Sensitization profile to sawtooth oak component allergens and their clinical implications

Abstract Objective The component allergens from sawtooth oak, which is a main cause of tree pollinosis in Korea, have not been extensively characterized except Que ac 1. This study was undertaken to characterize the allergenic components from sawtooth oak pollen and investigate the diagnostic values of each component allergen. Methods Transcriptomic analysis was performed to identify the birch pollen allergen homologues from sawtooth oak pollen. Recombinant Que ac 1, 2, 3, 6, 7, and 8 were produced in an E. coli expression system. IgE reactivity to each allergen was examined by ImmunoCAP and ELISA using the sera of 50 Korean tree pollinosis patients. Results Six birch pollen allergen homologues were identified using transcriptome analysis, as follows: Que ac 1 (54.8% identity to Bet v 1), Que ac 2 (79.7% to Bet v 2), Que ac 3 (24.9% to Bet v 3), 6 (71.3% to Bet v 6), Que ac 7 (80.9% to Bet v 7), and Que ac 8 (78.9% to Bet v 8). Que ac 1 sIgE was the most frequently recognized (84.0%), followed by Que ac 2 (12.0%), Que ac 3 (6.0%), and three other allergens (2.0% each). Que ac 1 was a dominant allergen affecting 83.7% of patients suffering from allergic rhinoconjunctivitis, and 92.9% of pollen food allergy syndrome patients. Conclusion Five novel IgE reactive components of sawtooth oak were characterized using transcriptome analysis. Que ac 1 is the single most important component allergen of sawtooth oak pollen.

Sawtooth oak, Quercus acutissima, showed the strongest allergenicity among the Korean oak species. 5 Que ac 1, which is homologous to Bet v 1, was the most potent allergen from sawtooth oak pollen. This molecule (Que ac 1) was recognized by IgE antibodies from 91.3% of the oak pollinosis patients. 6 However, we believe that there are more allergen molecules that have yet to be identified. Only four different allergen molecules from four different oak species are officially listed in the allergen nomenclature (www. aller gen.org), according to the guidelines of Allergen Nomenclature Subcommitee of the International Union of Immunological Societies, as follows: Que a 1 from Q. alba 7 ; Que m 1 from Q. mongolica 8 ; Que i 1 from Q. ilex 9 ; and Que ac 1 from Q. acutissima. 6 Furthermore, there is only one group of protein, pathogenesis-related protein 10 (PR-10), from oak pollen. In contrast, there are 7 allergens of different protein families that are described from one species of birch (Betula verrucosa), as follows: Bet v 1, PR-10 10 ; Bet v 2, profilin 11 ; Bet v 3, 4 EF hand polcalcin-like protein 12 ; Bet v 4, polcalcin 13 ; Bet v 6, phenyl coumaran benzylic ether reductase 14 ; Bet v 7, cyclophilin 15 ; Bet v 8, glutathione S-transferase. 16 In this study, we used transcriptome analysis of sawtooth oak pollen to identify allergens that are homologous to birch pollen allergens. Recombinant proteins were produced, and IgE reactivities were examined using ImmunoCAP. A plausible application of the component-resolved diagnosis of oak allergy was also evaluated.

| Subjects and serum samples
The serum samples were collected from 50 oak pollinosis patients (average 32 years, age range 7-62 years) who visited the Allergy-Asthma Center at Severance Hospital, Seoul, Korea (Table S1). The allergy diagnosis was based on patient history and IgE tests. Specific IgE reactivity to white oak was determined using an ImmunoCAP assay (ThermoFisher Scientific). Serum sample collection was approved by the Institutional Review Board of Severance Hospital (4-2017-1197).

| Transcriptome analysis of sawtooth oak pollen
Pollen grains were collected from sawtooth oak trees identified by a botanist. The total RNA was extracted with TRIzol reagent (Invitrogen) using the manufacturer's instruction. The RNA concentration, quality, and purity were assessed using a NanoDrop UV spectrophotometer (ThermoFisher Scientific) and Agilent

| Expression and purification of recombinant proteins
Que ac 2, 3, 6, 7, 8 clones were synthesized at Bioneer (Daejeon, Korea). The PCR-amplified DNA fragments with oligonucleotide ( Recombinant Que ac 1 was also produced by the same way as described previously. 6 In order to identify the recombinant proteins, they were confirmed by liquid chromatography-coupled electrospray ionizationtandem mass spectrometry (LC ESI MS/MS) after gel excision. In-gel tryptic digestion was performed for tandem mass spectrometry at ProteomeTech.

| Identification of allergen homologues by transcriptome analysis
We were able to identify 6 birch allergen homologous molecules (

| IgE reactivity and diagnostic sensitivity of the recombinant allergens
Six recombinant allergens were produced using the same expression system; pET6xHN-N vector and E. coli Rosetta-2 (DE3) (Figure 1). The molecular identity of the recombinant proteins was confirmed again using LC ESI MS/MS ( Notably, IgE reactivity to Que ac 1 was even stronger than IgE reactivity to Q. alba (t7) pollen extract, which is currently used for the diagnosis of oak allergy ( Figure 1B).
A possible change in IgE reactivity by the structural change of Que ac 3 induction was examined by ELISA with 10 mM CaCl 2 .
However, there was no significant change in IgE reactivity (data not shown).

| Sensitization profile and disease entities
The most common symptom from oak allergy was allergic rhinosinusitis (83.7%) (  Sensitization to a minor allergen component can also cause cross-reactivity. In particular, sensitization to polcalcin may lead to a diagnosis of allergy mirage. 19 One of the three Que ac 3 sensitized patients had positive skin tests to various trees, grasses, and weed pollens.   In conclusion, we characterized five new IgE reactive components (Que ac 2, 3, 6, 7, and 8) from sawtooth oak pollen and investigated their diagnostic value. Que ac 1 and Que ac 2 are useful to make a diagnosis of oak pollinosis. Most IgE reactivities are directed to Que ac 1. However, no significant sensitization pattern was correlated to any allergic diseases. The recombinant allergens that were identified in this study will be useful to improve allergy diagnostics.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article.