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Lee S.,Baylor College of Medicine | Augustin S.,University of Cologne | Augustin S.,Allergopharma Joachim Ganzer KG | Tatsuta T.,University of Cologne | And 4 more authors.
Journal of Biological Chemistry | Year: 2011

FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

A number of scientific publications have been published recently dealing with the use of single recombinant allergens for the detection of specific IgE antibodies in vitro. Primarily microarray-based systems have been used in this context. A prerequisite for a routine use of recombinant allergens is a comparable IgE-binding capacity as natural single allergens. Our investigation shows that natural and recombinant single allergens are indeed comparable regarding to their IgE antibody reactivities. Natural and recombinant allergens were pure since only one protein band was detectable by SDS-PAGE. Measurements performed by EAST-Inhibition revealed, that recombinant allergens show nearly similar allergenic potencies compared with natural allergens. Irrespectively of whether recombinant or natural allergens were used identical EAST-classes (class 0/≥1) were measured with sera obtained from different sensitized patients in most experiments. In some cases a little bit higher classes were measured with the native allergen discs. In summary the purity of both recombinant and native extracts were found to be comparable and may be employed for IgE measurements. © 2011 Dustri-Verlag Dr. Karl Feistle.

Herranz F.,Imperial College London | Herranz F.,CIBER ISCIII | Schmidt-Weber C.B.,Helmholtz Center Munich | Shamji M.H.,Imperial College London | And 3 more authors.
Contrast Media and Molecular Imaging | Year: 2012

In this study we report the development of a bioconjugate between superparamagnetic iron oxide nanoparticles and Phl p5a (one of the major allergens from grass pollen). The bioconjugate also contains an optical probe (Alexa647) conjugated to the nanoparticle via biotin-streptavidin association. We show that this conjugate has a range of features that makes it a very promising candidate to image the localization of this allergen in vivo: (a) upon conjugation to the iron oxide nanoparticles, the allergen retains its ability to interact with IgE antibodies; (b) the magnetic properties of the iron oxide core of this bioconjugate are suitable for MR imaging; and (c) Alexa647 fluorophore retains its emission properties once attached to the iron oxide nanoparticles, yielding a dual modality MRI-optical probe. © 2012 John Wiley & Sons, Ltd.

Matricardi P.M.,Charite - Medical University of Berlin | Kuna P.,University of Lodz | Panetta V.,Charite - Medical University of Berlin | Wahn U.,Charite - Medical University of Berlin | Narkus A.,Allergopharma Joachim Ganzer KG
Journal of Allergy and Clinical Immunology | Year: 2011

Background: Allergen-specific subcutaneous immunotherapy (SCIT) of seasonal allergic rhinitis (SAR) is usually considered a "second-line," slow-acting, disease-modifying treatment. Objective: We sought to test whether SCIT is as effective as antisymptomatic treatment in the control of symptoms in patients with SAR in the first year of treatment. Methods: We reviewed meta-analyses with 5 or more randomized, double-blind, placebo-controlled trials of SCIT or antisymptomatic treatment in patients with SAR. We then selected trials measuring the total nasal symptom score (TNSS), the total symptom score (TSS), or both during the first pollen season after treatment initiation. Efficacy was determined as the percentage reduction in TSSs and TNSSs obtained with active treatment compared with placebo (relative clinical impact [RCI]) and the standardized mean difference (SMD) of treatment verses placebo (effect size [ES]). Results: The weighted mean RCI of SCIT on TNSSs (-34.7% ± 6.8%) was higher than those of mometasone (-31.7% ± 16.7%, P <.00001) and montelukast (-6.3% ± 3.0%, P <.00001). The weighted mean RCI of SCIT on TSSs (-32.9% ± 12.7%) was higher than that of desloratadine (-12.0% ± 5.1%, P <.00001). The overall ES of SCIT in terms of TNSSs (SMD, -0.94; 95% CI, -1.45 to -0.43) was similar to that of mometasone (SMD, -0.47; 95% CI, -0.63 to -0.32; P >.05) and higher than that of montelukast (SMD, -0.24; 95% CI, -0.33 to -0.16; P <.05). The overall ES of SCIT in terms of TSSs (SMD, -0.86; 95% CI, -1.17 to -0.55) was comparable with that of desloratadine (SMD, -1.00; 95% CI, -1.68 to -0.32; P >.05). Conclusions: Our data provide indirect but consistent evidence that SCIT is at least as potent as pharmacotherapy in controlling the symptoms of SAR as early as the first season of treatment. © 2011 American Academy of Allergy, Asthma & Immunology.

Brehler R.,Universitatsklinikum Munster | Kahlert H.,Allergopharma Joachim Ganzer KG | Thum-Oltmer S.,Allergopharma Joachim Ganzer KG
Allergo Journal | Year: 2010

The characteristic feature of allergoids and recombinant hypoallergenic derivatives is their reduced allergenicity allowing the application of high allergen doses in abbreviated up-dosing periods without increasing the risk of adverse events. Their antigenicity and immunogenicity is retained, as is proven by in vitro experiments and serological data from clinical studies. The preparations can be used to treat allergic symptoms, the clinical efficacy is sustained and even has a disease-modifying long-term effect. Patients' compliance is high. In this review the hypoallergenic principal is described for allergoids and a folding variant of the recombinant birch pollen major allergen Bet v 1.

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