Primary identification, biochemical characterization, and immunologic properties of the allergenic pollen cyclophilin cat R 1

Abstract

Cyclophilin (Cyp) allergens are considered pan-allergens due to frequently reported cross-reactivity. In addition to well studied fungal Cyps, a number of plant Cyps were identified as allergens (e.g. Bet v 7 from birch pollen, Cat r 1 from periwinkle pollen). However, there are conflicting data regarding their antigenic/allergenic cross-reactivity, with no plant Cyp allergen structures available for comparison. Because amino acid residues are fairly conserved between plant and fungal Cyps, it is particularly interesting to check whether they can cross-react. Cat r 1 was identified by immunoblotting using allergic patients' sera followed by N-terminal sequencing. Cat r 1 (∼ 91% sequence identity to Bet v 7) was cloned from a cDNA library and expressed in Escherichia coli. Recombinant Cat r 1 was utilized to confirm peptidyl-prolyl cis-trans-isomerase (PPIase) activity by a PPIase assay and the allergenic property by an IgE-specific immunoblotting and rat basophil leukemia cell (RBL-SX38) mediator release assay. Inhibition-ELISA showed cross-reactive binding of serum IgE from Cat r 1-allergic individuals to fungal allergenic Cyps Asp f 11 and Mala s 6. The molecular structure of Cat r 1 was determined by NMR spectroscopy. The antigenic surface was examined in relation to its plant, animal, and fungal homologues. The structure revealed a typical cyclophilin fold consisting of a compact β-barrel made up of seven anti-parallel β-strands along with two surrounding α-helices. This is the first structure of an allergenic plant Cyp revealing high conservation of the antigenic surface particularly near the PPIase active site, which supports the pronounced cross-reactivity among Cyps from various sources.

Keywords: Allergen; Antibody; Antigen; Cloning; Cross-reactivity; Cyclophilin; Nuclear Magnetic Resonance (NMR); Protein Structure.

FIGURE 1.

IgE reactivity of allergic patients' sera to Western blots of C. roseus pollen extract. Immunoblots show different binding patterns between individual donors. Some sera recognized multiple bands, whereas others recognized only two protein bands). The band at 18 kDa (arrowhead) was consistently detected by all patients' sera. Lanes M and E (silver staining), molecular mass markers and crude C. roseus extract, respectively. Lane C, immunoblot using a control serum.

FIGURE 2.

Nucleotide and deduced amino acid sequence of Cat r 1. The 489 nucleotides encode a 172-amino acid protein. The N terminus of the protein, whose sequence was determined by Edman degradation, is underlined. The two potential N-linked glycosylation sites (Asn⁷⁸ and Asn¹¹⁵) are indicated in boldface type.

FIGURE 3.

a, SDS-PAGE and IgE-specific immunoblot of recombinant Cat r 1. Cat r 1 expressed in E. coli was subjected to 12% SDS-PAGE (lane 1, Coomassie stain; lanes 2 and 3, IgE-specific immunoblot using two individual patients' sera (lanes 2 and 3). b, recombinant Cat r 1 IgE-specifically triggers β-hexosaminidase release. FcϵR1-expressing RBL cells were incubated with individual patients' sera (P1–P5) and, subsequently, incubated with recombinant Cat r 1. The percentage of mediator release (vertical axis) was calculated considering total mediator release from lysed cells as 100% (total). Negative controls were mediator release using control sera (Con1–3); positive controls were β-hexosaminidase release following stimulation of anti-NP IgE-sensitized RBL cells with NP-HSA. Error bars, S.D.

FIGURE 4.

Peptidyl-prolyl cis-trans-isomerase assay. Cat r 1 (30 nm) catalyzes isomerization of the substrate N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (100 μm) and thus exhibits a functional activity typical for Cyps. Curve a, time course of the Cat r 1-catalyzed isomerization; curve b, thermal isomerization in the absence of Cat r 1 (control).

FIGURE 5.

Cross-reactivity between Cyps. a, to evaluate cross-reactivity between Cat r 1 and fungal Cyps, pooled serum from five Cat r 1-allergic subjects was preincubated with increasing amounts of either Asp f 11 or Mala s 6 and was added to Cat r 1-coated ELISA plates. Cat r 1 and BSA were used as positive and negative controls, respectively. Binding of serum IgE to plate-bound Cat r 1 was inhibited by fungal Cyps in the fluid phase, indicating their cross-reactivity to Cat r 1. b, rabbit antibodies against plant and animal Cyps recognized Cat r 1 and its loop deletion mutant ΔCat r 1 in immunoblots. SDS-PAGE lanes are as follows. Lane 1, marker; lane 2, Cat r 1; lane 3, ΔCat r 1. Immunoblot lanes are as follows. Cat r 1 (lanes 1 and 3) and ΔCat r 1 (lanes 2 and 4) were detected with rabbit antibody against plant (lanes 1 and 2) or animal (lanes 3 and 4) Cyp. IgE-specific immunoblotting showed that serum IgE from two Catharanthus-sensitized patients (patient 1 (lanes 7 and 10) and patient 2 (lanes 8 and 11)) but not the non-atopic control (lanes 9 and 12) recognized both cat r 1 (lanes 7–9) and its loop deletion mutant (lanes 10–12).

FIGURE 6.

a, far UV CD spectrum of recombinant Cat r 1, showing the characteristics of the folded protein. b, ¹H-¹⁵N HSQC spectrum of [U-¹³C- ¹⁵N]Cat r 1. The peaks are nicely disperse and fairly uniform in intensity. The expected number of peaks are found for this molecule.

FIGURE 7.

a, topological diagram showing the secondary structural elements of Cat r 1. The position of plant-specific loop RSGKPLH is shown. b, solved structures of Cat r 1 and Cyp proteins from bacteria, protozoa, fungi, plant, and animal sources, indicating the position of the loop specific for plant Cyps. This loop is also present in some protozoan and nematode Cyps.

FIGURE 8.

Cat r 1 compared with human Cyp. a and d compare the electrostatic surface of Cat r 1 (a) with the human Cyp (d) displayed in the same orientation with the substrate peptide. The color scale is in units of k_bT/e_c, where k denotes the Boltzmann constant, T is the absolute temperature, and e_c is the charge of the electron. In b and e, highly homologous residues of Cat r 1 (b) and human Cyp (e) are colored orange. The “plant loop” is colored magenta in b. c and f show ribbon diagrams of Cat r 1 and human Cyp, respectively, in the same orientation with the substrate peptide in the human Cyp shown in magenta. × (red), sites of potential N-glycosylation in c.

FIGURE 9.

Sequence alignment and structural elements of Cat r 1. Cyp sequences from bacteria, fungi, protozoa, nematodes, and mammals were aligned using ClustalX. The loop present predominantly in plant Cyp sequences is shown by red arrowhead. Red dot, Cat r 1 sequence. The active site residues involved in PPIase activity are shown in red. Regions R1 and R2 were previously indicated to be potential candidates for cross-reactivity.

FIGURE 10.

a, dendrogram showing the evolutionary relationship between Cyp proteins from bacteria, protozoa, fungi, plant, and animal sources. Uniprot accession numbers have been shown. b, sequence conservation mapped on the surface of Cat r 1 based on a multisequence alignment grouping plants with fungi, grouping mammals with plants, and grouping fungi with mammals. The highest degree of conservation was found adjacent to the active site. The sequences diverged through evolution but show considerably conserved antigenic surface.