Animals and cells
Female Sprague‒Dawley rats were purchased from SLC Inc., Japan and maintained under specific pathogen-free (SPF) conditions at the animal facility of Juntendo University. To perform experiments under the same conditions without bias, all animals were allowed to acclimate for 7 days before they were assigned work. All animal studies were performed according to the National Institute of Health Guidelines for Care and Use of Laboratory Animals. The experimental protocol was approved by the Animal Experimentation Committee of Juntendo University (Registration Number 1181, Approval Number 260229). Mouse mast cells (PT-18), generous gifts from Dr. C. Ra (Department of Immunology, Nippon University, Tokyo, Japan), were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA) supplemented with 10% heat-inactivated FCS, 3 mM L-glutamine, 1 mM sodium pyruvate with penicillin (100 U/ml), and streptomycin (100 g/ml) and maintained in a humidified incubator (5% CO2) at 37 °C.
Monoclonal antibodies
SPE-7 IgE (anti-DNP murine IgE antibody) and SPE-7 IgE F(ab’)2 were purchased from Sigma‒Aldrich Co. LLC (USA) and Immuno-Biological Laboratories Co., Ltd. (Gunma, JAPAN), respectively. The rat monoclonal antibody HMK-12 (specific for murine IgE) was used as described previously30,37. Rat IgG anti-murine κ antibodies and HRP-labelled goat anti-rat IgG were purchased from BioLegend (CA, USA) and Jackson ImmunoResearch (PA, USA), respectively. Highly purified Fab fragments of HMK-12 were prepared using Ficin, a cysteine protease isolated from fig latex. Briefly, HMK-12 from rat ascites was purified by combined stepwise caprylic acid and ammonium sulfate treatment. The purified HMK-12 was dialyzed against 0.1 M citrate buffer (pH 6.0), and then n-octyl-β-D-glucoside (DOTITE) and L-cysteine (Wako) were added to bring their concentrations to 0.5 mM and 25 mM, respectively. HMK-12 (1 mg/ml) then twice digested 0.5 ml of the immobilized Ficin resin slurry (Thermo Scientific prod. # 44881) in the presence of 25 mM cysteine/citrate buffer. The samples were then centrifuged and purified with vivaspin (1,000,000) (Sartorius AG) to remove resin or undigested IgG. The resulting HMK-12 Fab fragments were further purified with Protein G columns (Thermo Fisher Scientific Inc.).
PCA reactions
PCA reactions were performed as previously described38,39. Several sites of freshly shaved skin of 2 rats were injected intradermally with 1 μg/ml SPE-7 IgE. Two days later, 5 μg/ml doses of HMK-12 Fab or anti-k Fab were injected before challenge with DNP-BSA in saline (1 mg/ml) containing 0.5% Evans blue.
Extravasation of Evans blue from blood vessels into tissues was quantified by measuring the absorbance at 620 nm.
Flow cytometry analysis
Various concentrations of Alexa 488-labelled SPE-7 IgE were premixed with an excess (2 μM) of HMK-12 Fab or IgG2a Fab and incubated for 15 min at 37 °C. These mixtures were added to PT-18 mouse mast cells. After incubation for 20 min at 37 °C, the cells were washed twice with PBS and analysed using a FACSCelesta flow cytometer (Becton-Dickenson, CA, USA).
To investigate the dissociation efficiency of preformed IgE-FcεRI complexes by HMK-12 Fab, 1 x 107 PT18 cells were preincubated with 0.2 mM Alexa 488-labelled SPE-7 IgE for 15 min at 37 °C. The cells were washed twice to remove excess unbound antibodies and incubated with HMK-12 Fab or IgG2a Fab at various temperatures (2, 25 and 37 °C) for 5, 15 and 75 min. The cells were then washed and analysed using a FACSCelesta flow cytometer.
To further confirm the dissociation of IgE-FcεRI complexes, 1 x 107 PT18 cells were preincubated with 0.2 mM Alexa 488-labelled SPE-7 IgE for 15 min at 37 °C. After a washing step, they were incubated with HMK-12 Fab or IgG2a Fab at 37 °C for 15 and 60 min, and the fluorescence intensity was determined using a FACSCelesta flow cytometer. To measure the level of dissociated SPE-7 IgE, culture supernatants were added to DNP-BSA-coated plates to capture SPE-7 IgE/HMK-12 Fab immune complexes and assessed by ELISA using HRP-labelled anti-rat IgG (Fab’)2 secondary antibodies.
Western blot analysis
SPE-7 IgE (1 μg) was reduced with 2.5% 2-mercaptoethanol (2-ME) and run on 7.5% SDS‒PAGE sample buffer, transferred to PVDF membranes (Millipore Corp.) and probed with HMK-12 Fab, 6HD5 Fab and anti-rat l chain, followed by secondary antibodies (HRP-labelled anti-rat IgG for HMK-12 Fab and 6HD5 Fab, HRP-labelled anti-goat IgG for anti-rat l chain). Signals were developed using Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific), and immunoreactivity was detected with an ImageQuant LAS 4000 (GE Healthcare). To examine how HMK-12 Fab reacts with IgE during the process of reduction, SPE-7 IgE was treated with a serial dilution of dithiothreitol (DTT: 5-20 mM) for 60 min at room temperature. The samples were then incubated with 5 ml of 1.5 M Tris-HCl (pH 8.8) and 10 ml of iodoacetamide (IAA) for 30 min on ice. After dialysis, the samples were subjected to SDS‒PAGE under nonreducing conditions, followed by Western blot analysis. To define the epitopes on the IgE dimers recognized by HMK-12 Fab, SPE-7 IgE was digested with pepsin at pH 3.8 or pH 4.5 and then analysed for HMK-12 Fab reactivity with SDS‒PAGE under nonreducing conditions followed by Western blot analysis.
Crystallization
The peak fractions in Extended Data Fig. 1C containing HMK-12 Fab/IgE F(ab’)2 complexes were mixed and used for crystallographic studies. After the addition of sodium azide at a final concentration of 0.001% (w/v), mixed fractions were concentrated to 13.3 mg/ml by a Viva spin with MWCO 10,000.
Crystallization was performed by the hanging drop vapour diffusion method. Droplets were prepared by mixing 0.1 µl of the concentrated complex sample with 0.1 µl of a reservoir solution (14% (w/v) PEG3350, 0.09 M MES-NaOH (pH 6.5), 0.09 M magnesium acetate, and 0.09% (w/v) β-octylglucoside, 0.1 M NDSB-256) and equilibrated against a 50 µl reservoir solution at 4 °C. Prior to crystal freezing for X-ray diffraction experiments, 0.2 µl of cryoprotectant solution (15% (w/v) PEG3350, 0.1 M MES-NaOH (pH 6.5), 0.1 M magnesium acetate, and 0.1% (w/v) β-octylglucoside, and 0.1 M NDSB-256, 40% (v/v) ethylene glycol) were added to crystallization droplets containing needle-like microcrystals with dimensions of several tens of µm long and a few µm thick.
Diffraction experiments and structure analysis
X-ray diffraction experiments and diffraction image processing were performed automatically with BL32XU/SPring-8 using the ZOO system in the multicrystal data collection mode40. The X-ray wavelength was 1.0 Å, and diffraction images were collected for three hundred forty-eight crystals maintained in a 100 K N2 gas stream. Processing was performed on diffraction intensity data at 2.9 Å resolution with a CC0.5 of 0.899 at the highest resolution shell from 3.00 Å to 2.90 Å. Details of diffraction intensity data are provided in Extended Data Table 1.
The molecular replacement method using the program phenix.phaser with initial models of PDB ID 1i9i for IgG Fab, PDB ID 2vxq for IgE Fab and PDB ID 1o0v for IgE Cε2 was employed for initial phase calculation40. Structural refinement with phenix.refine and phenix.rosetta_refine and manual model modification using COOT were performed iteratively until the crystallographic R and Rfree factors converged to 0.228 and 0.267, respectively41,42. Details of the statistics of structural refinement are given in Extended Data Table 1. The structural coordinates and the structure factors were deposited in the protein data bank with PDB ID 8H2P. PyMOL was used to prepare figures for structural models in this work.