Recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites

Abstract

Background: N-linked glycans present in venoms, pollen and mites are recognized by IgE antibodies from >20% of allergic patients but have low or no allergenic activity.

Objectives: To engineer recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from venoms, pollen and mites which can discriminate carbohydrate-specific IgE from allergenic, peptide-specific IgE.

Methods: One or two N-glycosylation sites were engineered into the N-terminus of the non-allergenic protein horse heart myoglobin (HHM) using synthetic gene technology. HHM 1 and HHM 2 containing one or two N-glycosylation sites were expressed in baculovirus-infected High-Five™ insect cells and a non-glycosylated version (HHM 0) was obtained by mutating the glycosylation motif. Recombinant HHM proteins were analyzed regarding fold and aggregation by circular dichroism and gel filtration, respectively. IgE reactivity was assessed by ELISA, immunoblotting and quantitative ImmunoCAP measurements. IgE inhibition assays were performed to study cross-reactivity with venom, plant and mite-derived carbohydrate IgE epitopes.

Results: HHM-glycovariants were expressed and purified from insect cells as monomeric and folded proteins. The HHM-glycovariants exhibited strictly carbohydrate-specific IgE reactivity, designed to quantify carbohydrate-specific IgE and resembled IgE epitopes of pollen, venom and mite-derived carbohydrates. IgE-reactivity and inhibition experiments established a hierarchy of plant glcyoallergens (nPhl p 4 > nCyn d 1 > nPla a 2 > nJug r 2 > nCup a 1 > nCry j 1) indicating a hitherto unknown heterogeneity of carbohydrate IgE epitopes in plants which were completely represented by HHM 2.

Conclusion: Defined recombinant HHM-glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites were engineered which made it possible to discriminate carbohydrate- from peptide-specific IgE reactivity.

Keywords: Allergen; Allergy; Component-resolved diagnosis; Cross-reactive carbohydrate determinant; Molecular allergology; Recombinant glycoprotein.

Fig. 1

Construction plans for recombinant horse heart myoglobins (HHM) with and without carbohydrate epitopes. (A) Sequences and (B) schematic overviews of HHM (blue) derivatives with one (HHM1), two (HHM2) N-glycosylation sites (green) or without N-glycosylation sites (HHM 0: N4Q mutation; brown). Spacers are indicated in black. Hexa-histidine tags are indicated in red.

Fig. 2

Purification and IgE reactivity of glycosylated horse heart myoglobin derivatives. (A) Coomassie-blue stained SDS-PAGE containing insect cell-expressed and purified recombinant HHM 0, HHM 1 and HHM 2. Nitrocellulose-blotted purified HHM 0, HHM 1 and HHM 2 detected with anti-His-tag antibodies (B), with biotinylated lectin AAL (C) or serum IgE from a patient (i.e., patient 1) containing carbohydrate-specific IgE (D). (E) Coomassie-stained SDS-PAGE (upper part) and IgE immunoblot (lower part, patient 1) containing HHM 0, HHM 1 and HHM 2 treated with (+) and without (−) PNGase A. Molecular weights are indicated in kDa.

Fig. 3

Nitrocellulose-blotted HHM 0, HHM 1 and HHM 2 were incubated with sera from allergic patients or buffer without serum (b). Bound IgE was detected with ¹²⁵Iodine-labeled anti-human IgE and visualized by autoradiography. Molecular weights are indicated in kDa.

Fig. 4

Biochemical and biophysical characterization of HHM variants. (A) Coomassie-stained SDS-PAGE with HHM derivatives separated under reducing and non-reducing conditions. Molecular weights are indicated in kilo Dalton (kDa). (B) Size exclusion analysis of HHM variants using a Bio-Rad Gel Filtration Standard. Elution volumes (x-axis: mL) and absorbance units (y-axis) are shown. (C) Circular dichroism analysis of HHM variants at room temperature (RT) (dashed line), 95 °C (black line) and after cooling back to RT (gray line). Molecular ellipticities (y-axes) at different wavelengths (x-axes) are shown. (D) Thermal denaturation curves (y-axis: molecular ellipticities; x-axis: temperature) of HHM variants measured at 220 nm.

Fig. 5

Correlation of CCD-specific IgE levels measured by ELISA (y-axis: OD values) and ImmunoCAP (x-axis: kUA/L). Pearson's correlation coefficient and p value is indicated. Pearsons r = 0.84, p = .0022.

Fig. 6

HHM 2 inhibits allergic patients IgE-binding to carbohydrate epitopes in different allergen sources. (A) Sera from three bee and wasp sensitized patients (2, 3, 4) were pre-incubated with rBet v 1, non-glycosylated rApi m 1, HHM 2 or rVes v 5 and tested for IgE-reactivity to blotted bee (upper part) and wasp venom (lower part) extracts. (B) Sera from three grass pollen sensitized patients (5, 6, 7) were pre-incubated with HHM 2, nPhl p 4, rPhl p 1 or rBet v 1 and tested for IgE-reactivity to blotted grass pollen extract. (C) Sera from three HDM sensitized patients (6, 8, 9) were pre-incubated with HHM 2 or rBet v 1 and tested for IgE-reactivity to blotted house dust mite extract. (D) Sera from Blomia tropicalis-sensitized patients (6, 10, 11) were pre-incubated with rBet v 1, HHM 0 or HHM2 and tested for IgE-reactivity to blotted Blomia tropicalis extract. (E) Sera from two Alternaria-sensitized patients (1, 8) were pre-incubated with HHM 2 or Bet v 1 and tested for IgE-reactivity to blotted Alternaria alternata extract. Molecular weight is indicated in kDa (Table 1).

Fig. 7

HHM 2 selectively inhibits patients' IgE binding to natural glycosylated micro-arrayed allergens. (A) Screen shot of IgE reactivities of a serum (patient #13, Table E1) with (right) and without (left) preincubation with HHM 2. Natural glycosylated allergens are indicated by white boxes, recombinant non-glycosylated insect venom allergens by yellow boxes. (B) Hierarchy of carbohydrate-specific IgE reactivities (y-axis: ISU IgE; mean values indicated) to natural glycosylated micro-arrayed plant allergens for all patients from Table 2 for which a >90% inhibition with HHM 2 was obtained (left panel), those with venom sensitization only (middle panel) and those with sensitization to venoms and respiratory and/or food allergens (right panel) (x-axis). Significant differences are shown. **P < .01 and ***P < .001.