Solution Structure, Dynamics, and Hydrodynamics of the Calcium-bound Cross-reactive Birch Pollen Allergen Bet v 4 Reveal a Canonical Monomeric Two EF-Hand Assembly with a Regulatory Function

Abstract

Birch pollinosis is one of the prevailing allergic diseases. In all, 5 -20% of birch pollinotics mount IgE antibodies against the minor birch pollen allergen Bet v 4, a Ca 2þ -binding polcalcin. Due to IgE cross-reactivity among the polcalcins these patients are polysensitized to various plant pollens. Determination of the high-resolution structure of holo Bet v 4 by heteronuclear NMR spectroscopy reveals a canonical two EF-hand assembly in the open conformation with interhelical angles closely resembling holo calmodulin. The polcalcin-specific amphipathic COOH-terminal a-helix covers only a part of the hydrophobic groove on the molecular surface. Unlike the polcalcin Phl p 7 from timothy grass, which was recently shown to form a domain-swapped dimer, the hydrodynamic parameters from NMR relaxation, NMR translational diffusion, and analytical ultracentrifugation indicate that both apo and holo Bet v 4 are predominantly monomeric, raising the question of the physiological and immunological significance of the dimeric form of these polcalcins, whose physiological function is still unknown. The reduced helicity and heat stability in the CD spectra, the poor chemical shift dispersion of the NMR spectra, and the slightly increased hydrodynamic radius of apo Bet v 4 indicate a reversible structural transition upon Ca 2þ binding, which explains the reduced IgE binding capacity of apo Bet v 4. The remarkable structural similarity of holo Bet v 4 and holo Phl p 7 in spite of different oligomerization states explains the IgE cross-reactivity and indicates that canonical monomers and domain-swapped dimers may be of similar allergenicity. Together with the close structural homology to calmodulin and the hydrophobic ligand binding groove this transition suggests a regulatory function for Bet v 4.

Three-Dimensional Structure of the Cross-Reactive Pollen Allergen Che a 3: Visualizing Cross-Reactivity on the Molecular Surfaces of Weed, Grass, and Tree Pollen Allergens

“…Characteristic features specific for polcalcins are a short linker connecting the two EF-hand domains and a rather hydrophobic helix C-terminal of the second calcium-binding domain. Recently, the three-dimensional structures of the polcalcins from timothy grass, Phl p 7, and birch, Bet v 4, were solved (14,15).…”

Treatment of Peak Intensity Uncertainties in NMR Relaxation Data Analysis Can Lead to Severe Artifacts

“…Recombinant 15 N-enriched Bet v 4 was prepared as described elsewhere [24]. NMR-experiments for determining the longitudinal and transversal 15 N relaxation rates were recorded on a Bruker Avance DRX 600 spectrometer at 298 K in 5 mM ammonium acetate buffer, 25 mM calcium chloride, 0.02% sodium azide, and 10% D 2 O at pH 6 (uncorrected meter reading) with a protein concentration of 1.5 mM.…”

Polcalcins as pollen panallergens in allergic rhinitis

“…Approximately 15% to 30% of pollen-allergic individuals are sensitized to profilin, and about 5%, and according to some authors up to 20%, are sensitized to polcalcins. In less than 2%, sensitization occurs to both panallergens in the same individual 12,19 . IgE sensitization to polcalcin and profilin usually occurs in regions with high grass pollen exposure, and this leads to the induction of a broad IgE antibody repertoire specific for grass pollen with numerous molecular allergen components 12,20,21 .…”

“…These cross-reactive molecular allergen components have been described as panallergens in various pollen sources (grass, tree and weed pollen), not in plant foods or latex (polcalcin expression seems to be restricted to pollen), are highly conserved among species and their amino acid sequences share a high degree of sequence identity across species, of about 60%-90% 23,27,28 . Rarely occurring as sole sensitizers in patients with clinical symptoms, polcalcins are minor allergens, only about 5-20% of pollen sensitized individuals having IgE responses to these cross-reactive allergenic molecules 12,19 . The clinical relevance of Unauthenticated Download Date | 5/8/18 8:27 AM polcalcin sensitization is linked to geographical factors and to the level of exposure to different allergenic sources.…”

“…Two conformational states of calcium-binding proteins can be distinguished, the closed calcium-free "apo", and the open calcium-associated "holo" forms. It was suggested that the apo-forms are less stable to thermal denaturation and display decreased IgE-reactivity when compared to their calcium-bound counterparts 19,23,26 . Moreover, changes in polcalcin quaternary structure, termed molecular metamorphosis, are induced and controlled by a combination of EF-hand rearrangements and domain swapping 30 .…”