Crystallization and preliminary diffraction studies of prostaglandin E₂-specific monoclonal antibody Fab fragment in the ligand complex

Prostaglandin E₂ is a major lipid mediator that regulates diverse biological processes. To elucidate how prostaglandin E₂ is recognized specifically by its antibody, the Fab fragment of a monoclonal anti-prostaglandin E₂ antibody was prepared and its complex with prostaglandin E₂ was crystallized. The stable Fab–prostaglandin E₂ complex was prepared by gel-filtration chromatography. Crystals were obtained by the microbatch method at 277 K using polyethylene glycol 4000 as a precipitant. A diffraction data set was collected to 2.2 Å resolution. The crystals belonged to space group P2₁2₁2₁, with unit-cell parameters a = 70.3, b = 81.8, c = 82.2 Å. The asymmetric unit was suggested to contain one molecule of the Fab–prostaglandin E₂ complex, with a corresponding crystal volume per protein weight of 2.75 ų Da⁻¹.

1. Introduction

Prostanoids such as prostaglandins (PGs) and thromboxane (TX) have numerous and diverse biological activities under various physiological and pathological conditions. In the metabolic pathway of prostanoids, dioxygenases such as cyclooxygenase catalyze the conversion of arachidonic acid to prostaglandin H₂ (PGH₂), which is a unique and unstable intermediate from which all the prostanoids (PGD₂, PGE₂, PGF_2α, PGI₂ and TXA₂) are produced via specific synthases. PGE₂ is one of the most widely investigated prostanoids. Exhaustive studies have revealed biologically pivotal roles of PGE₂ in inflammation, pain, gastric mucosal integrity and the immune system (Willis & Cornelsen, 1973; Robert et al., 1976; Arvind et al., 1995). The distinct biological actions of PGE₂ are exerted by its binding to four distinct PGE₂ receptors, EP1–EP4 (Sugimoto & Narumiya, 2007).

PGE₂ (Fig. 1) is an unsaturated oxygenated product of arachidonic acid with a cyclopentane ring, α- and ω-chains attached to the ring and a carboxyl group. PGE₂ has two double bonds (5-cis and 13-­trans), 11-α and 15S hydroxyl groups and a 9-carbonyl group.

Figure 1 Chemical structure of PGE2.

The specific and sensitive quantification of PGE₂ in biological samples is crucial for biological and biochemical studies. Therefore, an enzyme-linked immunoassay system for PGE₂ has been developed by Shono et al. (1988). The anti-PGE₂ monoclonal antibody generated for this purpose was used in this study. The antibody has specific and high affinity for PGE₂ and 9-deoxy-9-methylene PGF_2α, which has been utilized as a stable PGE₂ mimic to raise anti-PGE₂ antibodies (Fitzpatrick & Bundy, 1978). The antibody has lower affinity towards other analogous prostanoids such as PGD₂, PGF_2α, 6-keto-PGF_1α, TXB₂ and metabolites of PGE₂ (Shono et al., 1988). The mechanism by which the antibody distinguishes PGE₂ from other PGs and the interactions between PGs and their receptors are not well understood. Therefore, it is indispensable to determine the atomic structure of the complex of the antibody with PGE₂. Here, we report the crystallization of and preliminary crystallographic studies on the complex of PGE₂ with the Fab fragment of the antibody. The findings from this work will support ligand design for drug discovery.

2. Materials and methods

2.1. Preparation of anti-PGE₂ antibody Fab fragment

Monoclonal anti-PGE₂ antibody was produced using a mouse hybridoma cell line established by Shono et al. (1988) in GIT serum-free medium (Nihon Pharmaceutical). The antibody that accumulated in the culture medium was collected and purified using Protein A Sepharose CL4B column chromatography (GE Healthcare). The collected fractions were concentrated to 1 mg ml⁻¹ protein in PBS buffer (10 mM sodium phosphate pH 7.4, 140 mM NaCl) using ultracentrifugation. The light-chain subtype of the mouse antibody was determined to be IgG1_κ using the Isotyping Monoclonal Antibodies Kit (GE Healthcare). The Fab fragment was prepared by papain digestion of the antibody (5 mg ml⁻¹) with 1:1000(w:w) papain (Calbiochem) for 3 h at 310 K in 75 mM sodium phosphate buffer pH 7.0 containing 20 mM cysteine–HCl, 75 mM NaCl, 2 mM EDTA and 5 mM NaN₃. The reaction was stopped by the addition of N-ethylmaleimide to a concentration of 20 mM. After incubation for 30 min at room temperature in the dark, the digested product was adsorbed onto HiTrap Protein G HP (GE Healthcare) equilibrated with 20 mM sodium phosphate buffer pH 7.0. After washing with the same buffer, the Fab fragment was eluted with 0.1 M glycine–HCl buffer pH 2.75. The collected Fab fraction was concentrated in Fab buffer (10 mM sodium phosphate pH 7.0, 140 mM NaCl) to a protein concentration of 10 mg ml⁻¹ with an ultrafiltration apparatus (Viva­spin 20, Sartorius).

2.2. Crystallization and data collection of the Fab–PGE₂ complex

20 µl 1 mg ml⁻¹ PGE₂ in ethanol (Cayman) was dispensed and dried in 1.5 ml sample tubes under a nitrogen-gas stream. After mixing the Fab fragment with a twofold molar excess of PGE₂, the Fab–PGE₂ complex was purified by Superose12 HR 10/30 gel-filtration column chromatography (GE Healthcare) equilibrated with Fab buffer. The eluted proteins were monitored by UV absorbance at 280 nm. The following standard proteins were used as molecular-weight markers (their molecular weights and elution volumes are given in parentheses): chymotrypsinogen A (25 000 Da, 14.8 ml), albumin (67 000 Da, 12.5 ml), catalase (232 000 Da, 11.6 ml) and thyro­globulin (669 000 Da, 8.14 ml) (GE Healthcare). The eluted Fab–PGE₂ complex fraction was concentrated using a centrifugal concentrator (Vivaspin 20, Sartorius). Initial crystallization was carried out by the oil-microbatch method (Chayen et al., 1990) using a screening kit designed for high-throughput protein crystallization (Sugahara & Miyano, 2002). TR plates (Takeda Rika Kogyo) were used in which 0.5 µl of each screening solution was mixed with 0.5 µl protein solution (38 mg ml⁻¹ protein in Fab buffer) and the mixture was covered with 15 µl paraffin oil; this was followed by incubation at 277 K. During optimization of the initial crystallization conditions, needle-shaped crystals appeared after a few days from a screening solution composed of 12.5%(w/v) PEG 4000, 50 mM MgCl₂ in 0.1 M Tris–HCl buffer pH 8.2. Thick needle-shaped crystals (Fig. 2) were transferred using a nylon loop (Hampton Research) from the crystallization drop into a cryoprotectant comprised of Paratone N and 10%(v/v) glycerol (Kwong & Liu, 1999). After removal of the aqueous solution from around the crystal, it was flash-frozen using a liquid-nitrogen gas stream at 100 K. X-ray diffraction data were collected on an ADSC Q210 CCD detector using synchrotron radiation on the RIKEN beamline BL44B2 at SPring-8. The native diffraction data were collected as 180 oscillation images with an exposure time of 20 s and an oscillation range of 1° using a camera distance of 200 mm. Images were processed using the programs MOSFLM (Leslie, 1992) and SCALA and the CCP4 suite of programs (Collaborative Computational Project, Number 4, 1994).

Figure 2 Needle-shaped crystals of the Fab–PGE2 complex. Nine graduations on the scale in this photograph correspond to 0.1 mm.

3. Results and discussion

Monoclonal anti-PGE₂ antibody was produced on a large scale by culturing an established hybridoma cell line (Shono et al., 1988) in serum-free medium. Approximately 7–8 mg IgG was obtained from 400 ml culture medium. 2 mg of the Fab fragment was purified from 20 mg IgG protein (Fig. 3). As shown in Fig. 4(a), the Fab complexed with PGE₂ showed a sharp elution peak at 43 kDa (Fig. 4c) in gel-filtration column chromatography. In sharp contrast, the apo Fab fragment eluted in two broad peaks from the same column (Fig. 4b), with the ratio of the peak heights varying from experiment to experiment. These eluates migrated at the same distance as the Fab–PGE₂ complex on SDS–PAGE (data not shown). Presumably, the purified Fab fragment exists in at least two forms hydrodynamically and the addition of PGE₂ converts these multiple forms to a single form which gives a single sharp peak as in Fig. 4(a). We attempted to crystallize both apo Fab and the Fab–PGE₂ complex. Crystals of the Fab–PGE₂ complex were obtained with dimensions of 0.3 × 0.01 × 0.01 mm after two weeks (Fig. 2), but the apo Fab could not be crystallized despite exhaustive trials. The unsuccessful crystallization of apo Fab may be a consequence of its multiple forms as mentioned above.

Figure 3 SDS–PAGE (12.5%) of the purified monoclonal anti-PGE2 antibody (lane 1), Fc fragment (lane 2) and Fab fragment (lane 3). Lane M, molecular-weight markers (labelled in kDa). The samples were prepared prior to electrophoresis by heating in the presence of 1 mM 2-mercaptoethanol.

Figure 4 Gel-filtration chromatograms. (a) Fab–PGE2 complex, (b) apo Fab. (c) Graph of elution time versus molecular weight for molecular-weight markers and the Fab–PGE2 complex.

The collected diffraction images (Fig. 5) were reduced to 24 732 reflections (the total number of reflections was 178 036) in the resolution range 58–2.2 Å with an R_merge of 11.4% (Table 1). Crystals of the Fab–PGE₂ complex belong to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 70.3, b = 81.8, c = 82.2 Å. The Matthews coefficient of 2.75 ų Da⁻¹ suggested the presence of one molecule of the Fab–PGE₂ complex (43 kDa) per asymmetric unit, corresponding to a solvent content of 55.3% (Matthews, 1968). As described above, we could obtain crystals from mixture of Fab and PGE₂ which gave a single sharp peak when subjected to gel filtration. The preparation was referred to as `PGE₂–Fab complex'. Our crystallographic analyses indicated the presence of PGE₂ in the crystal and a manuscript describing observations on the PGE₂–Fab complex structure is now in preparation.

Table 1
Crystal parameters and data-collection statistics

Values in parentheses are for the highest resolution shell.

X-ray source	Synchrotron (BL44B2 at SPring-8)
Wavelength (Å)	1.0000
Detector	CCD camera (ADSC Q210)
Space group	P212121
Unit-cell parameters (Å)
a	70.3
b	81.8
c	82.2
Mosaicity (°)	0.52
Wilson B factor (Å^2)	15.8
Resolution range (Å)	58–2.2 (2.32–2.2)
Total observations	178036 (25785)
Unique reflections	24732 (3523)
Multiplicity	7.2 (7.3)
Completeness (%)	100.0 (100.0)
Mean I/σ(I)	12.7 (5.0)
Rmerge† (%)	11.4 (42.1)

†R_merge = , where I_i(hkl) and 〈I(hkl)〉 are the observed intensity of the ith measurement and the mean intensity of the reflection with indices hkl, respectively.

Figure 5 Diffraction image of the Fab–PGE2 complex crystal.

Footnotes

‡These authors contributed equally to this work.