Lipocalins are among the most important indoor/outdoor groups of animal allergens. For some, the protein structure has been resolved, but their functions are still elusive. Lipocalins generally display a low sequence identity between family members ¹ , but the lipocalin allergens are usually well preserved and can present similar patches that, in addition to serum albumins, may contribute to allergic cross-reactions among furry animals ^{2– 4} . Rodents, especially mice and rats, are cosmopolitan species present in rural, periurban, and urban areas, and are most often considered as pests. In addition, the presence of these species as pets in homes and their permanent use as animal models in research laboratories have allowed constant exposure to their allergens, which is an important source of sensitization ^{5, 6} .

So far, only one rat allergen has been described, Rat n 1, which is a lipocalin formed by two fractions, a prealbumin and an α-2U-globulin, secreted by the liver and found in high concentrations in urine, but also in saliva and fur ^{7, 8} . Among patients allergic to rats, 87% reacted to Rat n 1 in dust ⁹ . This molecule is glycosylated and, up to now, was known to have two isoforms: Rat n 1.01 (21 kDa) and Rat n 1.02 (17 kDa). Its structure is like a conventional lipocalin with eight antiparallel β chains forming a single beta sheet and an α helix to create a pocket for ligand binding, very similar to that of other allergens, such as Mus m 1, the mouse's main allergen, with a highly conserved identity ^{5, 10, 11} . Four regions with potential immunodominant T cell epitopes have been described, and three of these are co-localized with the conserved regions of lipocalin, similar to the epitopes found in Bos d 2. No B cell epitopes have been reported for Rat n 1 ¹² .

Although its structure and sensitization capacity have been well described, little is known about its biological functions and how these may be related to hypersensitivity mediated by an IgE measured response and cross-reactivity with the main allergens of the most common domestic animals, dogs, cats, and horses ^{13, 14} . Therefore, the objective of this study was to explore cross-reactive epitopes among Rat n 1 and homologues in domestic pets through an in silico approach.

Animal allergens remain an important cause of sensitization and allergic diseases. Rodents such as rats are invasive cosmopolitan species that move between urban, periurban, and urban areas looking for favorable habitats and resources. The allergenicity of these species was first observed in animal caretakers and was considered an important source of occupational sensitization, affecting up to 15% of people in European countries with an active scientific community ¹² . Besides exposure in occupational settings, rodent exposure also occurs in domestic environments, as was shown in inner-city children with asthma in the USA, where rat sensitization rates were 19–21% ¹⁹ . In contrast, a recent study from Europe reported a very low prevalence of sensitization to rodents of 0.59% in urban atopic populations without occupational exposure ⁵ . Rat n 1 is the largest allergen of this species and has been well characterized. This protein belongs to the lipocalin family, a transport protein of hydrophobic ligands as lipid signaling molecules. A first approach in epitope prediction on Rat n 1 was made by Bayard et al. ⁷ , using in silico tool Chou Fasman, however, it was impossible to determine epitopes. On the other hand, with the use of synthetic octapeptides an IgE linear epitope was mapped spanning sequence STRGNLDVAKLNG, reported in this study as LE4. Authors are clear in that discontinuous epitopes were impossible to identify using same technology.

Several major allergens are members of the lipocalin family, including those from the mouse (Mus m 1), dog (Can f 1 and Can f 2), cat (Fel d 4 and Fel d 7), horse (Equ c 1), cow (Bos d 2 and Bos d 5), hamster (Phod s 1), and rabbit (Ory c 1 and Ory c 4), among others ³ . The factors that give rise to so many lipocalins becoming inducers of allergy are unclear. Among the allergens, cross-reactivity has been observed, mainly in organisms to which people are most exposed, such as cats, dogs, and horses.

In this study, we observed by in silico analysis possible crossbet-reactivity between Rat n 1, Can f 6, Equ c 1, and Fel d 4, with 62% identity among sequences compared. In previous studies, we found a high conservation state among Rat n 1, Equ c 1, and Fel d 4 (60% identity) and described possible residues with antigenic potential ^{15, 20} . Nilson et al. ²¹ found cross-reactivity between Can f 6, Fel d 4, and Equ c 1 by inhibition assays, especially in residues located on positions 29 to 73, which showed the highest identity. In these positions, Rat n 1 also presented the highest identity with the other three lipocalins, and here we found a possible lineal epitope (LE4: Start 24 – End 36) that could explain the cross-reactivity. Jeal et al. ¹² studied a population of individuals exposed to laboratory rats to determinate the proliferative response of peripheral blood mononuclear cells to Rat n 1. They found four regions as possible immunodominant T cell epitopes, and three of them were localized within the conserved regions of the lipocalins. One was also found in our study as a possible lineal epitope (LE2: Start 91 – End 97), with a high identity with Mus m 1, Equ c 1, Can f 6, and Fel d 4. The homologous allergens may contribute to multisensitization and symptoms in individuals allergic to different animals. Also, cross-reactivity to T cell epitope was found between Can f 1 and human tear lipocalin ²² . This could support the autosensitization and increased inflammatory response mediated by T lymphocyte CD4+. Also, first T cell epitope predicted in this study shares identity with Bos d 2, a major allergen from cow ²³ . This can explain cross reactivity among rat and others allergenic sources, such as: Can f 1 and Equ c 1, which has been related to share identity to T epitope level ^{24, 25} . However, Bos d 2 has been characterized as a weak inducer of immunological response ²⁶ .

The docking simulations demonstrated that the Rat n 1 cleft is big enough to accommodate the whole fatty acid molecule. Determining the capacity to bind some ligands by allergens is critical to understand their allergenic capacity. For Bet v 1, an allergen with capacity to link hydrophobic ligands, it has been determined that ligands such as lipids, iron, and calcium modulate its allergenicity capacity ^{27, 28} . When Bet v 1 is properly loaded with iron, it can promote Th2 response. A similar property is reported for Pru p 3, a peach lipid transfer protein. Results reveal that the ligand is recognized by a type of cellular receptor called CD1d in the cell surface where the antigens appear, that is, substances able to provoke an immune system response to produce antibodies. CD1d is responsible for presenting lipid antigen to activating cells of the immune system called invariant natural killer T (iNKT) cells. Once activated, these iNKT cells produce substances that cause the characteristic symptoms of allergic disorders ²⁹ . Since many allergens transport diverse compounds, the discovery of Pru p 3 lipid-ligand as an adjuvant to promote allergic sensitization through its recognition by CD1d expression opened new horizons ²⁹ .

Of the lipocalin family, also, Mus m 1 has been characterized for pheromone linking. Experimental assays revealed that 2-sec-butyl-4,5-dihydrothiazole (SBT), 6-hydroxy-6-methyl-3-heptanone (HMH), and (±)-dehydro-exo-brevicomin (DHB) are ligands for Mus m 1. This was a first step in determining ligands with allergenic capacity ³⁰ . Also, some ligands could influence the stabilization of IgE conformational epitopes ^{27, 31} . Here, we predicted three. So, experimental assays are needed to determine the impact of ligands in Rat n 1 on inducing allergic responses. Resolution of 3D structure of Rat n 1 by X-ray, it helped to understand structural basis for linking ligands. According to Bocskei et al. ³² , residues Tyr24, Val58, Ala107 and Phe94, are critical to lipocalin activity. However, none of these residues was identified in docking assay.

For other allergens, such as Fel d 1, lipid ligands enhance TLR4 activation and innate immune signaling and promote airway hypersensitivity reactions in diseases such as asthma ³³ . For Bla g 4, a lipocalin from Blattella germanica (German cockroach), a capacity to bind hydrophobic ligands such as: tyramine and octopamine has been characterized ³⁴ . But residues involved in linking ligands in Rat n 1 are not conserved in Bla g 4, this is relevant because suggest a capacity to link different kinds of ligands in both allergens.

The outcomes of the current work include (1) a comprehensive understanding of the structure of Rat n 1 protein and structural similarities and differences between Rat n 1 and other lipocalins, and (2) a structural and molecular basis for the identification of epitopes responsible for cross-allergenicity between rat and domestic animal allergenic lipocalins. These epitopes may contribute significantly to designing rational strategies for diagnosis of and immunotherapy for domestic animal allergies.

All data underlying the results are available as part of the article and no additional source data are required.