Cloning, Expression, and Immunological Characterization of Two Novel Profilins from Artemisia annua
The rapid increase in the incidence of allergic diseases in recent decades has become a significant public health concern worldwide, affecting both developed and developing countries. In China, pollen from Artemisia species, particularly Artemisia annua and Artemisia vulgaris, has been identified as one of the most important outdoor allergens. According to the first national pollen survey conducted across all provinces in China, Artemisia pollen is a major cause of seasonal asthma. Among the Artemisia species, A. vulgaris is predominantly found in western regions, while A. annua is abundant in the densely populated northern and eastern regions. Immunoglobulin E (IgE) antibodies play a central role in allergic diseases. These antibodies are synthesized and released by B cells and bind to high-affinity IgE receptors on mast cells or basophils. Upon exposure to allergens, these effector cells are triggered, leading to the release of inflammatory mediators and the onset of allergic inflammation. Allergen immunotherapy, which involves the administration of specific allergens to patients, is considered a promising and effective treatment for allergic diseases. Therefore, the identification of IgE-binding allergens is crucial for improving the diagnosis of allergic diseases and the application of immunotherapy. However, previous studies have primarily focused on pollen allergens from A. vulgaris, such as Art v 1–6. In contrast, only two allergens from A. annua have been identified: Art an 1 (a defensin-like protein) and Art an 7 (a putative galactose oxidase). This highlights the importance of further studying allergens from A. annua pollen.
In this study, two novel profilins from A. annua were identified using next-generation sequencing technology and the basic local alignment search tool (BLAST). The complementary DNAs (cDNAs) encoding profilin 1 and profilin 2 were amplified by polymerase chain reaction (PCR) using primers designed from non-coding region sequences obtained through transcriptome sequencing. Both profilins exhibited open reading frames of 399 base pairs, encoding 133 amino acids. The sequence data for these profilins have been deposited in GenBank under the accession numbers MN105099 and MN105100. Interestingly, the amino acid sequences of profilin 1 and profilin 2 were identical to those of Art v 4.0101 and Art v 4.0201 from A. vulgaris, respectively. Additionally, these profilins shared approximately 65% to 90% sequence identity with other profilins from different plant pollens. Phylogenetic tree analysis revealed that profilin 1 forms a close cluster with Amb a 8, while profilin 2 is closely related to Hel a 2. All these profilins were clustered into the same group, indicating their evolutionary conservation.
To isolate recombinant allergens for further analysis, the genes encoding profilin 1 and profilin 2 were subcloned into the pET-28a vector and expressed in Escherichia coli BL-21. Both profilins were expressed as inclusion bodies, which were purified by nickel affinity chromatography under denaturing conditions. The refolded proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), revealing a single band with an approximate molecular weight of 12,000 Da. This confirmed the successful expression and purification of the recombinant profilins.
The IgE-binding activity of the identified allergens is essential for initiating allergic inflammation. To evaluate this, 200 patients with allergic reactions to Artemisia pollen and 16 healthy subjects were included in the study. The clinical information of the patients is summarized in Supplementary Table 1. All participants provided written informed consent for the use of their blood samples. Enzyme-linked immunosorbent assay (ELISA) results showed that serum IgE reacted to profilin 1 in 65 of 200 patients (32.5%) and to profilin 2 in 54 of 200 patients (27%). These sensitization rates are similar to those of profilins from other pollens, such as Bet v 2 (22%) and Hel a 2 (30.5%), likely due to their structural conservation. The IgE-binding activities of the two profilins were further confirmed by Western blot analysis using serum samples from eight representative patients and one negative control. Of the eight positive serum samples, seven showed strong bands for profilin 1, and five showed bands for profilin 2. However, some ELISA-positive serum samples did not show bands in Western blotting, likely because some IgE-binding epitopes of profilin 1 and profilin 2 are conformational and can be disrupted during the Western blot process.
A competitive inhibition experiment was conducted to evaluate the immunoreactivity of profilin 1 and profilin 2 in comparison to the crude extract of A. annua pollen. The average inhibition rates of profilin 1 and profilin 2 were 23.6% and 18.6%, respectively. A further competitive inhibition experiment was performed to analyze the cross-reactivity between profilin 1 and profilin 2. The results showed that 54.57% of the IgE from serum No. 30 that bound to profilin 2 was inhibited by profilin 1, and 39.35% of the IgE that bound to profilin 1 was inhibited by profilin 2. This indicates a significant degree of cross-reactivity between the two profilins.
Comprehensive analysis of clinical symptoms and ELISA results revealed no significant difference in the immunoreactivity characteristics of profilins in patients with different allergic symptoms, such as asthma, rhinitis, eczema, hay fever, dermatitis, or conjunctivitis. Similarly, there was no significant difference in the immunoreactivity characteristics of profilins between asthma and non-asthma patient groups or between rhinitis and non-rhinitis patient groups. This suggests that sensitization to profilins from A. annua is not associated with specific clinical symptoms in Chinese Artemisia allergic patients, consistent with previous studies.
In summary, this study identified two novel profilins, named profilin 1 and profilin 2, from A. annua pollen. These profilins have identical amino acid sequences to those of Art v 4.0101 and Art v 4.0201 from A. vulgaris, respectively, but differ in their nucleotide sequences and non-coding regions. Alignment analysis showed that profilin 1 and profilin 2 share high amino acid sequence identity with previously reported profilin allergens from various species, which may contribute to cross-sensitization or polysensitization to different pollen or food allergens. The IgE-binding rates of profilin 1 and profilin 2 in Artemisia allergy patients were 32.5% and 27%, respectively, similar to those of profilins from other species. Additionally, the immunoreactivity of these profilins in Chinese patients with Artemisia allergy decreased with increasing age, although this observation requires further confirmation in patients from different ethnicities and races. Finally, the identified novel profilins have a significant influence on Chinese Artemisia allergic patients and hold potential value for component-resolved allergy diagnostics in the future.
doi.org/10.1097/CM9.0000000000001309
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