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 (T_m 42°C) and Der p 10 (T_m 44°C) 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.

Keywords: T cell; cross-reactivity; endolysosomal degradation; shrimp allergy; tropomyosin.

Figure 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 shrimp‐allergic patients (1‐17) and one healthy donor (C1) to demonstrate presence or absence of IgE co‐sensitization to invertebrate tropomyosins

Figure 2

Structural characterization of tropomyosins. Analysis of thermal stability of Pen m 1, Der p 10, Bla g 7, and Ani s 3 using (A) CD spectroscopy, observing changes in MRE at 222 nm, and (B) first derivative of MRE from 15 to 85°C. Analysis of thermal stability by using (C) DSF and (D) observing changes in heat capacity using (DSC) at pH 5.2 (dotted line) and pH 7.4 (solid line). E, Analysis of tropomyosins by SEC‐MALS indicated molar masses consistent with essentially full occupancy of dimer at pH 7.4. The RI chromatograms on the left axis (thin lines) while the evaluated molar masses shown with the right axis (thick horizontal lines)

Figure 3

Endolysosomal degradation of tropomyosins using the microsomal fraction of murine dendritic cells (JAWS II) to mimic degradation in antigen‐presenting cells. Coomassie‐stained SDS‐PAGE gel of time‐dependent digestion products of tropomyosins over 48 h; lane 1 indicates molecular ladder (A). Mass spectrometric sequencing of endosomal digestion‐generated tropomyosin peptides mapped against the whole amino acid sequence (numbered 1‐284) for tropomyosins under acidic condition at pH 5.2 (B) and pH 4.5 (C). Peptides generated at various time‐points are depicted in various colors (see color key). Regions corresponding to the immunoreactive Pen m 1 peptides; 67 and 82 are highlighted (dashed boxes)

Figure 4

Peptide heat map depicting the intensity at pH 5.2 (A) and speed (B) of generation of tropomyosin‐derived peptides during the endolysosomal degradation assay under acidic conditions. The heat map indicates the position and abundance of the generated peptides as sequenced by mass spectrometric analysis, mapped against the full‐length amino acid sequence of the different tropomyosins

Figure 5

Murine T‐cell epitope mapping of Pen m 1. Immunoreactive regions of Pen m 1 were mapped using overlapping 15‐mer peptides with an offset of 3 amino acids. A, Proliferating T‐cells were analyzed using CFSE dye‐dilution method. B, IL‐2 release in the supernatant was measured using ELISA. Data are shown as mean with standard error of mean (SEM) for three replicate cultures. The cutoff of three standard deviations above mean reactivity of medium‐only control wells is indicated by the dotted line (A, 3.98% proliferating cells or B, 103.2 pg/mL IL‐2). C, A model of tropomyosin representing the amino acid sequence conservation between Pen m 1, Der p 10, Bla g 7, and Ani s 3 generated using ConSurf conservation model in Chimera. The red shaded regions indicate the two T‐cell reactive regions of Pen m 1; peptide 67 and 82 selected for this study that are also highly conserved among the four allergenic tropomyosins

Figure 6

Murine T‐cell cross‐reactivity 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