Zora Markovic-Housley

Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier

Bet v 1l is a naturally occurring hypoallergenic isoform of the major birch pollen allergen Bet v 1. The Bet v 1 protein belongs to the ubiquitous family of pathogenesis-related plant proteins (PR-10), which are produced in defense-response to various pathogens. Although the allergenic properties of PR-10 proteins have been extensively studied, their biological function in plants is not known. The crystal structure of Bet v 1l in complex with deoxycholate has been determined to a resolution of 1.9A using the method of molecular replacement. The structure reveals a large hydrophobic Y-shaped cavity that spans the protein and is partly occupied by two deoxycholate molecules which are bound in tandem and only partially exposed to solvent. This finding indicates that the hydrophobic cavity may have a role in facilitating the transfer of apolar ligands. The structural similarity of deoxycholate and brassinosteroids (BRs) ubiquitous plant steroid hormones, prompted the mass spectrometry (MS) study in order to examine whether BRs can bind to Bet v 1l. The MS analysis of a mixture of Bet v 1l and BRs revealed a specific non-covalent interaction of Bet v 1l with brassinolide and 24-epicastasterone. Together, our findings are consistent with a general plant-steroid carrier function for Bet v 1 and related PR-10 proteins. The role of BRs transport in PR-10 proteins may be of crucial importance in the plant defense response to pathological situations as well as in growth and development.

Crystal structure of hyaluronidase, a major allergen of bee venom

BACKGROUND Hyaluronic acid (HA) is the most abundant glycosaminoglycan of vertebrate extracellular spaces and is specifically degraded by a beta-1,4 glycosidase. Bee venom hyaluronidase (Hya) shares 30% sequence identity with human hyaluronidases, which are involved in fertilization and the turnover of HA. On the basis of sequence similarity, mammalian enzymes and Hya are assigned to glycosidase family 56 for which no structure has been reported yet. RESULTS The crystal structure of recombinant (Baculovirus) Hya was determined at 1.6 A resolution. The overall topology resembles a classical (beta/alpha)(8) TIM barrel except that the barrel is composed of only seven strands. A long substrate binding groove extends across the C-terminal end of the barrel. Cocrystallization with a substrate analog revealed the presence of a HA tetramer bound to subsites -4 to -1 and distortion of the -1 sugar. CONCLUSIONS The structure of the complex strongly suggest an acid-base catalytic mechanism, in which Glu113 acts as the proton donor and the N-acetyl group of the substrate is the nucleophile. The location of the catalytic residues shows striking similarity to bacterial chitinase which also operates via a substrate-assisted mechanism.

Recombinant allergen-based IgE testing to distinguish bee and wasp allergy

BACKGROUND The identification of the disease-causing insect in venom allergy is often difficult. OBJECTIVE To establish recombinant allergen-based IgE tests to diagnose bee and yellow jacket wasp allergy. METHODS Sera from patients with bee and/or wasp allergy (n = 43) and patients with pollen allergy with false-positive IgE serology to venom extracts were tested for IgE reactivity in allergen extract-based tests or with purified allergens, including nonglycosylated Escherichia coli-expressed recombinant (r) Api m 1, rApi m 2, rVes v 5, and insect cell-expressed, glycosylated rApi m 2 as well as 2 natural plant glycoproteins (Phl p 4, bromelain). RESULTS The patients with venom allergy could be diagnosed with a combination of E coli-expressed rApi m 1, rApi m 2, and rVes v 5 whereas patients with pollen allergy remained negative. For a group of 29 patients for whom the sensitizing venom could not be identified with natural allergen extracts, testing with nonglycosylated allergens allowed identification of the sensitizing venom. Recombinant nonglycosylated allergens also allowed definition of the sensitizing venom for those 14 patients who had reacted either with bee or wasp venom extracts. By IgE inhibition studies, it is shown that glycosylated Api m 2 contains carbohydrate epitopes that cross-react with natural Api m 1, Ves v 2, natural Phl p 4, and bromelain, thus identifying cross-reactive structures responsible for serologic false-positive test results or double-positivity to bee and wasp extracts. CONCLUSION Nonglycosylated recombinant bee and wasp venom allergens allow the identification of patients with bee and wasp allergy and should facilitate accurate prescription of venom immunotherapy.

High-Affinity IgE Recognition of a Conformational Epitope of the Major Respiratory Allergen Phl p 2 As Revealed by X-Ray Crystallography

We report the three-dimensional structure of the complex between the major respiratory grass pollen allergen Phl p 2 and its specific human IgE-derived Fab. The Phl p 2-specific human IgE Fab has been isolated from a combinatorial library constructed from lymphocytes of a pollen allergic patient. When the variable domains of the IgE Fab were grafted onto human IgG1, the resulting Ab (huMab2) inhibited strongly the binding of allergic patients’ IgE to Phl p 2 as well as allergen-induced basophil degranulation. Analysis of the binding of the allergen to the Ab by surface plasmon resonance yielded a very low dissociation constant (KD = 1.1 × 10−10 M), which is similar to that between IgE and Fcε;RI. The structure of the Phl p 2/IgE Fab complex was determined by x-ray crystallography to 1.9 Å resolution revealing a conformational epitope (876 Å2) comprised of the planar surface of the four-stranded anti-parallel β-sheet of Phl p 2. The IgE-defined dominant epitope is discontinuous and formed by 21 residues located mostly within the β strands. Of the 21 residues, 9 interact directly with 5 of the 6 CDRs (L1, L3, H1, H2, H3) of the IgE Fab predominantly by hydrogen bonding and van der Waals interactions. Our results indicate that IgE Abs recognize conformational epitopes with high affinity and provide a structural basis for the highly efficient effector cell activation by allergen/IgE immune complexes.

Mutational Analysis of Amino Acid Positions Crucial for IgE-Binding Epitopes of the Major Apple (Malus domestica) Allergen, Mal d 1

Background: Individual amino acid residues of the major birch pollen allergen, Bet v 1, have been identified to be crucial for IgE recognition. The objective of the present study was to evaluate whether this concept was applicable for the Bet v 1-homologous apple allergen, Mal d 1. Methods: A Mal d 1 five-point mutant was produced by PCR techniques, cloned into pMW 172 and expressed in Escherichia coli BL21(DE3) cells. To evaluate the allergenic properties of the engineered protein compared to Mal d 1 wild-type IgE immunoblotting, ELISA, peripheral blood monocytes proliferation assays, and skin prick tests were performed. Results: The Mal d 1 mutant showed reduced capacity to bind specific IgE as compared to wild-ype Mal d 1 in in vitro assays in the majority of the sera tested. In ELISA, 10 out of 14 serum samples displayed an 88–30% decrease in IgE binding to Mal d 1 mutant compared to wild-type Mal d 1. Skin prick tests in apple-allergic patients (n = 2) confirmed the markedly decreased ability of the Mal d 1 mutant to induce allergic reactions in vivo. However, the relevant T cell epitopes were present in the mutated molecule according to peripheral blood mononuclear cell proliferation assays. Conclusions: Our findings suggest that it is possible to modulate the IgE-binding properties of allergens by single amino acid substitutions at crucial positions which might be useful for future immunotherapy of birch-pollen-associated food allergies which are not ameliorated by birch pollen immunotherapy.

Crystal Structure of the Major Allergen from Fire Ant Venom, Sol i 3

Fire ant venom is an extremely potent allergy-inducing agent containing four major allergens, Sol i 1 to Sol i 4, which are the most frequent cause of hypersensitivity reactions to hymenoptera in the southern USA. The crystal structure of recombinant (Baculovirus) major fire ant allergen Sol i 3 has been determined to a resolution of 3.1 A by the method of molecular replacement. The secondary-structure elements of Sol i 3 are arranged in an alpha-beta-alpha sandwich fold consisting of a central antiparallel beta-sheet surrounded on both sides by alpha helices. The overall structure is very similar to that of the homologous wasp venom allergen Ves v 5 with major differences occurring in the solvent-exposed loop regions that contain amino acid insertions. Consequently, the limited conservation of surface chemical properties and topology between Sol i 3 and Ves v 5 may explain the observed lack of relevant cross-reactivity. It is concluded that Sol i 3 recognizes immunoglobulin E antibodies with a distinct set of its own epitopes, which are different from those of Ves v 5. Indeed, the molecular area in Sol i 3 covered by non-conserved residues is large enough to accommodate four unique Sol i 3 epitopes.

Structure of the major carrot allergen Dau c 1

Dau c 1 is a major allergen of carrot (Daucus carota) which displays IgE cross-reactivity with the homologous major birch-pollen allergen Bet v 1. The crystal structure of Dau c 1 has been determined to a resolution of 2.7 A, revealing tight dimers. The structure of Dau c 1 is similar to those of the major allergens from celery, Api g 1, and birch pollen, Bet v 1. Electron density has been observed in the hydrophobic cavity of each monomer and has been modelled with polyethylene glycol oligomers of varying length. Comparison of the surface topology and physicochemical properties of Dau c 1 and Bet v 1 revealed that they may have some, but not all, epitopes in common. This is in agreement with the observation that the majority of carrot-allergic patients have Bet v 1 cross-reactive IgE antibodies, whereas others have Dau c 1-specific IgE antibodies which do not recognize Bet v 1.

Crystallization and preliminary X-ray analysis of γ-aminobutyric acid transaminase☆

Abstract γ-Aminobutyric acid transaminase from pig liver, an α2 dimeric enzyme of Mr, 110 100, has been crystallized by the vapour diffusion method with polyethylene glycol as precipitant. The crystals are monoclinic, space group P21, unit cell dimensions a = 82.1 A , b = 230.0 A , c = 70.3 A , β = 123.9 ° and diffract to 2.5 A resolution. There are two dimers per asymmetric unit.

Crystal Structures of Thioredoxins f and m from Spinach Chloroplasts

Thioredoxins are ubiquitous small-molecular weight proteins with a redox-active disulfide bridge in a highly conserved active site sequence (1). Plant cells contain at least three different types of thioredoxins characterized by their function, location and primary structure (2). Two types are found in the chloroplasts where they serve as regulatory proteins in carbon assimilation and a third type is present in the cytoplasm. The two chloroplast thioredoxins have been distinguished by their specificity towards target enzymes. Thioredoxinf(TRf) displays high specificity towards chloroplast fructose 1, 6-bisphosphatase and several other Calvin cycle enzymes whereas thioredoxin m (TRm) interacts very efficiently with the NADP-dependent malate dehydrogenase. The primary structures of the two chloroplast thioredoxins show relatively little resemblance with only 30% identity. TRm, which is of prokaryotic origin (3), is similar to the thioredoxin from E.coli with 46% identity. The f-type thioredoxin is different and of eukaryotic origin. The active sites in the chloroplast thioredoxins are strictly conserved as well as a few residues that have been shown to be important for structure and catalysis in the E.coli protein. The three dimensional structures of the E. cou (1) and the human thioredoxins (4,5) have been extensively studied. From photosynthetic cells two structures have been reported, the crystal structure of thioredoxin-2 from Anabaena (6) and the NMR-structure for thioredoxin h, the cytoplasmic thioredoxin from Chlamydomonas (7). So far no structure of a chloroplast thioredoxin has been reported. Here we report the crystal structures of the oxidized spinach chloroplast TRf at 1.9 A resolution, of the oxidized TRm at 2.1 A resolution and of the reduced TRm at 2.3 A resolution.

Crystallization and preliminary X-ray analysis of the β-isoform of glutamate decarboxylase from Escherichia coli

Glutamate decarboxylase (GAD) is a vitamin B6 enzyme which catalyzes the alpha-decarboxylation of L-glutamate to gamma-aminobutyric acid (GABA). Escherichia coli cells coexpress two highly homologous enzyme isoforms, GADalpha and GADbeta. Well diffracting crystals of GADbeta were obtained by taking advantage of the possibility of expressing each isoform separately. They belong to space group P31 or P32 with the unit-cell dimensions a = b = 115.6 and c = 206.6 A and contain one GAD hexamer in the asymmetric unit. High-resolution synchrotron data were collected at 100 K for the native protein and a potential heavy-atom derivative.