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Patterns of allergen recognition in Ligustrum polysensitized patients: An immunoproteomics approach

2024; Wiley; Volume: 79; Issue: 7 Linguagem: Inglês

10.1111/all.16110

1398-9995

Raúl Porras‐Gutiérrez‐de‐Velasco, Blessy M. Mani, Juan Carlos Vizuet‐de‐Rueda, Josaphat Miguel Montero‐Vargas, Luis M. Terán,

Asthma and respiratory diseases

Members of the Oleaceae family, including Ligustrum (privet), Olea (olive), and Fraxinus (ash), are recognized as important causes of allergenic respiratory diseases worldwide. In Mexico City, nearly 37% of allergic patients have been reported to show sensitization to Ligustrum lucidum pollen, correlating with sensitization to Fraxinus, a very abundant tree in the city.1 Lig v 1, a homolog of Ole e 1 and Fra e 1, was the first allergen identified in Ligustrum vulgare using sera from olive-sensitized subjects, initially considered the only allergen in this pollen.2 However, our group reported six IgE-binding proteins in patients monosensitized to Ligustrum, revealing unexplored allergen diversity in this pollen.3 Interestingly, Lig v 1 was not detected in patients sensitized only to Ligustrum, which made us wonder if it is indeed a Ligustrum-specific allergen. Therefore, we investigated Ligustrum polysensitized patients (Group A) employing an immunoproteomics discovery approach (methods at Data S1). At this time, a group of Ligustrum-sensitized patients co-sensitized with Fraxinus (group B) was included in order to investigate whether they show a different allergen profile including Lig v 1 differential expression in comparison with Ligustrum-sensitized patients without co-sensitization with Fraxinus. Interestingly, we observed two distinct allergen recognition patterns among the study subjects: Ligustrum-polysensitized patients without co-sensitization to Fraxinus revealed 6 immunoreactive protein spots, while polysensitive patients to both Ligustrum and Fraxinus exhibited 23 (Figure 1A–C). Patients of group B also showed a higher proportion of positive skin tests, indicating major sensitivity, which may explain this difference in the number of recognized reactive proteins. MS analysis indicated that group A patients recognized the same 6 proteins previously identified in Ligustrum-monosensitized patients, suggesting their possible role as marker proteins for sensitivity to Ligustrum. In contrast, the 23 immunoreactive spots in group B corresponded to 26 different proteins, including those found in group A. Interestingly, among the 26 proteins, 21 have been previously associated with allergic responses in other sources, while the remaining five have not (Table 1). These immunoreactive spots belong to diverse protein families with various biological functions. Involved with ATP processes, we identified ATP synthase subunits (s1-2), fructokinase (s6), and phosphoglycerate kinases (s9, s12, and s14). These proteins share homology with the Bet v 1 family, known for allergic reactions from pollen inhalation4 and plant ingestion.5 Surprisingly, actin, a major mollusk allergen, was also identified (s9).6 An adenosine kinase (s10), not previously linked to allergic reactions, was found, though several arginine, serine, or creatine kinases have been reported as allergens. Another interesting class of IgE-binding proteins identified were β-glucosidases (s4-5 and s8-9), reported as allergens through inhalation, ingestion, and contact. Due to their high homology, they are associated with triggering cross-reactions in patients with latex-fruit syndrome(e7). A well-characterized allergen from wind-pollinated plants and food sources was also identified as profilin (s23), recently listed as Lig v 2(e8). Enolase (s5) was another clinically relevant allergen identified, recognized as a pan-allergen for its highly conserved amino acid sequence present in molds, animals, and plants(e9). MDH and GAPDH (s6 and s9) were also identified as allergenic proteins; they are extensively reported as allergens by ingesting plant-derived foods, pollen, and common molds(e10). In s11, a nuclear RNA export factor was identified, part of the pathogenesis-related protein family like PR-10, showing homology with a reported chickpea allergen(e11). Isoflavone reductases (s15 and s17), present in various pollen sources and foods, were identified(e12). Another protein identified with oxidoreductase activity was an aldo-keto reductase (s13) that was only linked to asthma and rhinitis in animal models(e13). In the same spot, two mutases were revealed, including a pollen development protein previously reported as an IgE-binding protein in other pollen sources(e14). Two pectinesterases (s9 and s11) were identified; both proteins were recognized as allergens in olive(e15). An alanine aminotransferase, previously reported as an IgE-binding protein in maize seeds, was identified (s3)(e16). Interestingly, we detected a 40S ribosomal protein (s20) associated with cross-reaction between mushroom allergies, which has not yet been reported in pollen(e17). Phenylcoumaran benzylic ether reductases, implicated in inducing cross-reactivity across various pollens, plants, and foods, were also detected (s6 and s15)(e18). The last immunoreactive spot was a RING-type domain-containing protein, showing similarity to aeroallergens found in wheat and grass. Interestingly, we identified five proteins not previously linked to allergic sensitization, including an aldo-keto reductase, an uncharacterized mitochondrial protein, a flavanone 3-hydroxylase, acetyl-CoA C-acetyltransferase, and an F-box domain protein. Although most identified proteins have been reported in previous work as IgE-binding proteins (Table 1), they require subsequent validation employing recombinant proteins to elucidate their role in developing respiratory allergies as IgE-binding proteins. Phaphia textilef (e22) Littorina littoref (e23) Delonix regia a 6 Cannabis sativaa (e24) Quercus rubra a 4 Phoenyx sylvestrisa (e12) Lantana camara a (e25) Cocos mucifera a 5 Carya illionoinensisa (e10) Pla a 6 a Amb a 12 a Art si 12 a Hev b 9 c Cyn d 22 a Alt a 6 a Cla h 6 a Asp f 22 a Sal s 2 f Tri a 34 a Per a 13 a Pan h 13 f Ole e 9 a Ole e 10 a Heb v 2 c Mus a 5 f Ole e 12a Bet v 6a Pyr c 5f Dau c 5f Cry j IFRa Cor a 6a Senecio a (e26) Plantago lanceolataa (e27) Mala f 4c Asp f 37a Pan h 10f Bet v 6a Ole e 12a Pyr c 5f Dau c 5f Cryptomeria japonica a (e28) Ole e 1a Syr v 1a Fra e 1a Aca f 1a Pro j 1a Ama r 1a Sal k 5a Che a 1a Koc s 1a Pla l 1a Lol p 11a Phl p 11a Ole e 11a Sal k 1a Act d 7f Plantago lanceolataa (e27) Cannabis sativaa (e10) Amb a 8a Ama r 2a Amb t 8a Aca f 2a Sal k 4a Lig v 2a Cap a 2f Fra a 4f Mal d 4f Pru p 4f Pyr c 4f Delonix regia a 6 Prosopis velutina a (e14) Alt a 5a Alt a 12a Asp f 8a Cla h 5a Fus c 1a Tri a 41a Phl p 5a An interesting finding was that only Ligustrum-sensitized patients co-sensitized to Fraxinus exhibited IgE-binding activity to recombinant Lig v 1 (Figure 1D–F), unlike those without Fraxinus co-sensitization. This observation was confirmed in experiments using sera from Fraxinus-sensitive patients (Figure S1). Lig v 1 was discovered using sera from olive-sensitized subjects, and it was believed to be a Ligustrum-specific allergen.2 These findings suggest that Fraxinus-sensitive patients recognize Lig v 1, while Ligustrum-allergic patients do not. Indeed, WB showed that 90% of Ligustrum-sensitized patients co-sensitized to Fraxinus, and 87.5% of Fraxinus-sensitized were positive to Lig v 1 in our validation experiments. The three patients with a negative result suffered mild allergic disease and were on no medication, suggesting mild disease. Alternatively, these patients could have been allergic to other Fraxinus allergens. Consistent with this observation, it has been reported that sensitized patients to Fraxinus bound Fra e 1 with a prevalence of 75%(e19). Similarly, olive-sensitive patients evaluated by skin tests showed a prevalence of recognizing different recombinant Ole e-like close to 80%, but never 100%(e20). The identified isoform of Lig v 1 by MS corresponded to Lig v 1.0102; we do not rule out that this isoform is prevalent in Mexican trees because this is the main isoform amplified in cDNA in our laboratory. As reported for pollens and foods, the isoforms could be relevant in the IgE-binding and immunogenicity(e21). A potential limitation of applying immunoproteomics is that sometimes, an allergen may co-migrate with other non-allergic proteins in the 2-DE gel, making it difficult to identify the immunoreactive allergen. In our study, however, 21 of the 26 allergen proteins have previously been identified as allergens, suggesting they may play an important role in allergy disease. On the other hand, immunoproteomics has shown to be an invaluable tool in allergen discovery. The finding that Ligustrum-sensitized patients co-sensitized to Fraxinus, but not those patients without Fraxinus co-sensitization, exhibited IgE-binding activity to recombinant Lig v 1 supports this observation. These results show that the recognition patterns of IgE-binding proteins in Ligustrum-sensitized subjects depend on their co-sensitivity to Fraxinus. To our knowledge, this is the first report demonstrating a differential pattern of allergen recognition in allergic patients polysensitized to Ligustrum pollen. Investigation: RPGdV & BMM; Methodology: RPGdV, JCVdR & JMMV; Formal analysis: JCVdR & JMMV; Conceptualization: LMT; Writing—Original Draft Preparation: RPGdV, JCVdR & JMMV; Writing—Review & Editing: JMMV & LMT. This work was supported by the National Institute of Respiratory Diseases Ismael Cosío Villegas (INER, Mexico). Raúl Porras-Gutiérrez-de-Velasco is a doctoral student from the Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and has received CONAHCYT fellowship number 882984. We greatly acknowledge the technical support of MS Emmanuel Ríos Castro from the Genomics, Proteomics, and Metabolomics Unit (LaNSE, Cinvestav-IPN, Mexico) for the processing and analysis of the samples by Mass Spectrometry. The authors declare that they have no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.