Der p 1-based immunotoxin as potential tool for the treatment of dust mite respiratory allergy

Immunotoxins appear as promising therapeutic molecules, alternative to allergen-specific-immunotherapy. In this work, we achieved the development of a protein chimera able to promote specific cell death on effector cells involved in the allergic reaction. Der p 1 allergen was chosen as cell-targeting domain and the powerful ribotoxin α-sarcin as the toxic moiety. The resultant construction, named proDerp1αS, was produced and purified from the yeast Pichia pastoris. Der p 1-protease activity and α-sarcin ribonucleolytic action were effectively conserved in proDerp1αS. Immunotoxin impact was assayed by using effector cells sensitized with house dust mite-allergic sera. Cell degranulation and death, triggered by proDerp1αS, was exclusively observed on Der p 1 sera sensitized-humRBL-2H3 cells, but not when treated with non-allergic sera. Most notably, equivalent IgE-binding and degranulation were observed with both proDerp1αS construct and native Der p 1 when using purified basophils from sensitized patients. However, proDerp1αS did not cause any cytotoxic effect on these cells, apparently due to its lack of internalization after their surface IgE-binding, showing the complex in vivo panorama governing allergic reactions. In conclusion, herein we present proDerp1αS as a proof of concept for a potential and alternative new designs of therapeutic tools for allergies. Development of new, and more specific, second-generation of immunotoxins following proDerp1αS, is further discussed.

In this context, immunotoxins arise as a promising approach beyond cancer treatment, revealing themselves as promising candidates against inflammatory diseases such as allergies, mainly because of their specific ability to promote cell death. Immunotoxins are chimeric molecules that combine a targeting domain-usually antibody based-fused to a toxin domain providing the molecule with its cytotoxic activity 8,9 . Their mode of action comprises three main steps: (1) recognition of the antigen by the targeting domain; (2) cell internalization of the complex; and (3) subsequent intracellular release of the active toxic domain which catalytically causes target cell death 10,11 .
Regarding type I hypersensitivity reactions, the obvious suitable targeting domain for immunotoxin development would be the inclusion of an allergen, specifically directed to IgE presented on the cell surface of effector (basophils, mast cells) and B cells [12][13][14] . In this sense, an early example of this strategy has been used in animal models. Ovalbumin (OVA)-sensitized BALB/c mice were treated with an OVA-diphtheria toxin chimera, which resulted in protection from anaphylactic shock after OVA re-challenge 15 and simultaneous depletion of OVAspecific IgE and IgG1 levels but increased IgG2a 16 .
In this work we report the development of a different design using the prevalent house dust mite (HDM) allergen Der p 1, as targeting domain. Der p 1-sensitization accounts for more than 80% in mite-allergic subjects, affecting to more than 20% of the global population and up to 85% of asthmatics allergic to HDM 17 . Der p 1 is a papain-like cysteine protease which is synthetized as a 34 kDa proenzyme (proDer p 1), consisting of a cysteine protease domain (222 residues) and an N-terminal pro-peptide (80 amino acids) that blocks its proteolytic activity 18,19 . Der p 1 pro-peptide also functions as an intramolecular chaperone ensuring its correct folding and extracellular secretion 20 . Importantly, this pro-sequence has been used for recombinant Der p 1 production both in tobacco plants and in the yeast P. pastoris [21][22][23] . Regarding the toxic domain, we have chosen the highly specific ribotoxin α-sarcin, an extracellular fungal RNase secreted by Aspergillus giganteus, which specifically cleaves a unique single bond of the larger rRNA located at the so-known Sarcin Ricin Loop (SRL) 24 . This cleavage impairs ribosome function and, thereby, causes protein biosynthesis inhibition, leading to programmed cell death by apoptosis 25,26 . α-Sarcin is the most powerful ribotoxin candidate known so far according to its functional properties and previously described antitumoral therapeutic applications [27][28][29][30][31] .
Herein we present the design and characterization of an innovative allergen-based immunotoxin, consisting in the recombinant fusion of the allergen proDer p 1 and α-sarcin, representing a proof of concept for studying the potential of these molecules as allergy treatment. The chimeric construct, named proDerp1αS, was produced in the yeast P. pastoris and evaluated on humanized basophil-like cells (humRBL-2H3) sensitized with Der p 1 allergic patients' sera. Potential non-specific cytotoxicity of the construct was also evaluated in other cell lines such as HeLa, Calu-3, LAD2 and Raw 264.7. Finally, the biological relevance of proDerp1αS was reported in terms of activation and cytotoxicity against ex vivo basophils from Der p 1 allergic patients and non-allergic individuals.

Results
proDerp1αS generation, production and purification. The corresponding proDer p 1 and α-sarcin cDNAs were fused by means of a glycine-glycine-arginine (GGR) linker to produce the proDerp1αS construction ( Fig. 1A and S1). This construction was then cloned in pPICZαA, downstream of the α-factor secretion signal sequence within the plasmid, and successfully produced in P. pastoris yeast extracellular culture medium (Fig. 1B, C). The protein was purified, after extensive dialysis against 50 mM sodium phosphate buffer, 0.1 M NaCl, pH 7.5, by means of a Ni 2+ -NTA affinity chromatography, taking advantage of its C-terminal six histidine Figure 1. Domain arrangement of proDerp1αS construct, along with SDS-PAGE, Western blot and mass spectrometry analysis of the purified immunotoxin. (A) Schematic representation of proDerp1αS cDNA, highlighting its structural and functional motifs, alongside its theoretical molecular mass. 3D-model structure of proDerp1αS is included as Figure S1. (B) Coomassie blue stained SDS-PAGE (CBS) and Western blot analysis using rabbit anti-α-sarcin (left) and anti-α-Der p 1 antisera (right). CBS protein molecular weight standards correspond to Bio-Rad Unstained SDS-PAGE low range Standards; while the prestained Bio-Rad Precision. Plus Dual Color Standards (Bio-Rad, Hercules, CA, USA) were used for Western blot. Images correspond to full-length gels and blots acquired and analyzed using the Gel Doc XR Imaging System and Quantity One 1-D analysis sofware (BioRad) or ChemiDoc-It (UVP) and VisionWorks LS, respectively.Full-length blots/gels are presented in Supplementary Figure S2. (C) Mass spectrometry analysis spectrum showing the corresponding peak to proDerp1αS construct. The difference between theoretical and empirical masses agrees with the four N-terminal extra amino acids of α-factor secretion signal that could remain after its processing by kex2 protease.
IgE recognition by HDM allergic human sera. Considering the strategy designed and the mechanism of action expected for proDerp1αS on effector cells, the IgE-epitopes binding ability of the allergen within the chimera was analyzed. With this purpose, HDM-allergic sera from human patients were assayed for the comparatively recognition of proDerp1αS, natural Der p 1 (nDer p 1) and recombinant form of proDer p 1 (rproDerp1) in identical conditions (Fig. 3). Allergen-coated indirect enzyme-linked immunosorbent assays (ELISA) using individual Der p 1 allergic and non-allergic patients' sera showed practically identical IgE recognition behavior against the three proteins in all cases tested (n = 15), controls included (C1 and C2) (Fig. 3A). Therefore, these results showed how the presence of α-sarcin did not impair the ability of specific IgE to recognize the chimeric proDerp1αS construct. Regarding inhibition ELISA assays, rproDer p 1 and proDerp1αS showed behaviors that, although corresponding to lower inhibition values than nDer p 1, were within the experimental error of these assays (Fig. 3B).
Cell toxicity. With the aim of testing the non-specific cytotoxic effect of proDerp1αS, somatic (HeLa and Calu-3) and immune (LAD2 and Raw 264.7) representative cell lines were incubated for 24 h with increasing tenfold concentrations of α-sarcin or proDerp1αS proteins (Fig. 4). In all cases, the cytotoxic effect of wild-type α-sarcin was significantly higher than that of the construct, most especially at the highest protein concentration assayed (1 μM) (Fig. 4). In agreement with this observation, the viability of the cultures was almost always over 90% independently of the proDerp1αS concentration assayed. The exception was Calu-3 cells, that presented a remarkable decrease in viability by proDerp1αS-already at 100 nM, but indistinguishable from the fungal natural toxin effect (Fig. 4B), suggesting therefore a non-specific origin. 1 serum sensitized-humRBL-2H3 cells (n = 6), measured 1 h after stimulation, was similar for rproDerp1 and proDerp1αS (Fig. 5, compare results corresponding to serum #1). The β-hexosaminidase release assay manifested an allergen bimodal dose dependent effect, since after showing and increment, degranulation was again reduced at the higher protein concentrations employed, basically above 500 nM. HumRBL-2H3 activation was exclusively observed when cells were previously sensitized with HDM allergic patient sera. Supporting the specificity of this effect, α-sarcin never caused degranulation, independently of the serum treatment used (Fig. 5). The specificity of the cytotoxic effect caused by proDerp1αS on sensitized humRBL-2H3 cells was analyzed in parallel experiments using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assays. In this case, the chimeric molecule showed high specific cytotoxicity, promoting cell death exclusively on humRBL-2H3 cells sensitized with Der p 1-allergic patients' sera, but not on those treated with non-allergic patients' sera ( Fig. 5). Comparatively, neither rproDer p 1 nor α-sarcin showed significant cytotoxicity on Der p 1-sensitized and non-sensitized humRBL-2H3 cells, thus validating the specificity of the allergen-toxin construction.
Human basophils degranulation. The biological relevance of proDerp1αS was studied in terms of degranulation using basophils from HDM allergic and non-allergic patients (Fig. 6). Basophil activation test (BAT) showed a similar trend in degranulation caused in a dose-dependent manner by nDer p 1, rproDer p 1 and proDerp1αS in the case of HDM allergic patients (n = 5), with no degranulation caused by α-sarcin in any patient analyzed (Fig. 6A). Non-allergic patients (n = 5) did not show any different activation effect respect to the controls (data not shown). According to the half maximal effective concentration promoting degranulation (EC 50 ), nDer p 1 (0.00187 ± 0.00267 nM) manifested an activity in the order of tenfold higher than those of rproDer p 1 (0.0123 ± 0.0134 nM) and proDerp1αS (0.0151 ± 0.0138 nM) (Fig. 6B). Between rproDer p 1 and proDerp1αS EC 50 median values statistically significant differences were not appreciated after the corresponding ANOVA analysis (P < 0.186).

Cytotoxic effect on basophils and IgE internalization after proDerp1αS stimulation. After
showing that proDerp1αS binds to specific-Der p 1 IgEs on basophils surface, with a similar behavior to that observed for nDer p 1, its cytotoxic effect on these cells was studied. As it is shown in Fig. 7A, 10 nM proDerp1αS treatment did not promote any significant increase in annexin V (apoptotic) or LIVE/DEAD (non-viable) basophil labelling respect to the non-treated cells, neither after 24 h nor after 72 h of incubation. Coherently, identical results were observed for 0.1 nM concentrations (data not shown). Higher proteins amount, up to 1 μM and after 72 h of incubation with either free wild-type α-sarcin or proDerp1αS, caused a significant increase in the percentage of apoptotic and non-viable basophils, with independence of patient sensitization subset (Fig. 7B). The assays analysing IgE-FcεRI complex internalization after proDerp1αS binding to IgE showed that basophils surface IgE levels did not change significantly in the next 4 h after immunotoxin stimulation of Der p 1-sensitized patients' basophils (#17) (Fig. 7C).
Thereby, according to our results, due to its lack of internalization after surface IgE-binding, proDerp1αS did not produced any specific cytotoxic effect on Der p 1-sensitized basophils, regardless of incubation time (24 or

Discussion
Adequate prevention of allergies, diagnosis and treatment are one of the great health challenges of the twenty-first century. Due to the variable effectiveness among patients and sometimes adverse secondary effects of AIT 32 , new therapeutic alternatives and complementary approaches to AIT have been developed in the last years 33 . Many of them are based on biological treatments, including immunomodulatory strategies such as IgE-blocking antibodies as Omalizumab 34 and those directed to membrane-linked IgEs (mIgE) on memory B cells 35 and against FcƐRI receptor on effector cells 36,37 .
In this work we present a new alternative approach based on the production, and structural and functional characterization of proDerp1αS, an allergen-directed immunotoxin targeted against IgE-and/or FcεRI-positive effector cells. This proDerp1αS chimera was produced as a homogeneous soluble polypeptide chain of 53 kDa, which integrates the ability to bind FcεRI-positive cells and the cysteine protease activity described for Der p 1 allergen and the ribonucleolytic catalytic activity of α-sarcin. Although proDer p 1 has been described as an inactive zymogen due to the blocking of the active site by its N-terminal pro-peptide, proDerp1αS presented proteolytic activity against the Boc-QAR-AMC substrate, albeit minor than nDer p 1 mature protease. An explanation to this fact could rely on a structural distortion of N-terminal pro-peptide induced by the α-sarcin domain, as in proDer p 1 three-dimensional structure the C-terminal segment of the pro-peptide is located very close to proDer p 1 C-terminus, where α-sarcin is fused in the construct 18,38 .
As a major rule, the cytotoxic specificity of immunotoxins depends on the proper functionality of its marker domain 9 . In this case, this function lies on the proDer p 1 domain, which is expected to direct proDerp1αS against the allergen-specific effector cells. Therefore, its correct functionality is defined based on its adequate recognition www.nature.com/scientificreports/ by Der p 1-allergic patients IgEs. Accordingly, all sera from 15 allergic individuals recognized proDerp1αS in a very similar way as nDer p 1 or rproDer p 1, being the inhibition ability of the chimera slightly lower than the corresponding to nDer p 1. This was expected because of the molecular differences existing between rproDer p 1, unprocessed and non-glycosylated in Asn132, and the natural form of Der p1 (nDer p 1) to which allergic patients have been naturally sensitized 18,21,38 . These results, together with the enzymatic characterization of proDerp1αS, suggested an optimal conformational features for the immunotoxin and, more notably, an important maintenance of the main IgE-binding regions presented on nDer p 1. proDerp1αS produced a dose-dependent degranulation on both allergic sera-sensitized humRBL-2H3 cells and Der p 1 allergic patient-derived basophils, but not on those humRBL-2H3 cells sensitized with control nonatopic sera or basophils from non-allergic individuals. These results indicate that proDerp1αS effectively binds to the surface receptors of these cells and, it occurs specifically through the IgE-FcεRI complex which cause the cross-linking of FcεRI receptors and ultimately the cell degranulation 12 .
Simultaneously, it was demonstrated that the cytotoxic effect of proDerp1αS on humRBL-2H3 cells was specific and strictly dependent on the presence of Der p 1-specific IgE on their cell surface, inhibiting cell viability only in those cells previously sensitized with Der p 1 allergic patients' serum. In these cases, although the concentration to promote a significant decrease in cell viability was high (IC 50   . Serum sensitized humRBL-2H3 degranulation and viability inhibition assays. HumRBL-2H3 cells sensitized with Der p 1-positive allergic (#1, 5, 6, 12, 13) and non-allergic (C1) individuals' sera were treated with proDerp1αS for degranulation and viability analysis, performed by β-hexosaminidase release (β-Hex. % of total) and MTT viability assays, respectively. α-Sarcin and rproDer p 1 were used as controls with a representative serum of each allergic and non-allergic groups (serum #1 and C1, respectively). Bars represent means of duplicates and its standard deviation for all the experiments. (*) indicates technical limitations by no longer availability for serum from this patient.
Scientific RepoRtS | (2020) 10:12255 | https://doi.org/10.1038/s41598-020-69166-w www.nature.com/scientificreports/ Unlike the humRBL-2H3 cells, proDerp1αS did not produce any specific cytotoxic effect on basophils of patients allergic to Der p 1. Since the binding of proDerp1αS to these cells was previously proven, the analysis of this outcome suggested three possible explanations: (1) the lack of cell internalization of the proDerp1αS-IgE-FcεRI complex, (2) the degradation of the toxic domain of the construct before its release to the cytoplasm and (3) the resistance of basophils to the cytotoxic mechanism of ribotoxins. Since the last two options have not been previously demonstrated in any cell kind 9,26 , the most feasible justification is the absence of IgE-FcεRI internalization in basophils after their allergen-mediated cross-linking in the plasma membrane. As a result, the radically divergent behavior of proDerp1αS specific cytotoxicity between humFcεRI RBL-2H3 cells and human basophils would be based on differences in the destiny of the cellular IgEs after allergen binding on those cells. In this sense, RBL-2H3 cells have been described to suffer a rapid internalization of these allergen-IgE-FcεRI complexes 39,40 , similar to which takes place in tissue mast cells 41 . On the other hand, although human basophils have shown to internalize serum IgE through FcεRI in an allergen-independent way 42 , this internalization has not been demonstrated after allergen binding; which is in line with the current understanding of circulating basophils as purely effector cells 43,44 . As an approximation to proDerp1αS-IgE-FcεRI complexes behavior in basophils of Der p 1 allergic patients, we showed that cell membrane IgE levels remained unchanged for at least 4 h after stimulation with the immunotoxin. This result, coupled with the absence of any specific cytotoxic effect during the 72 h following stimulation with proDerp1αS, suggests that circulating human basophils do not internalize the allergen once it is bound to IgE, holding the whole complex at the surface for longer periods of time, thus preventing from the cytotoxic effects coming from proDerp1αS.
Regarding the in vivo potential effect of proDerp1αS, mIgE-Der p 1 + memory B cells-producers of specific soluble IgEs-have been proposed as one of the most relevant alternative cellular targets of our strategy, whose elimination would provide a greater therapeutic impact on the course of the allergic disease 45 . In this case, these cells present an efficient internalization of natural allergens after their binding to mIgE (membrane-bound IgEs) 46,47 , providing a promising target for proDerp1αS cytotoxic actions. Furthermore, as bolus administration of therapeutic immunotoxins provides a potential systemic range of action 8,9 , these kind of molecules would allow memory cells depletion with independence of its tissue location.
In conclusion, proDerp1αS reveals itself as a suitable proof of concept example of an allergen-directed immunotoxin. Thus, the results presented here are consistent with the potential benefit of using a new type of biologic tools capable of provoking specific cell death on those involved in allergic reactions. Although this chimeric molecule could be considered as a potent cytotoxic agent against allergy-affecting cells, there is a great need of a deep in vivo characterization of its effect during the course and allergic reaction, in order to state categorically their therapeutic potential. In short, the existence of a wide set of molecular targets which are potentially suitable for the development of more immunotoxins against allergies 48 , the increasing relevance of therapeutic antibodies in the treatment of allergic diseases 49 and the excellent properties of these molecules in terms of specificity and cytotoxic potency 8,9 , make immunotoxins a family of biotechnological tools with great potential for the treatment of allergic inflammatory reactions.

Methods
proDerp1αS cloning in pPICZαA and electroporation of BG11 P. pastoris cells. Optimized proDer p 1 cDNA sequence for P. pastoris expression was acquired from IDT Technologies (Coralville, IA, USA). EcoRI and NotI restriction sites were included in 5′ and 3′ ends. After digestion, proDerp1-specific cDNA was cloned downstream of α-factor signal peptide, providing the effective secretion of the protein to the extracellular media after its being processed by kex2 protease, and upstream of α-sarcin sequence in vector pPICZαA. Sequence identity was analyzed by sequencing the whole construct including the 3-amino acids linker (Gly-Gly-Arg) between the two domains. PmeI-linearized pPICZαA/proDerp1αS construct was electroporated in Western blot analysis. After purification proceeding, the samples were examined by 15% (v/v) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The structural identity of the purified chimera was analyzed in Western blot by both anti-Der p 1 and anti-α-sarcin rabbit polyclonal antisera and then detected with a goat anti-rabbit antibody conjugated with horseradish peroxidase (GAR-HRP). Ribonucleolytic activity. The specific ribonucleolytic activity of ribotoxins hydrolyzing SRL is usually detected by the release of a 400 nt fragment (α-fragment) from eukaryotic ribosomes 51 . Thereby, we studied comparatively the ribonucleolytic activity of proDerp1αS against ribosomes contained in a rabbit cell-free reticulocyte lysate, as previously described 52   www.nature.com/scientificreports/ PBS, the selected sera, diluted tenfold in blocking buffer, were incubated for 2 h at 37 °C. Human serum IgEs were detected by using an anti-human IgE rabbit antiserum followed by a goat anti-rabbit antibody labelled with horse radish peroxidase (GAR-HRP). Between incubations, wells were washed with 0.1% (v/v) Tween 20-PBS. Development was performed by adding 100 μL/well of 0.1 M sodium citrate, pH 5.0, 4% (v/v) methanol, 3.5 mM OPD (1,2-phenylenediamine dihydrochloride), 0.16% (v/v) H 2 O 2 and stopped with 100 μL/well of 10 N H 2 SO 4 . Specific IgE recognition was quantified by measuring the optical density at 492 nm. Additionally, inhibition ELISA were performed for comparing IgE epitope affinity between the natural allergen, its recombinant form and the derived immunotoxin. Following the indirect ELISA protocol, microplate wells were coated with nDer p 1 (37 pmol) and incubated with sera inhibited with proDer p 1, proDerp1αS and nDer p 1. This inhibition was performed by using blocking buffer for 2 h at 37 °C under vigorous shaking. The results were shown as inhibition % according to the inhibitor concentration and relativized to non-inhibited sera. All assays included duplicates for each condition.

N-acetyl-β-D-hexosaminidase release. For activation through cross-linking to the IgE receptor,
humFcεRI expressing RBL-2H3 cells were sensitized overnight with 5% (v/v) of sera from allergic patients. Next day cells were stimulated with α-sarcin, rproDer p 1 or proDerp1αS at different concentrations. To increase and stabilize the secretion of β-hexosaminidase, 50% (v/v) D 2 O was added to the Tyrode's buffer used for the dilution of allergens 54 . For detection of the granular enzyme β-hexosaminidase, an enzymatic colorimetric assay was used as described previously 55 . Briefly, 1 h after stimulation of the cells, 30 μl of supernatant was transferred to a 96-well plate and mixed with 50 μl of substrate solution (3.5 mg/mL p-nitrophenyl-N-acetyl-β-d-glucosaminide dissolved in 40 mM citric acid, pH 4.5). In order to calculate the total β-hexosaminidase activity, cells were lysate with 100 μl of 0.1% (v/v) Triton X-100 solution and same procedure was done. The mixtures were incubated for 60 min at 37 °C. After incubation, 100 μl of glycine 400 mM pH 10.7, was added to each well, and the absorbance was measured at 405 nm. The percentage of β-hexosaminidase release was calculated as a percentage of the total β-hexosaminidase content in the cells.
Cell viability inhibition. The ribosome inactivation exerted by ribotoxins leads to cellular death 23 . Hence, in vitro MTT viability assays are widely used for studying the effect of immunotoxins on the metabolic activity rate of different cell lines. MTT assay for quantifying as culture viability according to the formation of insoluble purple formazan crystals by metabolic active cells by reducing the MTT yellow dye. We used this method for analyzing the effect of proDerp1αS on cultures viability with little modifications according to the cell type assayed.
HeLa, Calu-3, LAD2 and Raw 264.7 cells viability assays were performed following the general methodology described for previous immunotoxins 28 . Briefly, cells were seeded into 96-well plates in culture medium for 24 h. Then, the medium was removed and 200 μl of fresh free-FBS medium containing the assayed proteins was added. After 24 h, 100 μl of medium were removed and 20 μl of 5 mg/mL MTT added per well. Finally, after 4 h of incubation, the medium was softly removed, and formazan pellets were dissolved in 100 μl of DMSO:methanol (1:1). Colorimetric absorbance was measured at 570 nm subtracting 650 nm background. Two different experiments with independent duplicates were carried out.
RBL-2H3 cells expressing humFcεRI were plated at 20,000 cells per well in 96-well plates in 100 μl of medium including 5% (v/v) of allergic and non-allergic patients' sera. The next day, the media were removed and the assayed proteins were added at different dilutions in 100 μl of fresh medium. After 24 h, 20 μl of 5 mg/mL MTT were added per well and incubated in culture conditions for another 4 h. Finally, the media were removed and the colorimetric absorbance was measured as explained before.
Human basophils degranulation. The BAT assays were performed, as previously described 56 , including samples from D. pteronyssinus allergic patients and non-allergic individuals. First, 100 μl aliquots of fresh heparinized whole blood were stimulated for 10 min at 37 °C with 20 μl of stimulation buffer. Then, the assayed proteins, diluted in PBS, were added in 100 μl at serial tenfold dilutions from 2.9 × 10 -5 nM to 2.9 nM. Polyclonal anti-human IgE or PBS alone were used as controls. Cells were stained with anti-hCCR3-fluorescein isothiocyanate (FITC), anti-hCD203c-peridinin chlorophyll protein complex (PerCP) and anti-hCD63-phycoeritryn (PE) monoclonal antibodies, and erythrocytes were lysed. The stimulation buffer and all the antibodies used in this assay were obtained from BioLegend (San Diego, CA, USA). Basophils were gated as CCR3 + and CD203c + .
Scientific RepoRtS | (2020) 10:12255 | https://doi.org/10.1038/s41598-020-69166-w www.nature.com/scientificreports/ Basophil degranulation was determined according to CD63 cell surface expression. All parameters were recorded with a FACSCalibur and FACScalibur software (BD Biosciences, San Jose, CA, USA). Data were analyzed with FlowJo software (BD Biosciences, San Jose, CA, USA). In all cases, the percentage of CD63-positive basophils in the control sample was below 2.5%, setting the cut-off for a positive test to 5%. Basophil allergen threshold sensitivity for the different tested proteins was analyzed by dose response curves, using as indicator the lowest allergen concentration (EC 50 ) giving 50% of maximum upregulation of CD63.
Cytotoxic effect on human basophils. Human peripheral blood mononuclear cells (PBMCs) were isolated from whole venous blood from consenting volunteers allergic (n = 3) and non-allergic (n = 2) to HDM. Per each individual, 20 mL of blood were gently added over 4 mL of ficoll-paque (Histopaque-1077, Sigma-Aldrich, St. Louis, MO, USA) and centrifuged without brake during 30 min at 400 g, R.T. PBMCs fraction was recover and washed in a fresh tube with 30 mL of 0.9% (w/v) NaCl and then centrifuged for 10 min, 300 g, R.T. After a second wash with 50 mL of the previous solution and another centrifugation in the same conditions, the cells were resuspended in 2 mL of PBS containing 1 mM EDTA and 10% (v/v) FBS. Finally, the purified PBMCs from each patient were counted, evaluating its viability by mean of trypan blue, and cultured separately in 96-round well plates containing 2 × 10 5 cells/well in 250 μl of RPMI including 10% (v/v) FBS and the assayed stimulus (proDerp1αS, rproDer p 1 or wild-type α-sarcin) to a final concentration of 0.1, 10 or 1,000 nM. Apoptosis and cell viability were evaluated by mean of annexin V-FITC and LIVE/DEAD staining, respectively. Annexin V-FITC is commonly used in flow cytometry to detect apoptotic cells by its ability to bind to phosphatidylserine exposed on the outer leaflet of the plasma membrane, a marker of apoptosis. LIVE/DEAD kit (Invitrogen) is a viability cell assay used in flow cytometry for simultaneous fluorescence staining of viable and dead cell, as it contains calcein-acetoxymethyl and propidium iodide (PI) solutions. In this case non-viable cells were assessed by PI staining (LIVE/DEAD+), as it intercalates with the nuclear DNA just in those dead cells with areas of disordered cell membrane.
After incubating 30 min at R.T., the cells were washed with 200 μl of annexin V binding buffer and analyzed in a FACScalibur apparatus (BD Biosciences, San Jose, CA, USA). Respectively, basophils were gated as CCR3 + / CD203c + within PBMCs lymphocyte/basophil region (FSC/SSC selection) by mean of anti-hCCR3-PE (Bio-Legend, San Diego, CA, USA) and anti-hCD203c-PerCP antibodies. Single and double staining controls were performed for compensating the overlapping fluorescence.

Allergen-IgE-FcεRI complex internalization.
Using the same general procedure as for the BAT, 100 μL of whole blood aliquots were incubated for 10 min at 37 °C after adding 20 μL of stimulation buffer. Then, proDerp1αS was added to a final concentration of 10 nM and incubated during 20 min and 4 h at 37 °C or directly in ice for non-internalization control. After desired times, the cells were fixed and labelled with anti-hCCR3-FITC, anti-hCD203C-PerCP and anti-hIgE-PE antibodies (BioLegend, San Diego, CA, USA). Samples were tested in a FACScalibur analyzer (BD Biosciences, San Jose, CA, USA), gating on basophils as CCR3 + / CD203c + , and following anti-hIgE-PE fluorescence indicative of IgE-FcεRI complex internalization.
Patient work-up. HDM-allergic patients included in this work presented a history of persistent allergic rhinitis or/and asthma to D. pteronyssinus, having specific IgE levels to D. pteronyssinus higher than 0.35 kU/L (ImmunoCAP-FEIA). Non-allergic subject group was included as a control (specific IgE < 0.35 kU/L). Levels of specific IgE to D. pteronyssinus crude extract in sera from allergic patients and non-allergic donors are included in Supplementary Table S1.
Sample collection, processing and storing. The biological samples from allergic and non-allergic patients were conducted in accordance with the Declaration of Helsinki. The study was approved by the local ethics committee (Comité de Ética de la Investigación provincial de Málaga; Servicio Andaluz de Salud, Consejería de Salud Andalucía, España). All participants signed informed consent forms before the study began.
Peripheral blood samples were collected from patients and processed immediately. PBMCs, isolated by Ficoll-Paque density gradient (Pharmacia Biotech, Barcelona, Spain), and fresh blood aliquots were treated for BAT and cytotoxicity assays as described above.
Serum was collected for specific IgE determination, and stored at − 20 °C until further use. Samples were managed by the Málaga Hospital-IBIMA Biobank that belongs to the Andalusian Public Health System Biobank. Specific IgE determination. Levels of specific IgE to D. pteronissynus were determined by ImmunoCAP-FEIA in serum samples, according to the manufacturer's instructions (Thermo Fisher Scientific, Massachusetts, USA).
Statistical analysis. ANOVA with a post hoc analysis by the Student-Newman-Keuls' test was used, within each test, to compare the results obtained with the different constructions for each concentration in the different assays. All values were expressed as arithmetic means ± sem (standard error of the media). Differences between experimental groups were considered statistically significant at P < 0.05. Scientific RepoRtS | (2020) 10:12255 | https://doi.org/10.1038/s41598-020-69166-w www.nature.com/scientificreports/ Equipment and settings. Gel image from Figs. 1B and 2B was acquired and analyzed using the Gel Doc XR Imaging System provided with Quantity One 1-D v4.6 analysis software (Bio-Rad, Hercules, CA, USA; www. bio-rad.com). Blots images from Fig. 1B, were acquired and analyzed using UVP ChemiDoc-It TS2 provided with UPV VisionWorks LS 7.0 analysis software (www.uvp.com). No high-contrast gels or blots has been displayed. When processing of brightness and contrast of gel and blot images has been made it was applied to the entire image including controls. No cropped gels neither juxtaposing images were displayed. SigmaPlot-11.0 Scientific Data Analysis and Graphing Software (Systat Software Inc.; www.sigma plot.co.uk) was used for statistical analysis and graphing of experimental data in Figs. 1C, 2A, 3, 4, 5, 6 and 7.

Data availability
All experimental data generated or analyzed during this study are included in the article.