Proteomic and immunochemical characterization of glutathione transferase as a new allergen of the nematode Ascaris lumbricoides

Abstract

Helminth infections and allergy have evolutionary and clinical links. Infection with the nematode Ascaris lumbricoides induces IgE against several molecules including invertebrate pan-allergens. These antibodies influence the pathogenesis and diagnosis of allergy; therefore, studying parasitic and non-parasitic allergens is essential to understand both helminth immunity and allergy. Glutathione transferases (GSTs) from cockroach and house dust mites are clinically relevant allergens and comparative studies between them and the GST from A. lumbricoides (GSTA) are necessary to evaluate their allergenicity. We sought to analyze the allergenic potential of GSTA in connection with the IgE response to non-parasitic GSTs. IgE to purified GSTs from Ascaris (nGSTA and rGSTA), house dust mites (rDer p 8, nBlo t 8 and rBlo t 8), and cockroach (rBla g 5) was measured by ELISA in subjects from Cartagena, Colombia. Also, multidimensional proteomic approaches were used to study the extract of A. lumbricoides and investigate the existence of GST isoforms. We found that among asthmatics, the strength of IgE levels to GSTA was significantly higher than to mite and cockroach GSTs, and there was a strong positive correlation between IgE levels to these molecules. Specific IgE to GSTA was found in 13.2% of controls and 19.5% of asthmatics. In addition nGSTA induced wheal and flare in skin of sensitized asthmatics indicating that it might be of clinical relevance for some patients. Frequency and IgE levels to GSTA were higher in childhood and declined with age. At least six GST isoforms in A. lumbricoides bind human IgE. Four isoforms were the most abundant and several amino acid substitutions were found, mainly on the N-terminal domain. In conclusion, a new allergenic component of Ascaris has been discovered; it could have clinical impact in allergic patients and influence the diagnosis of mite and cockroach allergy in tropical environments.

Figure 1. Identification of the 23 kDa component in the Ascaris extract.

(A) SDS-PAGE and immunoblotting using pooled sera from asthmatic patients sensitized to Ascaris. The arrows indicate the IgE binding component of ∼23 kDa in the extract. (B) Reconstructed total ion current chromatogram of A. lumbricoides extract and mass spectra of the 23 kDa component identified as GST.

Figure 2. IgE reactivity of human sera to A. lumbricoides GST.

(A) IgE levels to the natural Ascaris GST (nGSTA) and the recombinant Ascaris GST (rGSTA) in 29 subjects sensitized to the source (26 asthmatics and 3 non-asthmatic controls). The allergens rABA-1 and tropomyosin (rAsc l 3) are presented as positive control for the reactivity of these sera to other Ascaris allergens. (B) IgE levels to purified mite GSTs (rDer p 8, nBlo t 8 and rBlo t 8) in sera from 29 individuals sensitized to Ascaris. Each dot indicates an individual serum. ** p<0.001, *** p<0.0001. The dotted line indicates the cut-off value for positivity to GSTs (OD₄₀₅≥0.13). Solid bar indicate median. (C) Correlation between specific IgE levels to Ascaris and mite GSTs (n = 36); r =  Spearman coefficient. (D) Correlation of specific IgE levels between the nGSTA and the rGSTA. (E) Correlation of specific IgE levels between the nGSTA and nBlo t 8. For figures D and E, red dots indicate individuals with dual-sensitization; blue dots indicate individuals mono-sensitized to nGSTA and black dots non-sensitized subjects.

Figure 3. Predicted tertiary structures of Ascaris and non-parasitic GSTs.

(A) Ribbon representations of the predicted structures of Ascaris GST1, cockroach GST (Bla g 5) and house dust mite GSTs (Der p 8 and Blo t 8). (B) Superposition of A. suum GST1 with Bla g 5, Blo t 8 and Der p 8. Patches of solvent-accessible and conserved residues are shown in surface view. (C) 180° rotation (Y axis) of all structures to visualize other conserved regions.

Figure 4. Frequency and strength of the IgE reactivity to rGSTA.

(A) Specific IgE levels to rGSTA according to age groups. Green line represents asthmatics and blue line represents non-asthmatic controls. Error bars indicate 95%CI of the median IgE levels (B) A comparison between the strength of specific IgE levels to rGSTA and the cockroach GST (Bla g 5) in asthmatic patients sensitized to Ascaris (ImmunoCAP® >0.35 kU/l, n = 128). IgE levels to rBla g 2 and rDer p 2 were used as reference for the strength of IgE binding to non-GST allergens. Each dot indicates an individual serum.

Figure 5. Intact monoisotopic masses of the A. lumbricoides GST isoforms.

(A) Chromatogram and spectrum of the nGSTA. Injection volume: 15 µl (partial loop); column: 50×1 mm i.d. ProSwift® PS-DVB monolith; eluent A: water +0.05% TFA; eluent B: acetonitrile +0.05% TFA; gradient: 20–50% B in 45 min; flow rate: 60 µl/min; temperature: 60°C. (B) Deconvoluted mass spectrum containing the intact monoisotopic masses of the GST and their relative labeling based on the monoisotopic natural GST. (C) Periodic acid Schiff's staining of the Ascaris extracts in the range of 20–27 kDa. The extract of Artemisia vulgaris and the purified natural Art v 13 were used as positive controls of glycosylated allergens. (D) Enhanced chemiluminiscence analysis of glycosylation in the nGSTA and nBlo t 8. The recombinant allergen pBlo t 12.0101 produced in Pichia pastoris was used as positive control of glycosylated allergen.

Figure 6. Polymorphic residues of the natural A. lumbricoides GST.

(A) SDS-PAGE of the affinity purified nGSTA used for mass spectrometry and mapped peptides on the sequence of A. suum GST1 (UniProt P46436). Peptide matches are shown in yellow. Aminoacid positions with polymorphic residues are shown in their corresponding GST sequences (B) Two-dimensional electrophoresis of nGSTA and the mapped peptides from isoforms 1 to 4 on the sequence of A. suum GST1 (UniProt P46436). (C) Immunoblotting of the nGSTA with sera from Ascaris sensitized patients (n = 3) as primary antibodies. The relative positions of the GST isoforms after two-dimensional electrophoresis are presented in the upper panel.