Molecular, proteomic and immunological characterization of isoforms of arginine kinase, a cross-reactive invertebrate pan-allergen, from the house dust mite, Dermatophagoides farinae

Abstract

Rationale Arginine kinases (AK) were recently identified as major allergens in shrimp and the Indianmeal moth. Analysis of the mite proteome (Bi et al., ICACI 2003) revealed the presence of AK in its extracts. In this study, we cloned and expressed the AK from Dermatophagoides farinae (Der f) and examined its allergenicity. Methods Mass spectrometry and expressed sequence tag catalogues were used to identify this pan-allergen from Der f. IgE binding frequencies of the recombinant Der f AK were evaluated using sera from 49 Der f sensitive individuals, with or without shrimp allergy. Results AK was among the most abundant protein identified from the mite proteome. Mass spectrometry results indicated the presence of at least two isoforms. De novo peptide sequencing showed that the isoforms differed by one amino acid substitution (Gly161Glu162 substituting Tyr161), changing the protein charge from pI 6.33 to 6.54 without changing its molecular weight. Full length cDNA clones (showing 78% homology to shrimp AK) were obtained and the recombinant proteins expressed in E. coli were found to bind IgE in 12/49 (24%) of the sera tested. Significantly higher proportion of shrimp allergic individuals were sensitized to Der f AK (39% vs 12%, p<0.05). The pattern of reaction and inhibition studies however suggests that AK from shrimp and dust mites have both unique and cross reactive epitopes. Conclusions This study reports the first instance of the identification of native allergen isoforms by de novo peptide sequencing and the IgE reactivity of pan-allergenic arginine kinases from dust mites.