Mammalian derived lipocalin and secretoglobin respiratory allergens strongly bind ligands with potentially immune modulating properties

Abstract

Allergens from furry animals frequently cause sensitization and respiratory allergic diseases. Most relevant mammalian respiratory allergens belong either to the protein family of lipocalins or secretoglobins. Their mechanism of sensitization remains largely unresolved. Mammalian lipocalin and secretoglobin allergens are associated with a function in chemical communication that involves abundant secretion into the environment, high stability and the ability to transport small volatile compounds. These properties are likely to contribute concomitantly to their allergenic potential. In this study, we aim to further elucidate the physiological function of lipocalin and secretoglobin allergens and link it to their sensitizing capacity, by analyzing their ligand-binding characteristics. We produced eight major mammalian respiratory allergens from four pet species in E.coli and compared their ligand-binding affinities to forty-nine ligands of different chemical classes by using a fluorescence-quenching assay. Furthermore, we solved the crystal-structure of the major guinea pig allergen Cav p 1, a typical lipocalin. Recombinant lipocalin and secretoglobin allergens are of high thermal stability with melting temperatures ranging from 65 to 90°C and strongly bind ligands with dissociation constants in the low micromolar range, particularly fatty acids, fatty alcohols and the terpene alcohol farnesol, that are associated with potential semiochemical and/or immune-modulating functions. Through the systematic screening of respiratory mammalian lipocalin and secretoglobin allergens with a large panel of potential ligands, we observed that total amino acid composition, as well as cavity shape and volume direct affinities to ligands of different chemical classes. Therefore, we were able to categorize lipocalin allergens over their ligand-binding profile into three sub-groups of a lipocalin clade that is associated with functions in chemical communication, thus strengthening the function of major mammalian respiratory allergens as semiochemical carriers. The promiscuous binding capability of hydrophobic ligands from environmental sources warrants further investigation regarding their impact on a molecule's allergenicity.

Keywords: Cav p 1 crystal structure; farnesol; fluorescence-quenching assays; lipocalin; mammalian respiratory allergens; protein-ligand interactions; secretoglobin.

Figure 1

Recombinant allergens are of high purity and show a comparable IgE-binding to their native counterpart. (A) SDS-PAGE and silver stain of eight purified recombinant mammalian respiratory allergens and the human homolog LCN1 (200 ng protein per well). Intact masses and amino acid sequence identity of recombinant allergens and LCN1 were analyzed by mass spectrometry. (B) Comparison of specific IgE-binding of allergic patient sera to recombinant allergens and their native counterpart by ELISA (n = 9–13 patients). Median IgE-reactivity was compared between the recombinant and the native form by Multiple Mann-Whitney test (^**P-value = 0.0028). Statistical analysis using Wilcoxon matched-pairs signed rank test between recombinant and native allergens resulted in no discoveries [FDR (Q) = 1%]. No native Fel d 4 was available. Lower limit of detection (LOD) ≤ 0.32 kU/L.

Figure 2

Cav p 1.0101 crystal structure. (A,B) Cartoon representation of Cav p 1.0101 in two perpendicular orientations highlighting the stacking of helices observed in the crystal, with the five monomers of one asymmetric unit in bright colors. (C) Overall fold of Cav p 1.0101 with α-helix in blue and β-strands in green. (D) Same representation as (C) with residues defining the central cavity shown as sticks, the β5β6 loop is in red, and the electron density (Fo-Fc map at 2 sigma level) observed in central cavity of monomer E as a black mesh.

Figure 3

Circular dichroism spectroscopy confirmed the beta-sheet and alpha-helical structures of respective lipocalins and secretoglobins and revealed a high thermal stability of allergens. Examples of circular dichroism spectra at 20°C (green) and after temperature ramping at 95°C (red) and 20°C (blue) of guinea pig lipocalin Cav p 1.0101 (A) and rabbit secretoglobin Ory c 3 (B).

Figure 4

Lipocalin and secretoglobin allergen-fluorochrome complexes emit high fluorescence intensities with protein specific emission maxima. Framed allergen-fluorochrome complexes are chosen for ligand competition assays based on their-signal-to-noise-ratio. Emission spectrum of 5 μM allergen complexed with 5 μM fluorochrome were recorded in duplicates between 420 and 560 nm for (A) 1-AMA and (B) 1,8-ANS (λ_ex = 380 nm) and between 380 and 500 nm for (C) 1-NPN (λ_ex = 337 nm).

Figure 5

Example of dose-dependent fluorochrome titration curves with guinea pig lipocalin Cav p 1 and dog lipocalin Can f 6. (A) Mean fluorescence intensity (λ_ex = 380 nm; λ_em = 485 nm) of 5 μM Cav p 1.0101 titrated with 1-AMA (0–60 μM) in a 1.5 dilution series. Equilibrium dissociation constants (K_d) and EC₅₀ values were calculated by non-linear regression using a one-site fit specific binding model. Total and non-specific binding are depicted. (B,C) Mean fluorescence intensity (λ_ex = 337 nm; λ_em = 395 nm) of 5 μM Can f 6 titrated with 1-NPN (0–200 μM) in a 1.5 dilution series. K_d and EC₅₀ values were calculated by one-site fit models of specific binding (B) and of total and non-specific binding (C).

Figure 6

Competition assays with selected allergen-fluorochrome complexes and forty-nine ligands. Mean fluorescence inhibition of 5 μM allergen-fluorochrome complexes by 50 μM competitive ligands in percent (n = 2) for (A) Can f 1 (C) Cav p 1.0101 and (E) Ory c 3. Dissociation constants (K_i) of most effective ligands were calculated by non-linear regression of logarithmized dose-dependent titration curves with (B) 0–100 μM arachidonic acid and tetradecanoic acid for Can f 1, (D) 0–60 μM farnesol and 1-decanol for Cav p 1.0101, (F) 0–50 μM 1-dodecanol and 1-decanol for Ory c 3.

Figure 7

(A) Principle component analysis (PCA) identified three clusters of mammalian respiratory allergens with similar ligand binding behavior based on fluorescence inhibition values of forty-nine ligands (thirty-two for 1-NPN). Cluster 3 contains 2 proteins families, lipocalins and secretoglobins, that are unrelated in sequence. (B) Phylogenetic tree represents the relationships between eight mammalian respiratory allergens and human LCN1 based on a multiple amino acid sequence alignment. Amino acid sequence identity between cluster members is presented in percentage values.

Figure 8

Chemical structures of fluorochromes and strongest binding ligands of mammalian respiratory allergens. Allergens were clustered by similarities in ligand binding profiles and sequence identities.

Figure 9

Heat map highlighting universal ligands with high affinities toward four lipocalin and two secretoglobin mammalian respiratory allergens and human LCN1 from dark (high affinity) to faint red (low affinity). Bar on the left represents the number of carbon atoms for each ligand in dark violet < 9, blue 9–12, green 13–16 and yellow > 16. The heat map was created using mean fluorescence inhibition (n = 2) of 5 μM allergen-fluorochrome complexes by 50 μM (100 μM for Fel d 1) of forty-seven competitive ligands.

Figure 10

Alignment of lipocalin models with the Cav p 1.0101 structure. Residues structurally aligned with a 2 Å cutoff in PyMol are shown in red. The blue arrows and red rectangle define secondary structures of Cav p 1.0101. Residues with their sidechain contributing to the cavity are highlighted in yellow. Allergen structures with the acronym AF were predicted with AlphaFold v2.0.

Figure 11

Shapes of ligand-binding cavities of lipocalin and secretoglobin respiratory allergens and human LCN1. Protein structures are shown as gray cartoons in two perpendicular orientations. The ligand-binding cavities were identified using the CASTp webservice and are represented as red colored mesh structures using PyMol.