The first adverse reactions to cow’s milk were already described 2000 years ago. However, it was only 50 years ago that several groups started with the analysis of cow’s milk allergens. Meanwhile the spectrum of allergy eliciting proteins within cow’s milk is identified and several cow’s milk allergens have been characterized regarding their biochemical properties, fold and IgE binding epitopes. The diagnosis of cow’s milk allergy is diverse ranging from fast and cheap in vitro assays to elaborate in vivo assays. Considerable effort was spent to improve the diagnosis from an extract-based into a component resolved concept. There is still no suitable therapy available against cow’s milk allergy except avoidance. Therefore research needs to focus on the development of suitable and safe immunotherapies that do not elicit severe side effect.

IgE-mediated allergy to fish is a frequent cause of severe anaphylactic reactions. Parvalbumin, a small calcium-binding protein, is the major fish allergen. We have recently isolated a cDNA coding for carp parvalbumin, Cyp c 1, and expressed in Escherichia coli a recombinant Cyp c 1 molecule, which contained most IgE epitopes of saltwater and freshwater fish. In this study, we introduced mutations into the calcium-binding domains of carp parvalbumin by site-directed mutagenesis and produced in E. coli three parvalbumin mutants containing amino acid exchanges either in one (single mutants; Mut-CD and Mut-EF) or in both of the calcium-binding sites (double mutant; Mut-CD/EF). Circular dichroism analyses of the purified derivatives and the wild-type allergen showed that Mut-CD/EF exhibited the greatest reduction of overall protein fold. Dot blot assays and immunoblot inhibition experiments performed with sera from 21 fish-allergic patients showed that Mut-CD/EF had a 95% reduced IgE reactivity and represented the derivative with the least allergenic activity. The latter was confirmed by in vitro basophil histamine release assays and in vivo skin prick testing. The potential applicability for immunotherapy of Mut-CD/EF was demonstrated by the fact that mouse IgG Abs could be raised by immunization with the mutated molecule, which cross-reacted with parvalbumins from various fish species and inhibited the binding of fish-allergic patients’ IgE to the wild-type allergen. Using the hypoallergenic carp parvalbumin mutant Mut-CD/EF, it may be possible to treat fish allergy by immunotherapy.

The ribosome is a dynamic particle that undergoes many structural changes during translation. We show through chemical probing with dimethyl sulfate (DMS) that conformational changes occur at several nucleotides in the peptidyl transferase center upon alterations in pH, temperature, and monovalent ion concentration, consistent with observations made by Elson and coworkers over 30 years ago. Moreover, we have found that the pH-dependent DMS reactivity of A2451 in the center of the 23S rRNA peptidyl transferase region, ascribed to a perturbed pKa of this base, occurs only in inactive 50S and 70S ribosomes. The degree of DMS reactivity of this base in the inactive ribosomes depends on both the identity and amount of monovalent ion present. Furthermore, G2447, a residue proposed to be critical for the hypothesized pKa perturbation, is not essential for the conditional DMS reactivity at A2451. Given that the pH-dependent change in DMS reactivity at A2451 occurs only in inactive ribosomes, and that this DMS reactivity can increase with increasing salt (independently of pH), we conclude that this observation cannot be used as supporting evidence for a recently proposed model of acid͞base catalyzed ribosomal transpeptidation.T he dynamics of ribosome translation is a challenging problem for structural biologists. Structural changes occur during assembly of the subunits, tRNA binding, and translocation. The recent wealth of information from cryoelectron microscopy and x-ray structures of different ribosomal complexes provides clear evidence for various conformational changes during the ribosomal cycle (1-5). Biochemical evidence for structural switches in rRNA comes from mutagenesis studies on 16S rRNA in the 912 region (6) and from probing 23S rRNA from pre-and posttranslocational complexes (7), where recurrent changes in rRNA structure have been detected during elongation. In addition, experiments from more than 30 years ago suggested reversible conformational changes in the Escherichia coli 50S subunit, where, upon removal of monovalent cations, these particles could no longer perform peptidyl transfer or bind the peptidyl transferase inhibitor chloramphenicol (8-10). This phenomenon was thought to represent a conformational change near the peptidyl transferase center (Fig. 1A), and chemical footprinting with the peptidyl transferase inhibitor chloramphenicol later revealed this region to include 23S rRNA residue A2451 (11). A2451 is a universally conserved nucleotide whose interaction with peptidyl transferase substrates has been implicated by several methods (12)(13)(14).The recent publication of the high-resolution crystal structure of the Haloarcula marismortui 50S ribosomal subunit has placed A2451 at the center of attention, as it was shown to be the closest nucleotide to a peptidyl transferase inhibitor (C-C-dAphosphoramide-puromycin) designed to mimic the tetrahedral intermediate formed during transpeptidation (15-17). Based on this proximity, the authors proposed a mechanism for acid-base catalysis in which t...

Milk is one of the first components introduced into human diet. It also represents one of the first allergen sources, which induces IgE-mediated allergies in childhood ranging from gastrointestinal, skin, and respiratory manifestations to severe life-threatening manifestations, such as anaphylaxis. Here we isolated a cDNA coding for a major cow’s milk allergen, αS1-casein, from a bovine mammary gland cDNA library with allergic patients’ IgE Abs. Recombinant αS1-casein was expressed in Escherichia coli, purified, and characterized by circular dichroism as a folded protein. IgE epitopes of αS1-casein were determined with recombinant fragments and synthetic peptides spanning the αS1-casein sequence using microarrayed components and sera from 66 cow’s milk-sensitized patients. The allergenic activity of rαS1-casein and the αS1-casein-derived peptides was determined using rat basophil leukemia cells transfected with human FcεRI, which had been loaded with the patients’ serum IgE. Our results demonstrate that rαS1-casein as well as αS1-casein-derived peptides exhibit IgE reactivity, but mainly the intact rαS1-casein induced strong basophil degranulation. These results suggest that primarily intact αS1-casein or larger IgE-reactive portions thereof are responsible for IgE-mediated symptoms of food allergy. Recombinant αS1-casein as well as αS1-casein-derived peptides may be used in clinical studies to further explore pathomechanisms of food allergy as well as for the development of new diagnostic and therapeutic strategies for milk allergy.