The mature cysteine protease from Dermatophgoides pteronyssinus, Der p 1, is a major house dust mite allergen. Its enzymatic activity has been shown to have pro‐inflammatory effects that could also negatively influence efficacy of allergen‐specific immunotherapy. The aim of this study was to express recombinant pro‐Der p 1 (rpro‐Der p 1) in the yeast Pichia pastoris and to study its maturation. Expression was achieved at a concentration ranging from 45 mg·L−1 (methanol‐induced expression) to 168 mg·L−1 (constitutive expression). No significant spontaneous maturation of the secreted proenzyme was observed. rpro‐Der p 1 with a sequence‐based molecular mass of 34 kDa was hyperglycosylated by the yeast, migrating at 50–60 kDa on SDS/PAGE. Compared with its natural counterpart (nDer p 1), the recombinant proenzyme demonstrated decreased IgE reactivity, resulting in a 30‐fold lower capacity to induce histamine release from human basophils. Decreased immunoreactivity was also shown by competitive RIA and sandwich ELISA with Der p 1‐specific antibody reagents. CD spectra of rpro‐Der p 1 and nDer p 1 revealed significant structural differences. Deglycosylation of rpro‐Der p 1 with endoglycosidase H resulted in a decrease in apparent molecular mass from 50 kDa to 34 kDa, but did not affect nDer p 1. On removal of N‐glycans from rpro‐Der p 1, which harbours two putative N‐glycosylation sites in both propeptide and mature sequence, the mature rDer p 1 appeared. This suggests that hyperglycosylation hampers spontaneous maturation. Maturation of the recombinant pro‐enzyme was also achieved by addition of the active natural cysteine protease, nDer p 1. In conclusion, high‐level expression of rpro‐Der p 1 in P. pastoris results in a stable hypoallergenic proenzyme with potential for use in allergen‐specific immunotherapy.
Background: Grass pollen of the Poaceae grasses are known to be highly allergenic. Major allergens from the species Lolium, Phleum, Poa and Holcus have been cloned and expressed as recombinant proteins, but of the important species Dactylis glomerata no recombinants are available. Methods: Dac g 5 was cloned by PCR on the basis of homology with Lol p 5 and expressed in Pichia pastoris. Recombinant Dac g 5 (rDac g 5) was affinity purified and compared to natural Dac g 5 (nDac g 5) by immunoblot, radioallergosorbent test (RAST), RAST inhibition, basophil histamine release assay (HRA), competitive radioimmunoassay (RIA) and sandwich enzyme-linked immunosorbent assay (ELISA). In addition, N-terminal sequencing, concanavalin A (Con A) binding, circular dichroism spectrum measurements and matrix-assisted laser desorption ionization-time of flight mass-spectrometric analysis were performed. Results: Clones were obtained that coded for pro-Dac g 5 and two mature isoforms of Dac g 5; the deduced amino acid sequences of both isoforms differed by 4 amino acids. Both mature isoforms were expressed in Pichia at a concentration of ∼15 mg/l. SDS-PAGE analysis showed that rDac g 5 had an apparent Mr approximately 10 kD above nDac g 5. By mass spectrometry this difference was shown to be around 2.5 kD. Positive Con A staining suggested (O-linked) glycosylation as an explanation for this increase in Mr. Whereas both purified recombinants showed a tendency to dimerize, purified nDac g 5 contained a 12-kD peptide not observed for rDac g 5. RAST, RAST inhibition and HRA showed that the IgE reactivity of rDac g 5 was similar to that of nDac g 5. A small subgroup, however, clearly demonstrated decreased IgE reactivity to rDac g 5.02. Differences in immune reactivity of both isoforms were confirmed by monoclonal antibody (mAb)-based sandwich ELISA. Conclusions: Dac g 5 was successfully cloned and expressed in P. pastoris. Minor differences in primary structure between isoforms influence their immune reactivity.
Background: For the detection of allergen-specific IgE in serum, IgE-binding assays such as the radioallergosorbent assay (RAST) are commonly used. In this study, the applicability and sensitivity of the stripped basophil histamine release bioassay was investigated and compared to the RAST. Methods: Basophils were stripped of their IgE by an acidic buffer, sensitized by human serum and stimulated by allergen with or without interleukin (IL)-3. The histamine release was determined by fluorometric analysis. Results: We showed that for enhancement of the maximal histamine release and the sensitivity of the stripped basophil assay, the priming cytokine IL-3 can be added to the basophils simultaneously to the stimulus. Preincubation of the cells with IL-3, as described in other studies, was not necessary. The bioassay can be used to study the specificity of IgE-mediated reactions. Basophils sensitized by serum absorbed to a particular allergen did not respond to this allergen anymore. This method is very suitable to study cross-reactivity between allergens. The results obtained in the bioassay were comparable to those obtained in the RAST. Using the RAST, lower concentrations of allergen-specific IgE were detected than in the bioassay. However, sera containing IgE against minor allergenic components were negative in the RAST, but strongly positive in the basophil assay. Conclusions: The stripped basophil histamine release bioassay is useful to complement and extend serological detection of allergen-specific IgE. Especially with sera containing IgE against minor components, this assay is more suitable than the RAST. Furthermore, in this assay, the dependency of IgE and of allergen-specific IgE in reactions can be studied in more detail.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.