Thermal stability, ligand binding and allergenicity data of Mus m 1.0102 allergen and its cysteine mutants

The presented data were obtained with the lipocalin allergen Mus m 1.0102 and its cysteine mutants MM-C138A, MM-C157A and MM-C138,157A, whose structural features and unfold reversibility investigations are presented in the research article entitled “The allergen Mus m 1.0102: cysteine residues and molecular allergology” [1]. The data were obtained by means of a Dynamic Light Scattering-based thermal stability assay, a Fluorescence-based ligand-binding assay and a basophil degranulation test, and describe proteins’ fold stability, ligand binding ability and allergenic potential, respectively. Analysis of the collected data produced the temperatures corresponding to the onset of the protein unfolding, the dissociation constants for N-Phenyl-1-naphthylamine ligand and the profiles of β-hexosaminidase release from RBL SX-38 cells, sensitized with the serum of selected allergic patients and incubated with increasing antigens concentrations. These data allow for comparison of the lipocalin allergen Mus m 1.0102 with its conserved cysteines mutants and, with regard to their potential application in allergy diagnostics and immunotherapy, they contribute to the process of recombinant allergen characterization and standardization.

Value of the Data The data are useful to describe fold stability, ligand binding ability and allergenic potential of the recombinant lipocalin allergen Mus m 1.0102 and they allow for comparison with its conserved cysteines mutants. The data highlight the enhanced thermal stability of MM-C138A mutant, without a relevant modification of its binding function and in vitro allergenicity. The data contribute to the process of the recombinant allergen standardization, focused to its potential use in immunotherapy and diagnostics applications. Because of the high grade of conservation of lipocalin cysteines, these data suggest that a similar analytical approach might be applied for the preliminary characterization of any recombinant lipocalin allergen and its cysteine mutants.

Data description
The data presented in this article were obtained with the lipocalin allergen Mus m 1.0102 [1e3] and its cysteine mutants MM-C138A, MM-C157A and MM-C138,157A. The data derive from: 1) a Dynamic Light Scattering-based thermostability assay, 2) a Fluorescent-based ligand-binding assay and 3) an in vitro allergenicity test.
Dynamic Light Scattering data [4] of protein solutions are presented as scattering intensities (kilocounts per sec) during a temperature ramp from 25 to 80 C (Fig. 1), and report the protein transition from folded to unfolded state and aggregation. The temperature associated to an apparent signal increment (Tm onset ) corresponds to the onset of the protein unfolding, being the temperature at which the protein hydrodynamic radius starts to increase. The Tm onset values, presented in Fig. 1 caption, were calculated by a DLS dedicated software. Higher temperatures are indicative of aggregates formations.
The ligand binding test was performed by means of direct titration experiments, by increasing additions of N-Phenyl-1-naphthylamine (NPN) to protein solutions; the intensity of fluorescence emission of NPN probe, bound to Mus m 1.0102 and its mutants, was measured (Fig. 2). Fluorescence intensity data sets [4] were analysed by nonlinear regression to calculate the equilibrium dissociation constants, Kd, presented in Fig. 2 caption. Table 1 reports the diagnosis and serum IgE concentrations of ten allergic patients, selected to assess the proteins' allergenic potential. A degranulation assay was used as an in vitro allergenicity test. The assay used Rat basophilic leukemia cells, sensitized with the serum of the selected allergic patients, and measured the release of b-hexosaminidase in presence of increasing concentrations of Mus m 1.0102 and its cysteine mutants (Fig. 3) [4].

Thermal stability assay
For this set of experiments, recombinant proteins were dissolved in Tris-HCl 10 mM pH 7.2 at concentrations ranging from 45 to 240 mM and filtered (Anotop siringe filter 0.02 mm, Whatman) before use. Measurements were carried out using a Malvern Zetasizer mV instrument, the Temperature trend measurement type and a 2 mL quartz cuvette (ZMV1002, Malvern).
Thermal unfolding. To detect the onset of thermal unfolding, experiments were carried out using an automated 2.5 C incremental temperature ramp in the interval 25e80 C, with a 60 s equilibration time at each measurement step. As measurement progresses, the instrument software interpolates the most recent six points of the intensity trace in the aggregation point graph (Fig. 1) and calculates the slope; the temperature at which the slope exceeds the value of 1 corresponds to the melting onset temperature (Tm onset ).

Ligand binding assay
Direct titration experiments were performed with the fluorescent probe N-Phenyl-1naphthylamine (NPN, Sigma Aldrich), an effective probe for Mus m 1 proteins since it becomes strongly fluorescent when bound inside their hydrophobic pocket [5]. Measurements were performed on a JASCO FP-6200 spectrofluorometer, equipped with a Peltier-controlled cuvette holder. NPN binding was monitored by exciting at 337 nm and recording the emission spectra over the range 350e550 nm. Protein solutions, 20 nM in TrisHCl 10 mM pH 7.2, were titrated by increasing additions of NPN (up to the final concentration of 1500 or 2000 nM); fluorescence intensity was measured at 25 C, 20 min after the addition of NPN to allow for equilibrium to be reached. The per cent of maximum fluorescence intensity (recorded at l max ¼ 401 nm) was plotted versus increasing NPN concentration and a nonlinear regression analysis (SigmaPlot v. 11, Systat Software) was applied to calculate the dissociation constants of Mus m 1.0102 and its mutants.

Criteria for allergic patient selection
Ten subjects, in the 18e30 age range, were selected for the in vitro allergenicity test. All subjects fulfilled the following inclusion criteria: 1) they were diagnosed with allergic rhinoconjunctivitis with or without asthma by an experienced board-certified allergist, 2) their diagnosis was based on a clinical history of typical respiratory symptoms, appearing minutes or hours after exposure to the allergen source, according to the European Academy of Allergy and Clinical Immunology (EAACI) criteria, 3) minimum time from diagnosis was 2 years (only in one case this information was not available), 4) they had access to a standard pathogen-free animal facility for professional reasons.    Determination of allergen-specific IgE was performed by means of ImmunoCAP ISAC Immunoassay (Thermo Fisher/Phadia, Uppsala).
Patients 1e9 are allergic to Mus m 1, having Mus m 1-specific IgE titers in the range 0.82e65.39 ISU (ISAC Standardised Units); in addition, they have specific IgE molecules to at least one of the following allergens: Fel d 1, Equ c 1 and Can f 1.
Patient 10 was included as a control, being not sensitized against Mus m 1 allergen, but having Fel d 1, Equ c 1 and Can f 1-specific IgE molecules.
Informed consent for the use of sera was obtained from the allergic patients. Boston, MA, USA), stably expressing human FcεRI a, b and g chains, were cultured as previously described [6]. Following sensitization with IgE from a subset of human allergic donors, they were stimulated with allergen concentrations varying from 0.1 to 5.0 mM and a dose-response test of bhexosaminidase release was performed [7].
Experimental procedure and data analysis. To measure the amount of b-hexosaminidase released from the cells after the incubation with the allergen, p-Nitrophenyl-N-acetyl-b-D-glucosaminide substrate (p-NAG, Sigma-Aldrich) in 0.1 M citrate buffer (pH 6.2) was added to RBL-SX38 cell supernatants and incubated at 37 C for 120 min. The reaction was terminated using 0.1 M carbonate buffer pH 10 and the absorbance measured at 405 nm. The supernatant of non-stimulated cells was used as a negative control. The maximal stimulation was determined by using 100 ng of 4-hydroxy-3-nitrophenacetyl (NIP)-Bovine Serum Albumine (stoichiometric ratio NIP:BSA >10) (Biosearch Technologies, Vacaville, CA) as an IgE cross-linker [8], which reacted with RBL cells pre-incubated with a commercially available human recombinant IgE anti-NIP monoclonal antibody (Serotec, Milan, Italy) [9].
Negative control values consistently fell in the range of 15e25% of the total b-hexosaminidase content, assessed from the 0.05% Triton X-100 (Sigma-Aldrich) lysate of the cell monolayer. The extent of allergen reactivity (allergenicity) was expressed as percent of b-hexosaminidase release according to The authors ensure that the work described has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.