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The response to thermal stress is an important parameter relevant for characterizing the biological activity and long-term stability of recombinant proteins, which may show irreversible, pH dependent structural changes under these conditions. We selected the recombinant pollen allergen of mugwort (Artemisia vulgaris) rArt v 3.0201 as a relevant model to study structural changes due to thermal and pH stress by means of capillary zone electrophoresis (CZE)-UV and capillary zone electrophoresis (CZE)-electrospray ionization (ESI)-TOF-MS. Therefore, this recombinant protein was exposed to 95 °C under acidic (pH 3.4) and slightly alkaline (pH 7.3) conditions for up to 120 min. CZE-UV data showed a continuous degradation of the allergen accompanied by the gradual formation of several reaction products. Characterization of novel allergen variants occurring at longer migration times was done via CZE-ESI-TOF-MS using in-capillary transient capillary isotachophoresis (tCITP) preconcentration to facilitate the identification of minor variants. MS data revealed various modifications of rArt v 3.0201 in response to heating. Variants with deamidations and sulfur-related modifications including both yield and loss of sulfur were identified at increased migration times. Desulfurization produced allergen variants with up to four lanthionines that replaced initial disulfide bonds. In addition, mass spectra revealed shifts in the charge state distribution which indicate concomitant conformational alterations. Moreover, several low-abundant oxidized variants were identified. With extended thermal stress, the portfolio of variants increased and progressively shifted toward rArt v 3.0201 with high lanthionine content. The kinetics of conversion and the complexity of variant composition were pH dependent and increased under alkaline conditions.

Der Sammelbegriff „Kräuter“ bezeichnet sowohl Pflanzen, die als Küchenkräuter oder Heilpflanzen Verwendung finden, als auch die ökologisch anpassungsfähigen Beikräuter. In Europa werden allergische Reaktionen gegen Kräuter vorwiegend durch Pollen aus Traubenkraut, Beifuß, Spitzwegerich und Glaskraut ausgelöst. Bislang wurden 35 Allergene aus Kräutern identifiziert. Die relevantesten stammen aus der Proteinfamilie der Pektatlyasen, defensinähnlichen Proteine, nichtspezifischen Lipidtransferproteine und Ole-e-1-ähnlichen Proteine. Die Sensibilisierungshäufigkeit unterliegt geographischen Schwankungen und kann in bestimmten Regionen mehr als 50 % der Pollenallergiker betreffen. Aufgrund überlappender Blühzeiten, ähnlichen Habitaten, Polysensibilisierungen und kreuzreaktiven (Pan-)Allergenen ist eine genuine Kräuterpollensensibilisierung mit Extrakten schwierig zu diagnostizieren. Für alle wichtigen Kräuterpollen stehen jedoch Markerallergene für die Komponentendiagnostik zur Verfügung. Es sind dies Amb a 1 (Traubenkraut), Art v 1 (Beifuß), Pla l 1 (Spitzwegerich), und Par j 2 (Glaskraut). Die molekulare Allergiediagnostik kann hier konsequent genutzt werden, um den primären Auslöser zu identifizieren und die Auswahl des Kräuterextrakts für die spezifische Immuntherapie zu unterstützen. Zitierweise:Stemeseder T, Hemmer W, Hawranek T, Gadermaier G. Marker allergens of weed pollen — basic consideration and diagnostic benefits in the clinical routine. Part 16 of the Series Molecular Allergology. Allergo J Int 2014;23:274–80 DOI: 10.1007/s40629-014-0033-1

Nitration of the major birch pollen allergen Bet v 1 alters the immune responses toward this protein, but the underlying chemical mechanisms are not yet understood. Here we address the efficiency and site-selectivity of the nitration reaction of recombinant protein samples of Bet v 1.0101 with different nitrating agents relevant for laboratory investigations (tetranitromethane, TNM), for physiological processes (peroxynitrite, ONOO–), and for the health effects of environmental pollutants (nitrogen dioxide and ozone, O3/NO2). We determined the total tyrosine nitration degrees (ND) and the NDs of individual tyrosine residues (NDY). High-performance liquid chromatography coupled to diode array detection and HPLC coupled to high-resolution mass spectrometry analysis of intact proteins, HPLC coupled to tandem mass spectrometry analysis of tryptic peptides, and amino acid analysis of hydrolyzed samples were performed. The preferred reaction sites were tyrosine residues at the following positions in the polypeptide chain: Y83 and Y81 for TNM, Y150 for ONOO–, and Y83 and Y158 for O3/NO2. The tyrosine residues Y83 and Y81 are located in a hydrophobic cavity, while Y150 and Y158 are located in solvent-accessible and flexible structures of the C-terminal region. The heterogeneous reaction with O3/NO2was found to be strongly dependent on the phase state of the protein. Nitration rates were about one order of magnitude higher for aqueous protein solutions (∼20% per day) than for protein filter samples (∼2% per day). Overall, our findings show that the kinetics and site-selectivity of nitration strongly depend on the nitrating agent and reaction conditions, which may also affect the biological function and adverse health effects of the nitrated protein.

ABSTRACTMechanisms of host–pathogen interactions resulting in immunopathological responses upon human Campylobacter jejuniinfection are not completely understood, but the recent availability of murine infection models mimicking key features of campylobacteriosis helps solving this dilemma. During a screen for proteases expressed by C. jejuni, we identified a peptidase of the M24 family as a potential novel virulence factor, which was named PepP. The gene is strongly conserved in various Campylobacterspecies. A constructed deletion mutant ΔpepPof C. jejunistrain 81–176 grew as efficiently compared to isogenic wild-type (WT) or pepPcomplemented bacteria. To shed light on the potential role of this protease in mediating immunopathological responses in the mammalian host, we perorally challenged microbiota-depleted IL-10−/-mice with these strains. All strains stably colonized the murine gastrointestinal tract with comparably high loads. Remarkably, pepPdeficiency was associated with less severe induced malaise, with less distinct apoptotic and innate immune cell responses, but also with more pronounced proliferative/regenerative epithelial cell responses in the large intestine at d6post-infection. Furthermore, pro-inflammatory mediators were lower in the colon, ileum, and mesenteric lymph nodes of mice that had been challenged with the ΔpepPmutant compared to the WT or pepPcomplemented strains. This also held true for extra-intestinal organs including liver, kidneys, and lungs, and, strikingly, to systemic compartments. Taken together, protease PepP is a novel virulence determinant involved in mediating campylobacteriosis. The finding that apoptosis in the colon is significantly diminished in mice infected with the pepPmutant highlights the epithelial layer as the first and main target of PepP in the intestine.