Lori L. Edwards

The structure of the dust mite allergen Der p 7 reveals similarities to innate immune proteins.

BACKGROUND Sensitization to house dust mite allergens is strongly correlated with asthma. Der p 7 elicits strong IgE antibody and T-cell responses in patients with mite allergy. However, the structure and biological function of this important allergen are unknown. Allergen function might contribute to allergenicity, as shown for the protease activity of group 1 mite allergens and the interaction with the innate immune system by group 2 mite allergens. OBJECTIVE We sought to determine the crystal structure of Der p 7 and to investigate its biological function. METHODS X-ray crystallography was used to determine the Der p 7 structure. Nuclear magnetic resonance analysis and biochemical assays were used to examine the binding of Der p 7 to predicted ligands. RESULTS Der p 7 has an elongated structure, with two 4-stranded antiparallel beta-sheets that wrap around a long C-terminal helix. The fold of Der p 7 is similar to that of LPS-binding protein (LBP), which interacts with Toll-like receptors after binding LPS and other bacterially derived lipid ligands. Nuclear magnetic resonance and biochemical assays indicate that Der p 7 does not bind LPS but binds with weak affinity to the bacterial lipopeptide polymyxin B in the predicted binding site of Der p 7. CONCLUSIONS Der p 7 binds a bacterially derived lipid product, a common feature of some allergens. The finding that the group 7, as well as the group 2, mite allergens are structurally similar to different proteins in the Toll-like receptor pathway further strengthens the connections between dust mites, innate immunity, and allergy.

Identification of Maillard reaction products on peanut allergens that influence binding to the receptor for advanced glycation end products.

Recent immunological data demonstrated that dendritic cells preferentially recognize advanced glycation end product (AGE)‐modified proteins, upregulate expression of the receptor for AGE (RAGE), and consequently bias the immune response toward allergy.

Der p 5 crystal structure provides insight into the group 5 dust mite allergens.

Group 5 allergens from house dust mites elicit strong IgE antibody binding in mite-allergic patients. The structure of Der p 5 was determined by x-ray crystallography to better understand the IgE epitopes, to investigate the biologic function in mites, and to compare with the conflicting published Blo t 5 structures, designated 2JMH and 2JRK in the Protein Data Bank. Der p 5 is a three-helical bundle similar to Blo t 5, but the interactions of the helices are more similar to 2JMH than 2JRK. The crystallographic asymmetric unit contains three dimers of Der p 5 that are not exactly alike. Solution scattering techniques were used to assess the multimeric state of Der p 5 in vitro and showed that the predominant state was monomeric, similar to Blo t 5, but larger multimeric species are also present. In the crystal, the formation of the Der p 5 dimer creates a large hydrophobic cavity of ∼3000 Å3 that could be a ligand-binding site. Many allergens are known to bind hydrophobic ligands, which are thought to stimulate the innate immune system and have adjuvant-like effects on IgE-mediated inflammatory responses.

ADAM9 Inhibition Increases Membrane Activity of ADAM10 and Controls α-Secretase Processing of Amyloid Precursor Protein

Background: Raising ADAM10 α-secretase activity has been considered as an attractive therapeutic option in Alzheimer disease. Results: Administration of the prodomain of ADAM9 (proA9) prevents ADAM10 shedding and increases membrane α-secretase activity in a neuronal cell line. Conclusion: Use of proA9 is a means to modulate cellular ADAM10 activity. Significance: proA9 can be used in vivo to increase ADAM10 α-secretase activity. Prodomains of A disintegrin and metalloproteinase (ADAM) metallopeptidases can act as highly specific intra- and intermolecular inhibitors of ADAM catalytic activity. The mouse ADAM9 prodomain (proA9; amino acids 24–204), expressed and characterized from Escherichia coli, is a competitive inhibitor of human ADAM9 catalytic/disintegrin domain with an overall inhibition constant of 280 ± 34 nm and high specificity toward ADAM9. In SY5Y neuroblastoma cells overexpressing amyloid precursor protein, proA9 treatment reduces the amount of endogenous ADAM10 enzyme in the medium while increasing membrane-bound ADAM10, as shown both by Western and activity assays with selective fluorescent peptide substrates using proteolytic activity matrix analysis. An increase in membrane-bound ADAM10 generates higher levels of soluble amyloid precursor protein α in the medium, whereas soluble amyloid precursor protein β levels are decreased, demonstrating that inhibition of ADAM9 increases α-secretase activity on the cell membrane. Quantification of physiological ADAM10 substrates by a proteomic approach revealed that substrates, such as epidermal growth factor (EGF), HER2, osteoactivin, and CD40-ligand, are increased in the medium of BT474 breast tumor cells that were incubated with proA9, demonstrating that the regulation of ADAM10 by ADAM9 applies for many ADAM10 substrates. Taken together, our results demonstrate that ADAM10 activity is regulated by inhibition of ADAM9, and this regulation may be used to control shedding of amyloid precursor protein by enhancing α-secretase activity, a key regulatory step in the etiology of Alzheimer disease.

Serological, genomic, and structural analyses of the major mite allergen Der p 23

Der p 23 was recently identified in a European population as a major allergen and potentially a chitin binding protein.

Analysis of glutathione S-transferase allergen cross-reactivity in a North American population: Relevance for molecular diagnosis

BACKGROUND It is not clear whether cross-reactivity or cosensitization to glutathione S-transferases (GSTs) occurs in tropical and subtropical environments. In the United States, Bla g 5 is the most important GST allergen and lack of coexposure to GSTs from certain species allows a better assessment of cross-reactivity. OBJECTIVES To examine the molecular structure of GST allergens from cockroach (Bla g 5), dust mites (Der p 8 and Blo t 8), and helminth (Asc s 13) for potential cross-reactive sites, and to assess the IgE cross-reactivity of sensitized patients from a temperate climate for these allergens for molecular diagnostic purposes. METHODS Four crystal structures were determined. Sera from patients allergic to cockroach and mite were tested for IgE reactivity to these GSTs. A panel of 6 murine anti-Bla g 5 mAb was assessed for cross-reactivity with the other 3 GSTs using antibody binding assays. RESULTS Comparisons of the allergen structures, formed by 2-domain monomers that dimerize, revealed few contiguous regions of similar exposed residues, rendering cross-reactivity unlikely. Accordingly, anti-Bla g 5 or anti-Der p 8 IgE from North American patients did not recognize Der p 8 or Bla g 5, respectively, and neither showed binding to Blo t 8 or Asc s 13. A weaker binding of anti-Bla g 5 IgE to Der p 8 versus Bla g 5 (∼ 100-fold) was observed by inhibition assays, similar to a weak recognition of Der p 8 by anti-Bla g 5 mAb. Patients from tropical Colombia had IgE to all 4 GSTs. CONCLUSIONS The lack of significant IgE cross-reactivity among the 4 GSTs is in agreement with the low shared amino acid identity at the molecular surface. Each GST is needed for accurate molecular diagnosis in different geographic areas.

Targeting autocrine HB-EGF signaling with specific ADAM12 inhibition using recombinant ADAM12 prodomain.

Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, “decoy” antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling.

Refolding active human DNA polymerase ν from inclusion bodies

Human DNA polymerase nu (Pol nu) is a conserved family A DNA polymerase of uncertain biological function. Physical and biochemical characterization aimed at understanding Pol nu function is hindered by the fact that, when over-expressed in Escherichia coli, Pol nu is largely insoluble, and the small amount of soluble protein is difficult to purify. Here we describe the use of high hydrostatic pressure to refold Pol nu from inclusion bodies, in soluble and active form. The refolded Pol nu has properties comparable to those of the small amount of Pol nu that was purified from the soluble fraction. The approach described here may be applicable to other DNA polymerases that are expressed as insoluble inclusion bodies in E. coli.

Iron incorporation into MnSOD A (bacterial Mn-dependent superoxide dismutase) leads to the formation of a peroxidase/catalase implicated in oxidative damage to bacteria.

BACKGROUND Mn/Fe-superoxide dismutase (SOD) is a family of enzymes essential for organisms to be able to cope with oxygen. These enzymes bound to their classical metals catalyze the dismutation of the free radical superoxide anion (O2(-)) to H2O2 and molecular oxygen. E. coli has the manganese-dependent SOD A and the iron-dependent SOD B. METHODS Strains of E. coli overexpressing SOD A or SOD B were grown in media with different metal compositions. SODs were purified and their metal content and SOD activity were determined. Those proteins were incubated with H2O2 and assayed for oxidation of Amplex red or o-phenylenediamine, consumption of H2O2, release of iron and protein radical formation. Cell survival was determined in bacteria with MnSOD A or FeSOD A after being challenged with H2O2. RESULTS We show for the first time that the bacterial manganese-dependent SOD A when bound to iron (FeSOD A) has peroxidase activity. The in vivo formation of the peroxidase FeSOD A was increased when media had higher levels of iron because of a decreased manganese metal incorporation. In comparison to bacteria with MnSOD A, cells with FeSOD A had a higher loss of viability when exposed to H2O2. GENERAL SIGNIFICANCE The biological occurrence of this fundamental antioxidant enzyme in an alternative iron-dependent state represents an important source of free radical formation.

Characterization of an anti-Bla g 1 scFv: Epitope mapping and cross-reactivity

Bla g 1 is a major allergen from Blatella germanica and one of the primary allergens used to assess cockroach allergen exposure. The epitope of an anti-Bla g 1 scFv was mapped in order to better understand cross reactivity with other group 1 cockroach allergens and patient IgE epitopes. X-ray crystallography was used to determine the structure of the scFv. The scFv epitope on Bla g 1 was located by alanine scanning site-directed mutagenesis and ELISA. Twenty-six rBla g 1-GST alanine mutants were evaluated for variations in binding to the scFv compared to the wild type allergen. Six mutants showed a significant difference in scFv binding affinity. These mutations clustered to form a discontinuous epitope mainly comprising two helices of Bla g 1. The allergen-scFv complex was modeled based on the results, and the epitope region was found to have low sequence similarity with Per a 1, especially among the residues identified as functionally important for the scFv binding to Bla g 1. Indeed, the scFv failed to bind Per a 1 in American cockroach extract. The scFv was unable to inhibit the binding of IgE antibodies from a highly cockroach allergic patient to Bla g 1. Based on the surface area of Bla g 1 occluded by the scFv, putative regions of patient IgE-Bla g 1 interactions can be inferred. This scFv could be best utilized as a capture antibody in an IgE detection ELISA, or to differentiate Bla g 1 from Per a 1 in environmental exposure assays.