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.

In silico Design of Phl p 6 Variants With Altered Fold-Stability Significantly Impacts Antigen Processing, Immunogenicity and Immune Polarization

“…This is in line with the enhanced susceptibility to proteases of L89G observed in the degradome assay. We have previously found that immunodominant peptides from Bet v 1.0101 or tropomyosin (55,62) are quickly degraded in the degradome assay, but nevertheless are effectively presented by BMDCs in vitro, supporting the concept of "bind first, cut later." Kim et al have shown that immunodominant epitopes are protected from further degradation by binding to MHC II, whereas epitopes sensitive to DM-mediated dissociation are destroyed by cathepsins (57,63).…”

Effects of Extraction Buffer on the Solubility and Immunoreactivity of the Pacific Oyster Allergens

“…The blocked-nitrocellulose membrane was incubated overnight with a pooled serum from five shellfish-allergic patients ( Table 2 ) diluted 1:20 in PBST with added casein. After the washing step, secondary anti-human IgE (1:10,000 dilution, DAKO Corporation, Lincoln, NE, USA) was added and incubated for 1 h. The membrane was subsequently incubated for 35 min with donkey anti-rabbit IgG antibody conjugated infrared IRDye 800CW (1:10,000 dilution, LI-COR, Lincoln, NE, USA), and IgE antibody binding was visualised using the Odyssey ® CLx Imaging System (LI-COR Biosciences, Mulgrave, VIC, Australia) [ 17 ]. IgE reactive spots were annotated to the protein profile on SDS-PAGE, and corresponding bands cut out, tryptic digested and analysed using mass spectrometry.…”

Abundance and Stability as Common Properties of Allergens

“…Even with this expanded understanding it is important to remember that exceptions will always exist. For instance, a survey of tropomyosin allergens from both food (Pen m 1, Shrimp; Ani s 3, fish parasite) and environmental (Der p 10, dust mite; Bla g 7, cockroach) sources revealed significant variation in both thermodynamic stability and endosomal degradation kinetics ( 110 ). However, neither measure of stability was found to correlate with allergenicity.…”