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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 Å3 Da−1.
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, PGF2α, 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.
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 PGF2α, 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, PGF2α, 6-keto-PGF1α, 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.
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 IgG1κ 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).
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 ).
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. 4 c) in gel-filtration column chromatography. In sharp contrast, the apo Fab fragment eluted in two broad peaks from the same column (Fig. 4 b), 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.
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 Å3 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.
We thank T. Hori and T. Shimamura for valuable comments.