Effect of structural stability on endolysosomal degradation and T‐cell reactivity of major shrimp allergen tropomyosin

Abstract Background Tropomyosins are highly conserved proteins, an attribute that forms the molecular basis for their IgE antibody cross‐reactivity. Despite sequence similarities, their allergenicity varies greatly between ingested and inhaled invertebrate sources. In this study, we investigated the relationship between the structural stability of different tropomyosins, their endolysosomal degradation patterns, and T‐cell reactivity. Methods We investigated the differences between four tropomyosins—the major shrimp allergen Pen m 1 and the minor allergens Der p 10 (dust mite), Bla g 7 (cockroach), and Ani s 3 (fish parasite)—in terms of IgE binding, structural stability, endolysosomal degradation and subsequent peptide generation, and T‐cell cross‐reactivity in a BALB/c murine model. Results Tropomyosins displayed different melting temperatures, which did not correlate with amino acid sequence similarities. Endolysosomal degradation experiments demonstrated differential proteolytic digestion, as a function of thermal stability, generating different peptide repertoires. Pen m 1 (Tm 42°C) and Der p 10 (Tm 44°C) elicited similar patterns of endolysosomal degradation, but not Bla g 7 (Tm 63°C) or Ani s 3 (Tm 33°C). Pen m 1–specific T‐cell clones, with specificity for regions highly conserved in all four tropomyosins, proliferated weakly to Der p 10, but did not proliferate to Bla g 7 and Ani s 3, indicating lack of T‐cell epitope cross‐reactivity. Conclusions Tropomyosin T‐cell cross‐reactivity, unlike IgE cross‐reactivity, is dependent on structural stability rather than amino acid sequence similarity. These findings contribute to our understanding of cross‐sensitization among different invertebrates and design of suitable T‐cell peptide‐based immunotherapies for shrimp and related allergies.


| INTRODUC TI ON
The tropomyosin protein family is one of the largest allergen families containing over 60 identified and characterized allergens. 1 Tropomyosin exhibits a high degree of structural conservation between species. 2 Several studies have shown clinical cross-reactivity between crustaceans, mollusks, insects, mites, and nematodes is due mainly to shared IgE (B-cell) epitopes of tropomyosin. [3][4][5] However, there is a lack of understanding whether this high degree of structural and sequence conservation among tropomyosins would also lead to cross-reactive T-cell epitopes. Currently, T-cell epitopes have only been elucidated for shrimp tropomyosin with little or no data for other allergenic sources, making analysis of T-cell cross-reactivity challenging. [6][7][8] In the tropomyosin family, crustacean and mollusk shellfish tropomyosins are major allergens, particularly shrimp tropomyosin (Pen m 1), with more than 80% of shrimp-allergic patients being sensitized to this allergen. These major food allergens have been shown to be extremely thermostable and to withstand food-processing activities. 9,10 Some other invertebrate tropomyosins from sources such as mites and insects are considered only as minor allergens.
Tropomyosins are alpha-helical coiled-coil proteins that generally exist as stable dimers. Structural stability of allergenic proteins has been shown to have a direct impact on their allergenicity through differential endolysosomal degradation and subsequent generation of allergen-derived peptides for MHC Class II presentation. 11 It remains unclear, whether the different structural properties of the elicited similar patterns of endolysosomal degradation, but not Bla g 7 (T m 63°C) or Ani s 3 (T m 33°C). Pen m 1-specific T-cell clones, with specificity for regions highly conserved in all four tropomyosins, proliferated weakly to Der p 10, but did not proliferate to Bla g 7 and Ani s 3, indicating lack of T-cell epitope cross-reactivity.

Conclusions:
Tropomyosin T-cell cross-reactivity, unlike IgE cross-reactivity, is dependent on structural stability rather than amino acid sequence similarity. These findings contribute to our understanding of cross-sensitization among different invertebrates and design of suitable T-cell peptide-based immunotherapies for shrimp and related allergies.

K E Y W O R D S
cross-reactivity, endolysosomal degradation, shrimp allergy, T cell, tropomyosin

G R A P H I C A L A B S T R A C T
Allergenic tropomyosins have dissimilar structural stabilities despite having high amino acid sequence similarity. Tropomyosins are differentially degraded in the endolysosomal compartment of antigen-presenting cells due to differences in their structural stability.
Tropomyosin T-cell cross-reactivity, unlike IgE antibody cross-reactivity, is dependent on structural stability rather than amino acid sequence similarity.
various closely related tropomyosins also would affect their T-cell cross-reactivity.
In this study, four allergenic tropomyosins, from shrimp (Pen m 1), house dust mite (Der p 10), cockroach (Bla g 7), and Anisakis (food borne parasite) (Ani s 3) were investigated for their thermal and proteolytic stability, and their processing into peptides was analyzed in vitro and in vivo. T-cell cross-reactivity of Pen m 1-derived peptides to other tropomyosins was assessed in a murine model. The tropomyosins, although heat-stable, showed different melting temperatures that were pH-dependent. Exposure to endolysosomal proteases demonstrated that tropomyosins with similar thermal stabilities also showed a similar speed and pattern of degradation. In a murine model using Pen m 1 peptide-specific T-cell clones with conserved sequence identity, we found only limited T-cell cross-reactivity with Der p 10, and none with Bla g 7 and Ani s 3. Highly conserved invertebrate tropomyosins may share IgE epitopes leading to clinical cross-reactivity; however, presence of shared identical T-cell epitopes seems to be dependent on similarities in structural stability as opposed to amino acid sequence identity. Our findings have implications for understanding possible modes of sensitization as well as the design of suitable tropomyosin preparations for specific immunotherapy for shrimp and related allergies.

| Patient recruitment and IgE binding analysis
To analyze IgE binding to the different tropomyosins, 17 shellfishallergic patients were recruited at The Alfred Hospital Allergy Clinic, Melbourne, Victoria, Australia, with a positive serum shrimp-specific IgE (≥0.35 kU/L; ImmunoCAP ® , Phadia). Serum from a nonatopic donor recruited at the Translational Research Facility, James Cook University, was used as a negative control (Table S1). Written informed consent was obtained from all participants, and patient anonymity was preserved. Ethics approval was obtained from the Ethics Committees of James Cook University (Project numbers H4313 and H6829), The Alfred Hospital (Project number 192/07), and Monash University (MUHREC CF08/0225). IgE recognition of the different tropomyosins was investigated using grid immunoblotting 12 as described in Appendix S1.

| Biophysical characterization of allergenic tropomyosins
The alpha-helical structure and thermal denaturation of tropomyosins were compared and analyzed using circular dichroism (CD) spectroscopy. The effects of different pH conditions on protein melting temperatures was analyzed using differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF). Protein molecular mass under different pH conditions was determined using size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS). The detailed methodology for biophysical characterization of tropomyosins is given in the Appendix S1.

| Endolysosomal degradation of tropomyosins
Endolysosomal degradation assays were performed as described previously. 13 Briefly, 5 µg of purified protein was mixed with 8 µg of isolated microsomal fraction from the JAWSII cell line in 50 mmol/L citrate buffer (pH 5.2 or pH 4.5) and 2 mmol/L dithiothreitol. Bet v 1 was used as a nontropomyosin control allergen, and to confirm in vitro degradation assay condition reproducibility. Degradation was monitored over time using SDS-PAGE and Coomassie staining. The pool of peptides generated in the degradation assay was assessed by mass spectrometry using a Q-Exactive Orbitrap Mass Spectrometer (Thermo Fisher Scientific) and nano-HPLC (Dionex Ultimate 3000, Thermo Fisher Scientific). Detailed methods are provided in Appendix S1. To visualize the digestion-generated peptide data, the peptide sequences were mapped against the full-length amino acid sequences of the respective tropomyosins using MS tools. 14 The speed and intensity of peptide generation were visualized using plot.ly.

| Generation of Pen m 1 overlapping peptide library and mapping of murine T-cell epitopes of Pen m 1
To map the T-cell epitopes of Pen m 1, an overlapping peptide library was generated with 15-mer peptides with an offset of three amino acids spanning the entire length of Pen m 1 (Mimotopes). BALB/c mice were immunized with whole Pen m 1, and the splenocyte proliferation assay was performed using the overlapping peptide library.
T-cell reactive regions were mapped, based on CFSE-based T-cell proliferation and IL-2 release as correlates. The detailed methodology is provided in Appendix S1. were chosen based on (a) positive T-cell proliferation to these peptides and (b) high amino acid sequence similarity to corresponding regions in Der p 10, Bla g 7, and Ani s 3 ( Figure S1). The Pen m 1specific hybridomas were used as a tool to investigate whether the different tropomyosins share cross-reactive T-cell epitopes at these regions. The detailed methodology for peptide immunization and generation of hybridomas is provided in Appendix S1. C5 were incubated overnight with 2 × 10 4 GM-CSF BMDCs and concentrations of 50, 10, or 2 µg/mL of Pen m 1, Der p 10, Ani s 3, and Bla g 7 respectively, in triplicate. Control wells received either medium alone, peptides 67 and 82 at 10 µg/mL, or an equal volume of peptide diluent (DMSO). Culture supernatants thereof were removed and analyzed for IL-2 production as a correlate for T-cell activation using an ELISA MAX mouse IL-2 set (BioLegend).

| Allergenic tropomyosins are highly conserved proteins with frequent IgE co-sensitization in shrimpallergic patients
The tropomyosins selected for this study have a high degree of conservation with 70% or more amino acid sequence identity ( Figure 1A,B). These allergens were expressed in a bacterial expression system and purified as recombinant proteins using affinity and size exclusion chromatography F I G U R E 1 Invertebrate tropomyosins investigated in this study. A, A homology model of tropomyosin displaying the alpha-helical coiled-coil structure using a ribbon/space-fill model (orange) and the patterns of sequence conservation shown using ConSurf model. B, A phylogenetic tree and percent identity grid for invertebrate tropomyosins investigated in this study. C, SDS-PAGE Coomassie-stained gel profile of purified tropomyosins. D, Multiple sequence alignment using Clustal Omega algorithm of Pen m 1, Der p 10, Bla g 7, and Ani s 3 showing conserved amino acid residues. Pen m 1 IgE-binding epitopes are denoted by red boxes. 15 Yellow boxes indicate Pen m 1 peptides 67 and 82 selected for T-cell cross-reactivity experiments. E, IgE grid immunoblotting using serum from shrimpallergic patients (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) and one healthy donor (C1) to demonstrate presence or absence of IgE co-sensitization to invertebrate tropomyosins  Figure 1C). A multiple sequence alignment of the four tropomyosins revealed the high degree of conservation in the IgE epitopes of shrimp tropomyosin as characterized previously ( Figure 1D). 15 Five out of eight IgE epitopes from Pen m 1 were highly conserved in Der p 10, Ani s 3, and Bla g 7. To evaluate IgE antibody co-sensitization to the different tropomyosins, IgE grid immunoblotting was performed using sera from shrimp-allergic patients ( Figure 1E). Thirteen out of 17 subjects demonstrated IgE binding to all tropomyosins. Only one subject showed mono-sensitivity to Pen m 1. Interestingly, 8/17 subjects had stronger IgE binding to Der p 10 as compared to Pen m 1 based on densitometric analysis.

| Tropomyosins have differential structural stability and pH-dependent aggregation
Using CD spectroscopy, mean residual ellipticity (MRE) at 222 nm was monitored. Pen m 1 and Der p 10 had similar melting temperatures (inflection points) of 42°C and 44°C, respectively. Although Bla g 7 showed a higher melting temperature (63°C), loss of alpha-helical structure was initially observed from as early as 40°C (Figure 2A,B).
Interestingly, Ani s 3 showed the lowest melting temperature of 33°C with nearly complete loss of alpha-helical structure by 50°C.
DSF and DSC analysis of the tropomyosins confirmed similar melting temperatures for Pen m 1 and Der p 10, as well as the low melting temperature for Ani s 3 at neutral pH as compared to other tropomyosins ( Figure 2C, 2D).
The effect of two different pH conditions on melting temperatures was analyzed by DSC ( Figure 2D). The specific heat capacity (Cp) change during protein denaturation (mcal/°C) was measured from 10 to 85°C for the invertebrate tropomyosins at

| Allergenic tropomyosins demonstrate differential pH-dependent protease-mediated degradation
The in particular was stable to enzymatic digestion at this lower pH, with a native-sized protein persisting even after 48 hours ( Figure 3A).
Interestingly, the degradation patterns over time suggested uncoiling of the alpha-helical structure during temperature-dependent denaturation at lower pH as shown by DSC and CD analysis, correlating with more rapid peptide generation than for the other tropomyosins.

| D ISCUSS I ON
Tropomyosin is a highly conserved structural protein that has been the central focus of immunodiagnostic and therapeutic developments for shrimp allergy. Tropomyosin is the primary sensitizer in various crustacean and mollusk species, and a key player in our understanding of clinical cross-reactivity in shrimp-allergic patients on exposure to other invertebrate sources such as insects, 16,17 mites, 18 nematodes, 19 and more recently, even vertebrates such as fish. 20,21 However, it remains to be elucidated whether other invertebrate tropomyosins can also cross-react on a T-cell level.
In this study, we sought to understand the fundamental relation- (closely related to Pen m1 by sequence similarity), which showed highest stability among the tested tropomyosins, but showed lower resistance to proteolytic degradation that was similar to the least stable Ani s 3. Pen m 1 and Der p 10, which showed very similar thermodynamic stability, also displayed similar degradation patterns under both pH conditions. We concluded that similar structural stabilities rather than amino acid sequence similarities resulted in specific endolysosomal degradation patterns and peptide generation.
To further investigate whether differences in biophysical and immunochemical properties of tropomyosins would impact T-cell reactivity, murine T-cell cross-reactivity analysis was performed.

F I G U R E 6
Murine T-cell crossreactivity to allergenic tropomyosins. T-cell cross-reactivity of Pen m 1, Der p 10, Bla g 7, and Ani s 3 to T-cell hybridoma clones specific for peptide 67 and 82 was analyzed by measurement of IL-2 production upon exposure to protein, peptide controls or medium. Data are shown as mean with standard error of mean (SEM) for three replicate cultures Murine T-cell clones were generated specifically against regions that were T-cell reactive in Pen m 1 and highly conserved among all four tropomyosins. When BMDCs were cultured with these T-cell clones together with the four tropomyosins, Bla g 7 and Ani s 3 did not induce any significant response. Der p 10-induced T-cell stimulation only at the highest tested concentration. Clone 67-1.
A2 did not exhibit any stimulation even on exposure to whole Pen m 1. Surprisingly, peptide 67 was able to induce stimulation in T-cell clone 82.3.C5 indicating T-cell receptor (TCR) cross-reactivity between these two internal regions of Pen m 1. A single TCR has been shown to recognize more than one specific peptide. 25,26 We conclude that peptide 67 was only identified in the initial screening using whole Pen m 1 due to its cross-reactivity with the immunodominant region 244-254, as T-cell clones specifically raised against peptide 67 fail to be stimulated by all tested tropomyosins. In summary, invertebrate tropomyosins do not share similar stabilities as a function of amino acid sequence similarity. Protein stability differences may be the prime reason for differential degradation and antigen presentation of various allergenic tropomyosins, leading to generation of nonidentical T-cell epitopes. Our study concludes that T-cell cross-reactivity among tropomyosins may be associated to their structural stability rather than amino acid sequence similarity, which is more the case for IgE cross-reactivity of allergens. 4 Shellfish allergy, particularly to shrimps, affects more than 3% of the population and is frequently associated with life-long sensitivity and severe allergic reactions. [31][32][33] Exposure to other invertebrate sources such as house dust mites or insect-based food sources 34,35