Mapping Amyloid Regions in Gad m 1 with Peptide Arrays

Abstract

Amyloid formation is basically featured by a protein-protein interaction in which the reacting regions are the segments assembling into cross β-sheets. To identify these segments both theoretical and experimental tools have been developed. Here, we focus on the use of peptide arrays to probe the binding of several amyloid-specific probes such as the OC and A11 anti-amyloid conformation-selective antibodies and of monomers and preformed fibrils. These arrays use libraries containing partly overlapping peptides derived from the sequence of Gad m 1, the major allergen from Atlantic cod, which forms amyloids under gastrointestinal relevant conditions.

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

“…6a). Despite this similarity, the use of anti-amyloid conformational antibodies revealed structural differences among the amyloid aggregates 30 . Thus, gmPV1 and gmPV2 aggregates contained amyloid-fibril epitopes (OC reactivity), whereas those formed by sjPV2 contained solely βoligomer epitopes (A11 specificity) ( Fig.…”

“…Regions assembling into amyloids were predicted by AmylPred2, and they are depicted by arrows. Regions known to form amyloids in gmPV1 are depicted with pink arrows 25,30 .…”

“…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 .…”

Amyloid Assembly Endows Gad m 1 with Biomineralization Properties

“…The 109-residue chain is organized into three consecutive EF-hand motifs (AB, CD, and EF); only CD and EF contain acid loops with functional Ca 2+ binding. Regions B and D contain amyloid-promoting sequences, which are defined as sequences with alternating amyloid-forming and acid segments in the aggregated state; these sequences can function as Ca 2+ -nucleating centers for biomineralization events [ 26 , 27 ]. However, amyloids are polymorphic entities; studying their distinct aggregates could shed light on the features of the Ca 2+ -nucleating process.…”

“…In the cation-bound form, Gad m 1 displays a highly stable monomeric helical globular fold [ 24 ]. In contrast, removal of bound Ca 2+ at high protein concentrations triggers Gad m 1 amyloid aggregation through the regions forming helix B and helix D in the globular fold [ 25 , 26 , 27 ]. Once formed, amyloids partially dissociate into monomers upon Ca 2+ addition [ 25 ].…”