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 structural 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:Despite their conserved primary structure and consequent IgE co-reactivity, the invertebrate tropomyosins displayed different protein stabilities. Pen m 1 and Ani s 3, but not Der p 10 and Bla g 7 elicited differential melting temperatures that were pH-dependent. Endolysosomal experiments demonstrated differential degradation, as a function of stability, generating different peptide repertoires.Pen m 1 T-cell clones, with specificity for sequences highly conserved in all four tropomyosins, did not proliferate with Der p 10, Bla g 7 and Ani s 3, indicating that these peptides were not naturally produced for other invertebrate tropomyosins. Conclusions:Our data suggest that, although invertebrate tropomyosins exhibit a high degree of IgE cross-reactivity due to conserved B-cell epitopes, they do not necessarily share identical cross-reactive T-cell epitopes. This is likely due to differential endolysosomal processing as a function of different structural stabilities. Word Count: 3619 14.Kavan D, Man P. MSTools-Web based application for visualization and presentation of HXMS data. Int J Mass Spectrom. 2011;302(1):53-58. 15.Reese G, Viebranz J, Leong-Kee SM, et al. Reduced allergenic potency of VR9-1, a mutant of the major shrimp allergen Pen a 1 (tropomyosin).

Mollusk allergy: Not simply cross‐reactivity with crustacean allergens

“…We recently showed that T-cell cross-reactivity among tropomyosins is not dependent on amino acid sequence similarity, but instead as a function of structural stability. 6 This supports our findings that tropomyosins from different invertebrate sources may produce cross-reactive antibodies, even when sharing low amino acid sequence similarity 7 Our findings suggest that primary sensitization to tropomyosin from mollusk is possible, and inverse clinical cross-reactivity may occur to crustaceans in molluskallergic individuals.…”

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 ). Data on the protein dynamics from aMD simulations indicated that the introduction of glycin 89 enhanced the flexibility in the alpha helix (91–96) adjacent to the epitope ( Figure 4 ) facilitating access to additional cleavage sites ( Figures 6C,D ) and/or binding to MHC II.…”

Scientific Opinion on development needs for the allergenicity and protein safety assessment of food and feed products derived from biotechnology

“…In addition to resistance to extracellular digestion by gastrointestinal proteases, the resistance to endosomal degradation (i.e. digestion within the antigen‐processing and presenting cells (APC) of the immune system, such as dendritic cells) and its relationship with a protein’s capability to act as an allergen has been less studied (Foster et al, 2013; Machado et al, 2016; Soh et al, 2019; Kamath et al, 2020). To be recognised as an allergen, exogenous antigens must first be internalised into the endosome of APC and then are subjected to endosomal degradation, where they are exposed to cathepsin proteases under increasingly acidic and reducing conditions.…”