reacts specifically with epitope 3 shown by epitope mapping for a library of solid-phase-bound synthetic 15-mer peptides covering the entire sequence of Ara h 2. Such IgE-reactive epitopes are of high analytical relevance as they could constitute the basis for epitope-specific detection systems for use in quality control in the food industry or for forensic purposes in cases of fatal reactions to otherwise undetected peanut proteins.

• Maillard reaction and enzymatic browning affect the allergenicity of Pru av 1, the major allergen from cherry (Prunus avium).

  Authors: Patrick Gruber, Stefan Vieths, Andrea Wangorsch, Jörg Nerkamp, Thomas Hofmann

  Journal of agricultural and food chemistry. 07/2004; 52(12):4002-7.

  The influence of thermal processing and nonenymatic as well as polyphenoloxidase-catalyzed browning reaction on the allergenicity of the major cherry allergen Pru av 1 was investigated. After thermalThe influence of thermal processing and nonenymatic as well as polyphenoloxidase-catalyzed browning reaction on the allergenicity of the major cherry allergen Pru av 1 was investigated. After thermal treatment of the recombinant protein rPru av 1 in the absence or presence of carbohydrates, SDS-PAGE, enzyme allergosorbent tests, and inhibition assays revealed that thermal treatment of rPru av 1 alone did not show any influence on the IgE-binding activity of the protein at least for 30 min, thus correlating well with the refolding of the allergen in buffer solution as demonstrated by CD spectroscopic experiments. Incubation of the protein with starch and maltose also showed no effect on IgE-binding activity, whereas reaction with glucose and ribose and, even more pronounced, with the carbohydrate breakdown products glyceraldehyde and glyoxal induced a strong decrease of the IgE-binding capacity of rPru av 1. In the second part of the study, the effect of polyphenoloxidase-catalyzed oxidation of polyphenols on food allergen activity was investigated. Incubation of rPru av 1 with epicatechin in the presence of tyrosinase led to a drastic decrease in IgE-binding activity of the protein. Variations of the phenolic compound revealed caffeic acid and epicatechin as the most active inhibitors of the IgE-binding activity of rPru av 1, followed by catechin and gallic acid, and, finally, by quercetin and rutin, showing significantly lower activity. On the basis of these data, reactive intermediates formed during thermal carbohydrate degradation as well as during enzymatic polyphenol oxidation are suggested as the active chemical species responsible for modifying nucleophilic amino acid side chains of proteins, thus inducing an irreversible change in the tertiary structure of the protein and resulting in a loss of conformational epitopes of the allergen.

• Selective (R)-3-hydroxylation of FA by Stenotrophomonas maltophilia.

  Authors: Kerstin Weil, Patrick Gruber, Frank Heckel, Dag Harmsen, Peter Schreier

  Lipids. 04/2002; 37(3):317-23.

  Soil samples were screened for microorganisms selectively transforming FA. One of the isolated strains was identified as the bacterium Stenotrophomonas maltophilia by its phenotypic features andSoil samples were screened for microorganisms selectively transforming FA. One of the isolated strains was identified as the bacterium Stenotrophomonas maltophilia by its phenotypic features and genotypic characterization by sequencing the ribosomal RNA gene. Using linoleic acid as substrate resulted in the formation of two major compounds. After liquid chromatographic isolation and separation, their structures were elucidated by HPLC-tandem MS, GC-MS, and NMR techniques to be 3-hydroxy-Z6-dodecenoic acid and 3-hydroxy-Z5,Z8-tetradecadienoic acid. In additional experiments, other FA, such as a-linolenic, oleic, palmitoleic, myristoleic, and cis-vaccenic acids, were converted to 3-hydroxylated metabolites of shorter chain lengths as well. Determination of the enantiomeric composition revealed highly enriched (R)-hydroxylation (88-98% enantiomeric excess).

• Untersuchungen zur Modifizierung des allergenen Potentials von Kirsch- und Erdnussproteinen durch nicht-enzymatische Glycosylierung und enzymatische Bräunung

  Authors: Patrick Gruber

  The modulation of the IgE-binding of the recombinant allergens rPru av 1 from cherry, rAra h 2 from peanut as well as peanut lectin after thermal treatment, Maillard reaction an enzymatic browningThe modulation of the IgE-binding of the recombinant allergens rPru av 1 from cherry, rAra h 2 from peanut as well as peanut lectin after thermal treatment, Maillard reaction an enzymatic browning was studied. Sequences of Ara h 2, in which the amino acids cysteine, serine, lysine, and arginine were replaced by their thermal degradation or advanced glycation products, were synthesized and evaluated. Finally, multiple antigenic peptides (MAP) bearing epitopes of Ara h 2 were synthesized and used for the production of sequence specific antibodies.