Subcutaneous immunotherapy with purified Der p1 and 2 suppresses type 2 immunity in a murine asthma model.

BACKGROUND: Allergen-specific immunotherapy can induce long-term suppression of allergic symptoms, reduce medication use, and prevent exacerbations of allergic rhinitis and asthma. Current treatment is based on crude allergen extracts, which contain immunostimulatory components such as ß-glucans, chitins, and endotoxin. Use of purified or recombinant allergens might therefore increase efficacy of treatment. AIMS: Here, we test application of purified natural group 1 and 2 allergens from Dermatophagoides pteronyssinus (Der p) for subcutaneous immunotherapy (SCIT) treatment in a house dust mite (HDM)-driven mouse model of allergic asthma. MATERIALS AND METHODS: HDM-sensitized mice received SCIT with crude HDM extract, a mixture of purified Der p1 and 2 (DerP1/2), or placebo. Upon challenges, we measured specific immunoglobulin responses, allergen-induced ear swelling response (ESR), airway hyperresponsiveness (AHR), and inflammation in bronchoalveolar lavage fluid (BAL) and lung tissue. RESULTS: ESR measurement shows suppression of early allergic response in HDM-SCIT- and DerP1/2-SCIT-treated mice. Both HDM-SCIT and DerP1/2-SCIT are able to suppress AHR and eosinophilic inflammation. In contrast, only DerP1/2-SCIT is able to significantly suppress type 2 cytokines in lung tissue and BAL fluid. Moreover, DerP1/2-SCIT treatment is uniquely able suppress CCL20 and showed a trend toward suppression of IL-33, CCL17 and eotaxin levels in lung tissue. DISCUSSION: Taken together, these data show that purified DerP1/2-SCIT is able to not only suppress AHR and inflammation, but also has superior activity toward suppression of Th2 cells and HDM-induced activation of lung structural cells including airway epithelium. CONCLUSIONS: We postulate that treatment with purified natural major allergens derived from HDM will likely increase clinical efficacy of SCIT.

Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases.

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. The k(cat) and K(m) values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s(-1) and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK(a) of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.

Enzymic and structural studies on Drosophila alcohol dehydrogenase and other short-chain dehydrogenases/reductases.

Enzymic and structural studies on Drosophila alcohol dehydrogenases and other short-chain dehydrogenases/reductases (SDRs) are presented. Like alcohol dehydrogenases from other Drosophila species, the enzyme from D. simulans is more active on secondary than on primary alcohols, although ethanol is its only known physiological substrate. Several secondary alcohols were used to determine the kinetic parameters kcat and Km. The results of these experiments indicate that the substrate-binding region of the enzyme allows optimal binding of a short ethyl side-chain in a small binding pocket, and of a propyl or butyl side-chain in large binding pocket, with stereospecificity for R(-) alcohols. At a high concentration of R(-) alcohols substrate activation occurs. The kcat and Km values determined under these conditions are about two-fold, and two orders of magnitude, respectively, higher than those at low substrate concentrations. Sequence alignment of several SDRs of known, and unknown three-dimensional structures, indicate the presence of several conserved residues in addition to those involved in the catalyzed reactions. Structural roles of these conserved residues could be derived from observations made on superpositioned structures of several SDRs with known structures. Several residues are conserved in tetrameric SDRs, but not in dimeric ones. Two halohydrin-halide-lyases show significant homology with SDRs in the catalytic domains of these enzymes, but they do not have the structural features required for binding NAD+. Probably these lyases descend from an SDR, which has lost the capability to bind NAD+, but the enzyme reaction mechanisms may still be similar.

Isolation of protease-free alcohol dehydrogenase (ADH) from Drosophila simulans and several homozygous and heterozygous Drosophila melanogaster variants.

The enzyme alcohol dehydrogenase (ADH) from several naturally occurring ADH variants of Drosophila melanogaster and Drosophila simulans was isolated. Affinity chromatography with the ligand Cibacron Blue and elution with NAD+ showed similar behavior for D. melanogaster ADH-FF, ADH-71k, and D. simulans ADH. Introduction of a second Cibacron Blue affinity chromatography step, with gradient elution with NAD+, resulted in pure and stable enzymes. D. melanogaster ADH-SS cannot be eluted from the affinity chromatography column at a high concentration of NAD+ and required a pH gradient for its purification, preceded by a wash step with a high concentration of NAD+. Hybrid Drosophila melanogaster alcohol dehydrogenase FS has been isolated from heterozygous flies, using affinity chromatography with first elution at a high concentration NAD+, directly followed by affinity chromatography elution with a pH gradient. Incubation of equal amounts of pure homodimers of Drosophila melanogaster ADH-FF and ADH-SS, in the presence of 3 M urea at pH 8.6, for 30 min at room temperature, followed by reassociation yielded active Drosophila melanogaster ADH-FS heterodimers. No proteolytic degradation was found after incubation of purified enzyme preparations in the absence or presence of SDS, except for some degradation of ADH-SS after very long incubation times. The thermostabilities of D. melanogaster ADH-71k and ADH-SS were almost identical and were higher than those of D. melanogaster ADH-FF and D. simulans ADH. The thermostability of D. melanogaster ADH-FS was lower than those of D. melanogaster ADH-FF and ADH-SS. D. melanogaster ADH-FF and ADH-71k have identical inhibition constants with the ligand Cibacron Blue at pH 8.6, which are two times higher at pH 9.5. The Ki values for D. simulans ADH are three times lower at both pH values. D. melanogaster ADH-SS and ADH-FS have similar Ki values, which are lower than those for D. melanogaster ADH-FF at pH 8.6. But at pH 9.5 the Ki value for ADH-FS is the same as at pH 8.6, while that of ADH-SS is seven times higher. Kinetic parameters of Drosophila melanogaster ADH-FF, ADH-SS, and ADH-71k and Drosophila simulans ADH, at pH 8.6 and 9.5, showed little or no variation in K(m)eth values. The K(m)NAD values measured at pH 9.5 for Drosophila alcohol dehydrogenases are all lower than those measured at pH 8.6. The rate constants (kcat) determined for all four Drosophila alcohol dehydrogenases are higher at pH 9.5 than at pH 8.6. D. melanogaster ADH-FS showed nonlinear kinetics.

Modeling studies of conformational changes in the substrate-binding loop in Drosophila alcohol dehydrogenase.

Three-dimensional structures of seven short-chain dehydrogenases/reductases show that these enzymes share common structural features. Sequence alignment studies of Drosophila alcohol dehydrogenase (DADH), with an unknown 3D-structure, and four short-chain dehydrogenases/reductases with known X-ray structures suggest that DADH shares the same structural features. However, the substrate binding regions, which are located in the C-terminal region of these enzymes, share little sequence homology, because of the wide variety of substrates used. X-ray structures of short-chain dehydrogenases/reductases indicate that conformational changes occur in a loop, in the C-terminal region, upon substrate binding. This substrate-binding loop is located between a strand and a helix and may contain one or two small helices. Secondary structure predictions and modeling studies of this substrate-binding loop in DADH predict that the two helices may also be present in this enzyme. The naturally occurring variants of Drosophila melanogaster alleloenzymes ADH-S and ADH-F differ in a replacement of threonine by lysine at position 192, which is located at a central position in the substrate-binding loop. The positive charge of lysine may move significantly on substrate binding, resulting in a direct charge interaction with NAD+ in the enzyme-substrate complex, explaining a very strong influence of pH on the binding of ADH-S for the NAD+ analogue Cibacron Blue. This indicates that the ADH S/F polymorphism has a direct influence on the catalytic properties of the enzyme.

Comparison of three methods for competitive binding of monoclonal antibodies. The localization of antigenic sites for monoclonal antibodies on Panulirus interruptus hemocyanin.

The competitive binding of a panel of monoclonal antibodies against hemocyanin of Panulirus interruptus hemocyanin was investigated with three different methods. A competitive-binding immunoassay method was more successful in the determination of ternary complexes than gel electrophoresis and gel filtration. The latter two methods could only be applied with antibodies possessing a higher affinity. Eleven monoclonal antibodies were assigned to groups on the basis of their interactions with five antigenic regions.