In Silico Analysis of the Cross-Reactivity of Pacific White Shrimp ( Litopenaeus vannamei ) Allergens with Other Arthropod Species

This study provided new data in the area of cross-reactivity for Pacific white shrimp (Litopenaeus vannamei). Although the cross-reactivity between the most prominent allergenic proteins of shrimp and other crustaceans has been extensively studied, few data are showing the frequency of arthropod-shrimp Litopenaeus vannamei cosensitization in an in silico analysis. A comparative analysis of “typical and non-typical” shrimp L. vannamei allergens with other species was achieved, revealing new allergens and previously characterized shrimp allergens, which validated the comprehensive identification approach used in this study. Importantly, up to 192, amino acid sequences were identified that had matches to shrimp L. vannamei allergens that matched allergenic proteins in mites, insects, fish, bacteria, mammals, birds, and plants.


Introduction
Allergic processes have become a public health problem. The increase in these diseases is mainly due to complications caused by environmental and/or food triggers, as well as genetic problems of patients to predisposition (atopy) [1].
The allergy caused by shrimp proteins (allergens) is one of the main ones in terms of crustaceans and can occasionally have serious/fatal episodes of an anaphylactic process [2]. The symptoms that stand out from the anaphylactic process caused by shrimp are angioedema, laryngospasm, urticaria, itching, anaphylaxis, gastrointestinal discomfort, such as vomiting, stomach pain, and diarrhea that are usually confused with infectious problems, so the diagnosis of food allergy it is not easy [2] [3]. Countries like Australia have the highest prevalence of IgE-mediated food allergy with around 18% of their population affected. On the contrary, England and the United States have estimated between 12% and 17% [4]. While in Mexico, it is estimated that about 16% of the population suffers from a food allergy, of which 8% -9% corresponds to an allergy caused by shellfish. The most affected population is children with 4.3% and adults with 3.8% [3] [4] [5].
In recent years, species belonging to the crustacean phylum have been characterized as potential allergenic sources. Sensitization to allergenic crustacean proteins is complicated by the highly reactive nature of some of these proteins [6]. One of the known allergens from crustaceans (including Pacific white shrimp) is tropomyosin. Tropomyosin is one of the allergens with the highest percentage of cross-reactivity reported between mollusks and crustaceans, but also with other invertebrates [6] [7]. This allergen is highly stable to heat treatment, maintaining its allergenicity. There is a diverse group of different isoforms that are found primarily in muscle cells. About 60% of shrimp-allergic patients react to this protein. And it is highly conserved among various species of crustaceans, such as shrimp, lobster, and crab with 95% -100% identity [7] [8] [9].
Cross-reactivity between proteins from different species is proposed as one of the mechanisms that would explain the immediate hypersensitivity reactions observed in individuals exposed to an allergenic source for the first time [9]. Although exact data are not available, it is suspected that this mechanism would be involved in patients allergic to shrimp with a hypersensitivity reaction when they consume this food for the first time [10] [11]. The structural homology (amino acids) that exists between allergens can result in binding to antibodies, mainly of the IgE type, resulting in a process known as cross-sensitization, which occurs during exposure to an allergen and causes an animal or person is hypersensitive to another protein and cross-reactivity takes place resulting in an allergic process [11]. Cross-sensitization of food allergens can cause non-sensitized allergen-specific IgE resulting in a false-positive test (e.g., skin prick test) that is extremely important for clinical practice and differential diagnosis [11] [12] [13].
IgE cross-reactivity to shrimp allergens is increasingly important, not only tropomyosin but also the other outstanding allergens (myosin light chain, arginine kinase, and sarcoplasmic calcium-binding protein) and the less studied (hemocyanin, troponin C, ubiquitin, triosephosphate isomerase among others) [13]. Although the cross-reactivity between the most prominent allergenic proteins of shrimp and other crustaceans has been extensively studied [14] [15], few data are showing the frequency of arthropod-shrimp sensitization in an in silico analysis; most epidemiologic analyses have not evaluated the clinical significance of such sensitization [15]. Therefore, the main objective of this study was to analyze in silico the structural cross-reactivity of Pacific white shrimp (Litopenaeus vannamei) allergens with other arthropod species, as well as superficially describing the possible form of sensitization: consumption vs. cross-reactivity and the clinical relevance of sensitization to shrimp in patients sensitized to other allergens. and 5 sequences for the rest of the allergens.

Cross-Reactivity Analysis between Shrimp Allergens and Allergens from Other Species
Sequence cross-reactivity of Pacific white shrimp allergens was determined using free tools such as Allermatch (http://allermatch.org/). This tool provides comparative data on cross-reactivity and IgE-binding properties of clinically important shrimp Litopenaeus vannamei allergens.
Allermatch contains the entries from three databases of known allergen pro-

Phylogenetic Analysis
The Molecular Evolutionary Genetic Analysis (MEGA) program, version X, was used to obtain phylogenetic trees, using the taxa method with Bootstrap support with 1000 repetitions as a measure of reliability and robustness under the assumption of an evolutionary minimum. In topology, this model uses a comparative matrix to find similarities between amino acid sequences to establish evolutionary proximity between species. The array was constructed with all amino acid sequences of the allergens retrieved from the UniProt database and reported to the WHO/IUIS from the Allermatch software. Alignment for phylogenetic analysis was carried out using CLUSTAL W, which performs alignments. The A total of 67 crustacean allergens were identified for cross-reactivity among the four major allergens (Lit v1 -v4) of white shrimp (L. vannamei) ( Figure 1 and Figure 2  (A2V731). As well as an identity of 93% -94% for the shrimp "Metapenaeus ensis" and the Malaysian jumbo shrimp "Macrobrachium rosenbergii" (Table S6, red color). With the discovery of cockroach tropomyosins, Blattella germanica (Q9NG56), Periplaneta fuliginosa (Q8T6L5), Periplaneta americana (P0DSM7), and Gromphadorhina portentosa (A0A4P8D340); as well as the moth "Galleria mellonella" (A0A4P8D330) that showed that they have identities greater than 44% with the TM of L. vannamei, resulting in a moderate reactivity for cross-linking of the IgE receptor to occur and a process of anaphylaxis. The same occurs for mites, Dermatophagoides farinae (Q23939) Sarcoptes scabiei (A0A1M4PIX0), and the mosquito "Aedes aegypti" (Q17H75) ( Table S7, yellow color). And finally, low or null identities were found for some parasites (e.g. Ascaris lumbricoides), insects (e.g. Tenebrio Molitor), and some mammals such as humans and pigs. The amino acid sequences found by the Allermatch software and those reported by the WHO/IUIS for tropomyosins were used for the analysis of the phylogenetic tree, revealing that the TMs of crustaceans are mostly very similar to each other and amphipods (small crustaceans) as well as some mites. In contrast, the TM sequences of some mammals, insects, and microorganisms appear to be more distantly related to the TM of shellfish including white shrimp (L.
For the arginine kinase (AK) allergen, several species of crustaceans were identified, including Penaeus monodon, Penaeus Chinensis, Crangon Crangon, and Scylla paramamosain. The 4 amino acid sequences for AK mentioned above together with that of L. vannamei were very similar to each other, with more than 65% identity (Table S7, red color), which indicated a high possibility of cross-reactivity. These sequences are also mildly like AK allergens from Periplaneta americana cockroaches (A1KY39) and the mites Dermatophagoides farinae (A0A088SAW4) and Dermatophagoides pteronyssinus (B2ZSY4) (30% -39% identity). In contrast, these were different from allergens from some plants such as Betula pendula and some mammals such as Bos Taurus (Table S6, blue color). As with TM, the amino acid sequences found for AK from crustaceans are most closely related to each other, as well as to insects and mites, but not closely related to mollusks ( Figure 4).
As for the myosin light chain (MLC) allergen, only 5 almost identical amino acid sequences were identified. Of the 5, 2 are from the same shrimp (Penaeus monodon) with 88-89% identity (high potential for cross-reactivity; Table S8). And a low cross-reactivity was obtained for the cockroach (Blattella germanica) and the rooster (Gallus gallus). Molecular phylogenetic tree analyses on the MLC distance between crustaceans and the shrimp Litopeneaus vannamei are closely related, even with some bacteria. However, they are not closely related to the MLCs of insects such as butterflies and moths ( Figure 5).
For the sarcoplasmic calcium-binding protein allergen, a very similar amino acid sequence (100% identity) was identified between the shrimp Litopenaeus vannamei (C7A639) and Penaeus monodon (E7CGC4). And a medium identity (from 34% to 53%) for the crustaceans Penaeus monodon (H7CHW2), Astacus Leptodactylus (P05946), Scylla paramamosain (I2DDG2), and the Crangon crangon (D7F1P9) (Table S10; yellow color). And, finally, an identity (low reactivity) of less than 3% was identified for the Aedes aegypti mosquito (Q16XK7) (  SCPs obtained by Allermatch and WHO/IUIS in a phylogenetic tree analysis showed that all edible crustacean SCPs are closely related to other species within the same phylum, but distantly related between classes of insects and bacteria ( Figure 6). No allergen sequences were found for comparison for mites or mammals.
For troponin C and pyruvate kinase allergens, amino acid sequences with low or null identities (less than 10%) were found, which would not indicate a low probability of cross-reactivity with these allergens and therefore that they can trigger a series of anaphylactic processes. The only thing that can be saved for these allergens is that an amino acid sequence with medium identity was found for troponin C of the shrimp Penaeus merguiensis (S5ZHH2) and pyruvate kinase a sequence of low identity with the shrimp Penaeus monodon (E7CGC5).
For the allergen thioredoxin, no matches were found.
As it was observed, the tropomyosin allergen of the shrimp L. vannamei is the one that has the highest number of coincidences with other species and is mostly high and medium identities (greater than 80% and 40%, respectively).  Which would confirm the theory or hypothesis that tropomyosin is the major shrimp allergen. Figure 8 describes the 6 shrimp (L. vannamei) allergens and some of the species that had coincidences in common. As can be seen, tropomyosin is the one with the highest identities (red, yellow, and brown), followed by arginine kinase and myosin light chain. And in lower percentages of identity troponin C and hemocyanin (blue color). This would not indicate that   tropomyosin, arginine kinase, myosin light chain, and sarcoplasmic calcium-binding protein from other species (mainly crustaceans) could trigger an anaphylactic response in people sensitized to Pacific white shrimp.

Discussion
As previously mentioned, there are already studies on the cross-reactivity of  [20].
With the increase in clinical reports on anaphylactic processes to shellfish, shrimp being the main source of allergies, as well as the cross-reactivity of people sensitized to shellfish and who react to other species, there is already an analysis of potential allergens [21]. The results obtained in the present study on typical and non-typical allergens of L. vannamei indicate that sensitization to shellfish may be difficult due to the highly reactive nature of some allergenic proteins [21]. As it was possible to observe, the allergens of L. vannamei have several similarities with other species, which would indicate that the cross-reactivity of the Pacific white shrimp is wide and therefore the possibility of carrying out a process of respiratory, gastrointestinal, and even hypersensitivity. Skin is high ( Figure 9).
Tropomyosin is the primary allergen for clinically reported cross-reactivity among shellfish, but also cockroaches, mites, and even parasites [22] [23]. As previously reported, tropomyosin has epitopes that are exclusive to IgE in a linear fashion and that are of great importance in determining the degree of cross-reactivity in different types of crustaceans [23]. This allergen is highly conserved in various crustaceans such as lobsters, crabs, and shrimp with identities between 95% and 100%. Thus, cross-reactivity of mainly IgE antibodies is quite frequent [24] [25]. The 100% demonstrated identity percentage between L. vannamei and L. monodon has been previously reported and validates the in silico approach used in this study. Furthermore, it was shown for the first time that crustaceans such as M. ensis and M. rosenbergii also have 100% amino acid sequence identity with the TM of L. vannamei. And L. aztecus and   [28]. It is important to mention that most of the tropomyosin that resulted from the Allermatch software and that have coincidences with the L. vannamei tropomyosin are potentially allergenic. According to the IUIS nomenclature, and is allergen is a protein from the same species, or family of species, with the same biological function and similar biochemical properties, including greater than 67% amino acid sequence identity and similar size [29]. The high identity (<70%) between the amino acid sequences of tropomyosins from crustaceans (Table S7, red color) and Pacific white shrimp indicates a high probability of immunological cross-reactivity. As already reported, an identity in the amino acid sequence greater than 70% would demonstrate a very probable possibility of binding IgE antibodies with cross-reactivity to these allergens [30] [31] [32].
Here we have demonstrated conclusive molecular data on the amino acid sequence similarity of a major allergen (tropomyosin) of Pacific white shrimp with other invertebrate species [28] [29] [30]. The allergen arginine kinase (AK) is a monomeric phosphagen ATP phosphotransferase that is generally found in invertebrates and is the key to energy metabolism. It has been identified in more than 6 crustaceans including shrimp. It is heat unstable, but IgE-AK binding has been shown in cooked shrimp, which may be due to intact IgE epitopes on expressed AK [33]. Amino acid sequence similarity is high among crustaceans (70% -100%), indicating that patients allergic to Pacific white shrimp sensitized to arginine kinase would likely react to the other crustacean species (L. monodon, L. chinensis, C. crangon, and S. paramamosain). Arginine kinase is considered a pan-allergen [34] [35] and is important among insects and mites. In our results, about 37% of the amino acid sequences that had coincidences with the AK of L. vannamei are from insects and mites. These species then represent a medium identity (>30%) for cross-reactivity to be carried out by parts of the allergic patients. In addition, the similarities of the arginine kinases found for Pacific white shrimp of effort indicate that they are potential variants of is allergens because their PI is greater than 90% [29].
Myosin light chain (MLC) allergen is found primarily in smooth muscle in complexes with myosin heavy chain motor domains. It has a molecular weight of 17 to 20 kDa and is heat stable [36]. Its form of sensitization is by ingestion, it regulates the function of smooth muscle when it is phosphorylated by the kinase. 50% of shellfish-allergic patients have cross-reactivity against this protein. Light chain myosin from L. vannamei was reported as an allergen with a molecular weight of 20 kDa and was named Lit v 3 [37] [38]. The very low amino acid sequence PI (<20%), MLC1, and MLC 2 are not considered isoforms but rather two different proteins [39]. We mention in this study for the first time that the MLC allergen has (low) similarities with some birds among them (Gallus gallus), in addition to the previously reported MLC 2 allergen in L. vannamei and L. monodon shrimp. Furthermore, this study also suggests that MLCs of the cockroach (Blattella germanica) are probably MLC 2, respectively, which explains the close molecular phylogenetic relationship with crustaceans, but not with insects. Finally, the sarcoplasmic calcium-binding protein (SCP) allergen is a protein that promotes muscle relaxation by translocating calcium from the myofibrils to the endoplasmic reticulum. It has a molecular weight of 20 to 22 kDa. It is thermostable and resists treatment with acids and alkanes. Until now, three isoforms are known (SCP-I, II, III) with isoelectric points of 5.05, 4.90, and 4.75 respectively [40] [41]. We identified 100% amino acid sequences for the shrimp Penaeus monodon implying the presence of SCP is allergens. However, the significantly average abundance among the other crustaceans (30% -50%) concludes that the amino acid sequence is relatively unlikely to cross-react by allergic patients. Other identified muscle regulatory proteins include the protein troponin. Troponin is made up of three subunits, suffixed C, I, and T, and troponin C is listed as an allergen. Our study first demonstrated this allergen [42], together with hemocyanin for medium cross-reactivity in L. merguiensis and L. monodon shrimp.
In addition to the identified amino acid sequences, as well as the comparison of known crustacean allergens with Pacific white shrimp (L. vannamei), another type of allergen repertoire was also identified in this study. About 15 new potential shrimp allergens were successfully identified with low matches such as RNA-dependent polymerase, vitellogenin, and aldehyde dehydrogenase among others. These three proteins are allergens recorded in different species of mites and insects [43] [44]. Clinical studies frequently report cross-allergic patient reactions to crustaceans as well as mites and insects, but it remains unclear whether shrimp sensitization occurs through the respiratory or gastrointestinal tract, or, in other words, whether it is a secondary phenomenon following mite/insect/nematode sensitization or a true primary allergy [44].
In addition, we found allergens that are possibly responsible for the crossreactivity between shellfish and fish. Aldose-A allergen was found in low identities for fish (Pangasianodon hypophthalmus), this allergen was not included in the main results of this study as they are phylogenetically distant to Pacific white shrimp. However, there is emerging evidence that patients suffering from shrimp allergy are also sensitized to pollen and fish allergens [45].

Conclusions
This study provides new data in the area of cross-reactivity for Pacific white shrimp (L. vannamei). A comparative analysis of "typical and non-typical" shrimp L. vannamei allergens with other species was achieved, revealing new allergens and previously characterized shrimp allergens, which validated the comprehensive identification approach used in this study.
From previous studies, it is known that shrimp is one of the main causes of allergy and that it causes greater sensitization in patients with a high risk of atopy; however, the relationship of sensitization between these invertebrates and their relevance has not yet been explored clinic. Therefore, the difference in the proteomic abundance of different allergens between arthropod species, including the shrimp L. vannamei, may have clinical and diagnostic importance. Im- portantly, up to 192, amino acid sequences were identified that had matches to shrimp L. vannamei allergens that matched allergenic proteins in mites, insects, fish, bacteria, mammals, birds, and plants. These include shrimp proteins that have a high probability of being potential allergens such as beta-enolase and aldolase-A; however, these have not yet been identified as true shrimp allergens. Future studies will have to evaluate the abundance of proteins of known and potential allergens of shrimp L. vannamei, as well as analyses that allow evaluating, through inhibition tests, the role of allergens to examine the binding capacity of IgE antibodies and thus show clinical sensitization in patients with a shellfish allergy and determine its allergenic potential.