## 3 Results and Discussion
### Identification of High-Confidence Allergen Protein Families in Mango  
While approximately 1500 proteins demonstrated strong evidence of allergenicity and over 5200 proteins were considered to pose a lower allergenic risk, we applied an additional filter (\(\geq\)70% identity across an 80 amino acid linear window and \(\geq\)80 known allergen hits) to refine the candidate list and better categorize protein families. This approach reduced the dataset to 63 high-confidence allergens and 185 proteins with moderate evidence (Figure 4, Table S2). Our results showed that several proteins belonging to the cysteine protease family were the second-highest category in the mango genome, which indicated a considerable risk of allergies (Figure 3B). Several cysteine protease protein isoforms were significantly matched with kiwi (_Actinidia deliciosa_) Act d1 allergen (Figure 4A). The top three cysteine protease proteins, which are accession XP_04488586.1, XP_044511549.1, and XP_044509143.1, were aligned with Act d1 using multiple sequence alignment (Figure S1) and showed \(>\)82% amino acid sequence identity with Act d1 (Figure 5A). Furthermore, amino acid sequence alignment reveals a highly conserved B-cell epitope region (Figure S2), and the epitope scores of these cysteine proteases were higher than those of the known Act d1 (Figure S2). A newly developed AI-based allergen prediction tool called pLM4 Alg [26] further cross-validated the allergen potential for these cysteine proteases and confirmed the significant allergenity comparable to Act d1. Cysteine proteases are increasingly recognized as clinically important plant food allergens, with several members of this protein family known to trigger IgE-mediated hypersensitivity reactions [27; 28]. These enzymes, which play key roles in plant defense and ripening, are structurally stable and resistant to gastrointestinal digestion--features that contribute to their heightened allergenic potential [27; 29]. Well-characterized examples include Act d 1 from kiwi, Ana c 2 from pineapple, and Cari p 1 from papaya, all of which have been associated with severe systemic allergic responses [27]. Their conserved structure and proteolytic function are believed to support both direct immune sensitization and disruption of epithelial barriers, increasing the likelihood of allergen exposure [30; 31]. The  
Figure 3: In silico allergenicity classification of 54,010 mango proteins. (**A**) Pie chart showing strong (\(n=1489\); 3%), weak (\(n=5277\); 10%), and no (87%) allergen evidence. (**B**) The bar graph shows the number of proteins in the mango genome that fall into the strong and weak categories of known allergen family proteins. high sequence identity and epitope similarity observed between mango cysteine proteases and Act d 1 point to a significant risk of cross-reactivity in sensitized individuals, aligning with the broader clinical significance of this allergen family. Lipid transfer proteins (LTPs), which are primarily low-molecular-weight heat-resistant proteins, are among the most well-characterized plant allergens that can be found in substantial quantities in the fruits [32]. Interestingly, our findings demonstrated that only two non-specific lipid-transfer proteins (LTPs), which correspond to accession numbers XP_044477456.1 and XP_044465367.1 in the mango genome, were identified as allergens with strong evidence. Despite being matched to known allergens Jug r 8 and Cry j LTP, these LTPs did not meet our high confidence criteria. On the other hand, among the weakly evident allergen list, a total of 24 LTPs met our high confidence criteria (Table S2). Among these LTPs, a non-specific lipid-transfer protein 1-like (XP_044503745.1) protein matched with 192 known LTPs, showing 71.2% identity in a linear 80 aa window and 85.7% identity in a 3D epitope. Notably, this mango LTP showed the greatest match with the chestnut (_Castanea sativa_) Cas s8 allergen (Table S2, Figure 4B). Figure 4: High-confidence mango allergen candidates identified using refined thresholds.