Lipocalins are one of the groups of allergens derived from domestic animals with clinical importance for the development of allergic responses. They have been characterized in different animals. With allergenic capacity characterized, little is known about the epitopes involved in allergic responses. Here, potential antigenic regions involved in cross-reactivity among lipocalins were explored through bioinformatics tools. The amino acid sequences of several lipocalins from different domestic animals (mouse, dog, cat, bull, hamster, horse and pig) were used to determine the degree of kinship by phylogenetic studies. Groups with highest phylogenetic relation were obtained by using MEGA software. 3D models of lipocalins not reported in the protein data bank were modeled by homology to identify potential antigenic regions compromised in the cross-reactivity of this group of allergens. The alignment of the entire database of allergenic lipocalins and the inferred maximum likelihood tree segregate lipocalins into five monophyletic clades (referenced here as A, B, C, D and E). According to the multiple pairing analyzes, group C (Fel d 4, Rat n 1 and Equ c 1) showed the highest degree of identity among their amino acid sequences (58%). The analysis of conserved and exposed residues showed that group C shares three antigenic regions that could potentially contribute to its cross-reactivity. Potential antigenic sites were identified for the generation of cross-reactivity between the different lipocalins analyzed in this study. These studies support the need to carry out directed mutagenesis tests to confirm their relevance in the allergenic capacity of lipocalins.
The WAO defines the allergy as “A hypersensitivity reaction initiated by immunological mechanisms” [
An example of these allergens is lipocalins, which represent the most important group of allergens coming from furry animals. In addition, the growing number and diversity of pets in homes has allowed the increase in lipocalin exposure, leading to an increase in sensitization [
The identification of allergenic lipocalins from domestic animals has been increasing in the last decade and among patients allergic to pets, co-sensitization to different animals is frequent [
The amino acid sequences of lipocalins from seventeen domestic animals were selected based on allergenic capacity reported. The sequences were obtained from the uniprot database (https://www.uniprot.org/) (
The program Molecular Evolutionary Genetic Analysis (MEGA) version 7 was used for the construction of the trees, using the method of reconstruction of Neighbor-Joining with support by Bootstrap with 500 replications as a measure of reliability and robustness under the assumption of minimal evolution in the topology, this model uses a comparative matrix to find the similarity between amino acids of seventeen sequences to establish the evolutionary proximity between the species. The matrix was constructed with all amino acid sequences of lipocalins retrieved from uniprot database and reported in WHO/IUIS Allergen Nomenclature Sub-Committee (http://www.allergen.org). Thus, the more positive identity values are found among the sequences, the greater their relationship will be and they will be located in closer positions in the tree. All empty spaces were eliminated (full deletions). From the global comparison and the homologies, the sum of the length of branches (SBL) will be presented, which will determine the number of nodes and the position of the same, including the “clusters” of the evolutionarily closest sequences. Due to the number of sequences used, no phylogenetic sub-analyzes were performed. The alignment for the phylogenetic analysis was carried out through the CLUSTAL W. program, which performs alignments.
Allergens | Allergenic sources | Uniprot |
---|---|---|
Bos d 2 | Bos domesticus Bos taurus (domestic cattle) | Q28133 |
Bos d 5 | Bos domesticus Bos taurus (domestic cattle) | P02754 |
Can f 1 | Canis familiaris (dog) | O18873 |
Can f 2 | Canis familiaris (dog) | O18874 |
Can f 4 | Canis familiaris (dog) | D7PBH4 |
Can f 6 | Canis familiaris (dog) | H2B3G5 |
Cav p 2 | Cavia porcellus (guinea pig) | F0UZ11 |
Cav p 3 | Cavia porcellus (guinea pig) | F0UZ12 |
Cav p 6 | Cavia porcellus (guinea pig) | S0BDX9 |
Equ c 1 | Equus caballus (domestic horse) | Q95182 |
Fel d 4 | Felis domesticus (cat) | Q5VFH6 |
Fel d 7 | Felis domesticus (cat) | |
Mus m 1 | Mus musculus (mouse) | P02762 |
Ory c 1 | Oryctolagus cuniculus (rabbit) | |
Ory c 4 | Oryctolagus cuniculus (rabbit) | U6C8D6 |
Pho s 1 | Phodopus sungorus (Siberian hamster) | S5ZYD3 |
Rat n 1 | Rattus norvegicus (Rat) | P02761 |
The models of those lipocalins not reported in the protein data bank, were made by homology. The Swiss-model server (https://swissmodel.expasy.org/) was used. The quality of the models was analyzed by ProSA-web. The models were refined in Deep-View (energy minimization and rotamer replacements). Its quality was evaluated by several tools, including the Ramachandran graphs, WHATIF, QMEAN4 index and energy values (GROMOS96 force field). The relative values of the area of accessible solvent (r-ASA) were determined by ASA-view. The lipocalin sequences were aligned to identify conserved residues. Those preserved and the residues accessible to the solvent (rASA > 0.25) were located in the 3D model to identify pooled areas (>4 residues) and possible cross-reactivity.
A total of 17 sequence were included in the analysis with 152 positions in the final dataset. The sum of the branch length for an optimal tree was 16.5. When analyzed the lipocalin sequences, it was found that they formed five nodes with the highest phylogenetic relationship among them. According to the analyzes, group A contains the highest number of phylogenetically related lipocalins including Cav p 6, Can f 6, Bos d 5, Mus m 1 and Can f 2. Meanwhile, the group D, threw only two members, Bos d 2 and Can f 4. The group A presents the greatest relationship among the groups (
The 3D models of those lipocalins not reported in protein data bank were constructed by modeling homology (with the exception of Bos d 2, Can f 2 and Equ c 1, were retrieved from SDAP database (http://fermi.utmb.edu/ ). All generated models show classic folding of lipocalins, following the pattern of eight antiparallel β strands and an α helix, which help to form a cavity for the union of lipid ligands (
Multiple alignments of the lipocalins belonging to the different groups obtained from the phylogenetic analyzes were made. Lipocalins from Group A lipocalins have 30% identity between their amino acid sequences (
Groups of lipocalins | Residues preserved and antigenic capacity |
---|---|
A (Can f 2 as reference) | E32, E33, S35, G36, D49, T51, T52, H63, D82, G83, Q84, S89, T91, L145, S146, Q148, S173, D177, R178, C179. |
B (Can f 1) | D27, S32, G33, K34, A42, D43, V56, K55, G66, G68, Y98, T99, D117, I120, G139, R140, G160, L161, N163, Q164, E165, I165, L166, E173, T174, E175. |
C (Equ c 1) | D30, I31, S32, K33, S35, G36, E37, Y39, E49, K50, E52, E53, N54, A65, L67, D68, N69, S71, N81, G82, E83, L84, K93, T94, E97, D98, Y95, D96, G97, K151, E152, E153, K157. |
D (Bos d 2) | G30, K32, Y36, N41, D43, K44, P50, G80, C89, E97, G100, I127, K129, E143, E154, R155, G156, P173, N175. |
E (Cav p 3) | L20, D21, S23, G28, D37, N38, G46, D59, G60, T67, D73, G74, C76, L82, K85, Q86, R88, Q95, Y96, I105, A103, T119, K127, R135, L138, T139, E141, K145, G154, P156, Q171. |
For group B, a 28% identity was found between the amino acid sequences of the lipocalins analyzed (
In the present study, we describe potential antigenic regions shared among several lipocalins from domestic animals. Identification of epitopes is crucial to determine the role of cross-reactivity in sensitization to different allergenic sources. Several studies tested cross reactivity in this group of allergens [
Nilsson et al, found cross reactivity between Can f 6, Fel d 4 and Equ c 1, when inhibition assays were performed [
Bos d 2 has been characterized as a weak immunogen. Experimental studies in mice revealed that contains a T cell epitope located in C-terminal region and its amino acid sequence share homology with Can f 1 and Rat n 1 allergens [
In conclusion, we were able to identify some potential antigenic sites among some lipocalins; however, there is a low identity between these proteins from different species which shows that although cross-reactivity between them is possible, their frequency in most cases is low. These studies support the need to carry out mutagenicity tests to confirm their relevance in the allergenic capacity of lipocalins.
JS participated in its design and coordination and helped to draft the manuscript. AS and YE participated in the design of the study. MM conceived of the study and performed in silico analysis. All authors read and approved the final manuscript.
The authors declare no conflicts of interest regarding the publication of this paper.
Marlon, M., Andres, S., Jorge, S. and Yuliana, E. (2018) In Silico Analysis of Cross Reactivity between Lipocalin of Domestic Animals. Open Journal of Immunology, 8, 97-106. https://doi.org/10.4236/oji.2018.84006