Comparison of natural and recombinant forms of the major fish allergen parvalbumin from cod and carp

Abstract

Allergic reaction following fish consumption can trigger life-threatening reactions in predisposed individuals. Parvalbumins from different species have been identified as the major fish allergens. There are two distinct phylogenetic lineages of parvalbumins, alpha and beta. Most allergic reactions are caused by beta-parvalbumins. We cloned and expressed cDNAs encoding cod (Gadus morhua) and carp (Cyprinus carpio) beta-parvalbumins and purified natural cod beta-parvalbumin. CD spectra of the purified proteins showed that their overall secondary structure contents were very similar. No differences in thermal stability were monitored in the calcium-bound or calcium-depleted form of natural cod parvalbumin. IgE reactivity was assessed using 26 sera of fish allergic patients from Spain, The Netherlands, and Greece in immunoblot and ELISA experiments. Twenty-five of the 26 patients with IgE reactivity to native and recombinant cod parvalbumin also reacted to the recombinant carp parvalbumin. IgE inhibition assays were performed using cod and carp extracts and purified recombinant parvalbumin of cod and carp. High crossreactivity among cod and carp parvalbumins was observed in immunoblots as well as in fluid phase assays. Natural and recombinant parvalbumins gave comparable results when performing various in vitro diagnostic assays.

High-Throughput Proteomics: A New Tool for Quality and Safety in Fishery Products

“…Allergenomic studies in seafood products have been only sparingly applied using SDS-PAGE and immunoblotting [105] or these techniques combined with MS [106,107]. Other scientists investigated the allergenic mechanisms of the parvalbumin fraction with myofibrillar protein fraction using crystallography [108] or immunological tools [109] which are not proteomic techniques in sensu stricto [110].…”

Characterization of Seafood Proteins Causing Allergic Diseases

“…Edman sequencing was in the past the only technique to identify the amino acid sequence of the N-terminus of seafood allergens, which is necessary to design the appropriate primers for isolating the relevant gene and deoxyribonucleic acid (DNA) sequencing (Ayuso et al, 2008;Hamada et al, 2004;Ma et al, 2008). The Edman reaction is performed on the protein N-terminus, where the amine side of the N-terminal amino acid reacts with phenylisothiocyanate (PITC) to form a phenylthiocarbamyl (PTC) protein.…”

“…Instrumentally, most of this unambiguous characteristic information was generated by coupling MALDI ion source to the TOF mass analyzer, which can give the molecular weight of the purified intact protein with a margin of error of 0.1% (DeHoffmann & Stroobant, 2007) and perform the PMF experiments. The precision of MALDI-TOF is poorer with larger proteins, which provoked to use more advanced ion sources like ESI coupled with FTICR (Ma et al, 2008). The high resolution of MS analyzers are used to study seafood authentication by measuring the exact molecular weight of a marker protein (Mazzeo et al, 2008).…”

“…This method was used in the seafood allergen studies as a confirmatory technique to study some biochemical and biophysical properties by collecting the amino acid sequence of a few peptides (Perez-Gordo et al, 2011). Ma et al (2008) validated the using of high resolution MS profiles to evaluate the cross-reactivity of the allergens based on their structural similarities. The cross-reactivity between both the natural and recombinant forms of cod and carp parvalbumin was evaluated using patients' sera and the structure was also evaluated using both circular dichroism (CD) and nuclear magnetic resonance (NMR) (Ma et al, 2008).…”

“…Ma et al (2008) validated the using of high resolution MS profiles to evaluate the cross-reactivity of the allergens based on their structural similarities. The cross-reactivity between both the natural and recombinant forms of cod and carp parvalbumin was evaluated using patients' sera and the structure was also evaluated using both circular dichroism (CD) and nuclear magnetic resonance (NMR) (Ma et al, 2008). In Helleur's group, the tandem MS technique was the method for de novo sequencing of seafood allergens.…”