Solution and high-pressure NMR studies of the structure, dynamics, and stability of the cross-reactive allergenic cod parvalbumin Gad m 1

Moraes, Adolfo H.; Ackerbauer, Daniela; Kostadinova, Maria; Bublin, Merima; Oliveira, Guilherme R.; Ferreira, Fátima; Almeida, Fábio C. L.; Breiteneder, Heimo; Valente, Ana Paula

doi:10.1002/prot.24664

Cited by 25 publications

(27 citation statements)

References 43 publications

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“…Therefore, β-parvalbumin chains that are unable to form amyloids share a diminished IgE interaction. Third, the overlap of epitopes and amyloid folds in the AB motif, which is a hot spot for conformational exchange and lacks Ca 2+ -binding properties, suggests that amyloids could also form in the presence of Ca 2+ as a function of the chain sequence5354. Therefore, unless specifically removed by centrifugation, β-parvalbumin solutions may contain traces of amyloid species that, as function of their analytical use, could mislead reactivity assignments.…”

Section: Discussionmentioning

confidence: 99%

The amyloid fold of Gad m 1 epitopes governs IgE binding

Sánchez

Martínez

Castro

et al. 2016

Sci Rep

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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.

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Section: Discussionmentioning

confidence: 99%

The amyloid fold of Gad m 1 epitopes governs IgE binding

Sánchez

Martínez

Castro

et al. 2016

Sci Rep

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“…Interaction of [ 15 N- 13 C]-labeled rGad m1 and scFv-gco9 was monitored by analysis of 1 H- 15 N HSQC experiments. The production and the solution structure of the [ 15 N- 13 C]-labeled rGad m 1 was previously published by our groups [ 23 ].…”

Section: Methodsmentioning

confidence: 99%

“…1*| ΔδN| , where |ΔδH| and Δδ N| are the CSP of 1 H and 15 N nuclei. The molecular dynamics of free Gad m 1 and Gad m 1:scFv-gco9 were monitored by 15 N backbone relaxation experiments measuring the transversal (R 2 ) and longitudinal (R 1 ) relaxation rates measured as described in [ 23 ].…”

Section: Methodsmentioning

confidence: 99%

A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope

et al. 2015

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Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients’ sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.

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“…The protein structure of cod parvalbumin was first modeled on the basis of the X‐ray structure from carp parvalbumin. Recently, the NMR‐based protein structure was published revealing important characteristics on the folding and stability of parvalbumins . Gad m 1 has a six α‐helical protein fold which is a common feature of parvalbumins (Fig.…”

Section: The Proteinmentioning

confidence: 99%

EAACI Molecular Allergology User's Guide

Matricardi

Kleine-Tebbe²,

Hoffmann

et al. 2016

Pediatric Allergy Immunology

724

870

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The availability of allergen molecules ('components') from several protein families has advanced our understanding of immunoglobulin E (IgE)-mediated responses and enabled 'component-resolved diagnosis' (CRD). The European Academy of Allergy and Clinical Immunology (EAACI) Molecular Allergology User's Guide (MAUG) provides comprehensive information on important allergens and describes the diagnostic options using CRD. Part A of the EAACI MAUG introduces allergen molecules, families, composition of extracts, databases, and diagnostic IgE, skin, and basophil tests. Singleplex and multiplex IgE assays with components improve both sensitivity for low-abundance allergens and analytical specificity; IgE to individual allergens can yield information on clinical risks and distinguish cross-reactivity from true primary sensitization. Part B discusses the clinical and molecular aspects of IgE-mediated allergies to foods (including nuts, seeds, legumes, fruits, vegetables, cereal grains, milk, egg, meat, fish, and shellfish), inhalants (pollen, mold spores, mites, and animal dander), and Hymenoptera venom. Diagnostic algorithms and short case histories provide useful information for the clinical workup of allergic individuals targeted for CRD. Part C covers protein families containing ubiquitous, highly cross-reactive panallergens from plant (lipid transfer proteins, polcalcins, PR-10, profilins) and animal sources (lipocalins, parvalbumins, serum albumins, tropomyosins) and explains their diagnostic and clinical utility. Part D lists 100 important allergen molecules. In conclusion, IgE-mediated reactions and allergic diseases, including allergic rhinoconjunctivitis, asthma, food reactions, and insect sting reactions, are discussed from a novel molecular perspective. The EAACI MAUG documents the rapid progression of molecular allergology from basic research to its integration into clinical practice, a quantum leap in the management of allergic patients.

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Solution and high-pressure NMR studies of the structure, dynamics, and stability of the cross-reactive allergenic cod parvalbumin Gad m 1

Cited by 25 publications

References 43 publications

The amyloid fold of Gad m 1 epitopes governs IgE binding

The amyloid fold of Gad m 1 epitopes governs IgE binding

A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope

EAACI Molecular Allergology User's Guide

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