The amyloid fold of Gad m 1 epitopes governs IgE binding

Abstract

Amyloids are polymeric structural states formed from locally or totally unfolded protein chains that permit surface reorganizations, stability enhancements and interaction properties that are absent in the precursor monomers. β-Parvalbumin, the major allergen in fish allergy, forms amyloids that are recognized by IgE in the patient sera, suggesting a yet unknown pathological role for these assemblies. We used Gad m 1 as the fish β-parvalbumin model and a combination of approaches, including peptide arrays, recombinant wt and mutant chains, biophysical characterizations, protease digestions, mass spectrometry, dot-blot and ELISA assays to gain insights into the role of amyloids in the IgE interaction. We found that Gad m 1 immunoreactive regions behave as sequence-dependent conformational epitopes that provide a 1000-fold increase in affinity and the structural repetitiveness required for optimal IgE binding and cross-linking upon folding into amyloids. These findings support the amyloid state as a key entity in type I food allergy.

Reconstruction of fish allergenicity from the content and structural traits of the component β-parvalbumin isoforms

“…It must also be noted that K29, which is only present in sjPV1, was suggested to be part of an IgE conformational epitope in this protein 43 . All chains were produced as recombinant proteins and purified as Ca 2+ -bound forms using established protocols 25,45 .…”

“…Circular dichroism Spectroscopy. Circular dichroism (CD) measurements were performed with a Jasco J-820 spectropolarimeter (Jasco Inc., USA) as described previously 25,29 .…”

“…Fish β-PVs are made up of a single polypeptide chain of approximately 109 amino acids that contains three EF-hand motifs (AB, CD and EF), of which only two (the CD and EF) have functional cationbinding acid loops 3,4,16,17 . These chains display a complex pattern of IgE linear epitopes that involve intermotif spacers (residues 33-44 joining the AB and CD motifs, and 65-74 linking the CD and EF motifs), acid loops (residues 49-64 and 88-94 on the CD and EF motifs, respectively) and a salmonid-specific region (residues 1-18) 18,19,20,21,22,23,24,25 . Of these, the segment containing residues 33-44 has been described as highly immunoreactive in β-PV from several fish species 3,19,20,24,25 .…”

“…These chains display a complex pattern of IgE linear epitopes that involve intermotif spacers (residues 33-44 joining the AB and CD motifs, and 65-74 linking the CD and EF motifs), acid loops (residues 49-64 and 88-94 on the CD and EF motifs, respectively) and a salmonid-specific region (residues 1-18) 18,19,20,21,22,23,24,25 . Of these, the segment containing residues 33-44 has been described as highly immunoreactive in β-PV from several fish species 3,19,20,24,25 . Ca 2+ binding stabilizes the proteins in a helical globular fold, as shown by the 3D structures of cod (Gad m 1) and mackerel (Sco j 1) major β-PVs, among others 3,26,27 .…”

“…However, cation removal by either gastric pH or chelates at neutral pH triggers Gad m 1 aggregation into amyloid assemblies 25,29 . Gad m 1 amyloids are formed by the polymerization of the intermotif linker regions and cause the amplification of IgE binding 25,29,30 . This sequence and concentration-dependent conformational transition are not unique to this β-PV, having been described for a large number of proteins, some of which, such as β-lactoglobulin (Bos d 5), ovalbumin (Gal d 2) and κ-casein (Bos d 8), are food allergens 31,32 .…”