ANTIBODIES TO PHOSPHOLIPASES
The SP-Sephadex beads are then packed into a column on top of a fresh layer of SP-Sephadex C-25. The column is eluted with a salt gradient from 0 to 0.5 M NaCI. The proPLA2 peak elutes after the majority of the strongly absorbing colored material which forms the large peak seen in Fig. 4. At this stage the proPLA 2 is the major band on SDS-polyacrylamide gel electrophoresis. After dialysis, the proPLA2-containing fractions are further purified on a carboxymethyl-cellulose column at pH 6.0 in 5 mM acetate buffer. The resulting preparation is desalted and freed from traces of colored material on a Sephadex G-50 fine column in 10 mM acetic acid. Yeast proPLA 2 has the same turnover number for monomeric substrates as native proPLA 2 , although its activation peptide in the a-mating factor and in the G A L 7 constructs is 9 amino acids longer than in native proPLA2. Apparently the extra amino acids do not hinder the entrance of substrate to the active site; they are probably fully exposed to the solvent and do not form contacts with any other part of the enzyme. This is also evident from the fact that yeast proPLA2 is as rapidly cleaved by trypsin as is native proPLA 2 . The resulting yeast PLA2 is indistinguishable from authentic pancreatic PLA2, showing that a protein with as many as seven disulfide bridges per 124 amino acid residues is correctly processed by the yeast secretory apparatus. Yeast, rather than E. coli, may therefore be the organism of choice to express PLA 2 mutants with impaired folding properties.
 P r e p a r a t i o n o f A n t i b o d i e s to P h o s p h o l i p a s e s A 2 B y MAKOTO M U R A K A M I , KIYOSHI TAKAYAMA, MASATO U M E D A , ICHIRO K U D O ,
and KEIZO INOUE
Introduction P h o s p h o l i p a s e s A 2 play important roles in several biological phenomena such as membrane phospholipid turnover, production of lipid mediators (eicosanoids, platelet-activating factor, etc.),~ and the process of inflammation. 2,3 Phospholipases A 2 a r e classified into two groups in terms z H. van den Bosch, Biochim. Biophys. Acta 6114, 191 (1980). 2 p. Vadas and W. Pruzanski, Lab. Invest. 55, 391 (1986). 3 p. Vadas, S. Wasi, H. Z. Movat, and J. B. Hay, Nature (London) 293, 583 (1981).
METHODS IN ENZYMOLOGY,VOL. 197
Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any formreserved.
PHOSPHOLIPASE STRUCTURE--FUNCTION TECHNIQUES
of primary structure. 4,5 The group I family includes the enzymes from Elapidae and Hydrophidae snake venom or mammalian pancreas, while the group II family includes the enzymes from Crotalidae and Viperadae snake venom. Recently, phospholipases A2 belonging to group II have been detected in some mammalian nonpancreatic sources. 6These enzymes have been shown to share common characteristics, such as a low molecular weight (-14,000), a high content of basic and hydrophobic amino acid residues, stability to acids, a high affinity for heparin, and more effective Ca2+-dependent hydrolysis of phosphatidylethanolamine than phosphatidylcholine at alkaline pH. To gain an understanding of the physiological functions, distribution, dynamics, and structural characteristics ofphospholipases Az, application of antibodies might provide useful information. Such antibodies would also be useful for identification and purification of the enzymes. Reports dealing with antibodies to phospholipases A2 are listed in Table I. 7-9 In this chapter, we describe the preparation and possible application of antibodies to mammalian group II phospholipases A2 obtained from both rat platelets 7'8 and human rheumatoid synovial fluid. 9
Monoclonal Antibodies to Phospholipases A2 from Rat Platelets 7 or Human Rheumatoid Synovial Fluid
Preparation of Antigen Rat Platelet Phospholipase A2. Blood is taken by cardiac puncture from ether-anesthetized Wistar rats (>400 g body weight, male) through a 20-gauge butterfly needle. 7 To minimize platelet activation, blood is collected into a plastic syringe containing 3.8% (w/v) sodium citrate in a ratio of 1 part to 9 parts blood. The blood is gently transferred to plastic tubes and centrifuged for 10 rain at 270 g at room temperature to prepare platelet-rich plasma (PRP). After addition of a 1 : 5 (v/v) solution of 65 mM citric acid and 85 mM sodium citrate in 2% (w/v) glucose (ACD solution) to the PRP, platelets are separated from plasma by centrifugation at 1500 4 R. L. Heinrikson, E. T. Krueger, and P. S. Keim, J. Biol. Chem. 252, 4913 (1977). 5 E. A. M. Fleer, H. M. Verheij, and G. H. de Haas, Eur. J. Biochem. 82, 261 (1978). 6 I. Kudo, H. W. Chang, S. Hara, M. Murakami, and K. Inoue, Dermatologica 179, 72 (1989). 7 M. Murakami, T. Kobayashi, M. Umeda, I. Kudo, and K. Inoue, J. Biochem. (Tokyo) 104, 884 (1988). 8 M. Murakami, I. Kudo, Y. Natori, and K. Inoue, Biochim. Biophys. Acta 1043, 34 (1990). 9 K. Takayama, I. Kudo, S. Hara, M. Murakami, M. Matsuta, T. Miyamoto, and K. Inoue, Biochem. Biophys. Res. Commun. 167, 1309 (1990).
ANTIBODIES TO PHOSPHOLIPASES A 2
TABLE I ANTIBODIES TO PHOSPHOLIPASESA 2 Enzyme source
Pig, horse, ox, sheep pancreas Human pancreas Human seminal plasma Rat pancreas Swine intestine Porcine pancreas Naja naja venom Rat liver mitochondria Rat platelets Human rheumatoid synovial fluid
Polyclonal Polyclonal Polyclonal Polyclonal Polyclonal Polyclonal Polyclonal Monoclonal Monoclonal, polyclonal Monoclonal
a b, c d e f g h i j, k l
a H. Meijer, M. J. M. Meddens, R. Dijkman, A. J. Slotboom, and G. H. de Haas, J. Biol. Chem. 253, 8564 (1978). b j. Nishijima, M. Okamoto, M. Ogawa, G. Kosaki, and T. Yamano, J. Biochem. (Tokyo) 94, 137 (1983). c B. Sternby and B. Akerstrom, Biochim. Biophys. Acta 128, 788 (1985). d M. Wurl and H. Kunze, Biochim. Biophys. Acta 834, 411 (1985). e M. Okamoto, T. Ono, H. Tojo, and T. Yamamoto, Biochem. Biophys. Res. Commun. 128, 788 (1985). YF. Senegas-Balas, D. Balas, R. Verger, A. de Caro, C. Figarella, F. Ferrato, P. Lechene, C. Bertrand, and A. Ribet, Histochemistry 81, 581 (1984). g D. Bar-Sagi, J. P. Susan, F. McCormick, and J. R. Feramisco, J. Cell Biol. 106, 1649 (1988). h j. Masliah, C. Kadiri, D. Pepin, T. Rybkine, J. Etienne, J. Chambaz, and G. Bereziat, FEBS Lett. 222, 11 (1987). i j. G. N. de Jong, H. Amerz, A. J. Aarsman, H. B. M. Lenting, and H. van den Bosch, Eur. J. Biochem. 164, 129 (1987). J Ref. 7. k Ref. 8. t Ref. 9.
g for 10 min at room temperature. The cells are then gently resuspended in 5 parts HEPES-Tyrode buffer [137 mM NaCI, 2.7 mM KC1, 10 mM HEPES, and 0.1% (w/v) glucose] and 1 part ACD, then washed once. The washed platelets are resuspended in HEPES-Tyrode buffer at a final concentration of approximately 2 × 109 cells/ml. The platelets are then activated with 2.5 units/ml thrombin (Sigma, St. Louis, MO) in the pressure of 2 mM CaCI2 at 37 ° for 5 min. After platelet aggregation, the mixture is centrifuged at 3000 g for 5 min at 4 ° to obtain supernatant fluid. The supernatant fluid is used as an enzyme source after allowing it to stand at 4 ° for 5 hr and separating any fibrin clots by centrifugation (4000 g, 10 min). Starting from 70 rats, approximately 200 ml of supernatant fluid will be obtained.
PHOSPHOLIPASE STRUCTURE-FUNCTION TECHNIQUES
Phospholipase A 2 in the fluid is purified by the sequential use of column chromatography on heparin-Sepharose (Pharmacia, Uppsala, Sweden) and TSK gel G2000SW (Tosoh, Tokyo, Japan) gel filtration [high-performance liquid chromatography (HPLC)]. 10Fractions containing phospholipase A2 are pooled and used for immunization. Human Rheumatoid Synovial Fluid Phospholipase A 2 . Phospholipase A2 is purified to near homogeneity from the synovial fluid9 of patients with rheumatoid arthritis by the sequential use of column chromatographies on heparin-Sepharose, butyl-Toyopearl (Tosoh), and reversed-phase HPLC (Shiseido, Tokyo, Japan).1° Immunization by Intrasplenic Injection
Ten micrograms of purified phospholipase A2 is absorbed on a nitrocellulose filter (1 x 1 cm). The filter is then minced, suspended in 250/~1 of phosphate-buffered saline (PBS), pH 7.4 (137 mM NaC1, 2.7 mM KCI, 80 mM Na2HPO4, and 1.5 mM KH2PO4), and homogenized using a Polytron homogenizer (Kinematica GmbH, Luzern, Switzerland). The suspension is injected into a mouse by intrasplenic injection H as described below. A BALB/c mouse (8- to 12-week-old female) is anesthetized by intraperitoneal injection of 1 mg of Nembutal (Abbott Laboratories, North Chicago, IL), and the antigen suspension is injected with a 25-gauge butterfly needle into the spleen, which is exposed after laparotomy. After the injection, the spleen is replaced in the peritoneal cavity and the peritoneal wall is sutured with platinum staples. Production o f Monoclonal Antibodies by Hybridomas
The fusion procedure is that described by Galfre et al. 12 and Kohler and Milstein et al. 13with some modifications. Three days after the second intrasplenic injection (2-week interval), a spleen cell suspension is prepared by gently teasing the spleen while holding it with a pair of forceps in a petri dish. The cells are washed twice with Dulbecco's modified Eagle's medium (DMEM) (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) and resuspended in 10 ml of the same medium. Myeloma cells (P3X63Ag.8.653) are collected at the logarithmic growth phase, washed twice, and resuspended in DMEM. Then 108 spleen cells and 107 myeloma cells are mixed and pelleted at 200 g for 5 min at room temperature. The i0 S. Hara, H. W. Chang, K. Horigome, I. Kudo, and K. Inoue, this volume . N M. Spitz, this series, Vol. 121, p. 33. 12 G. S. Galfre, C. Howe, C. Milstein, G. W. Butcher, and J. C. Howard, Nature (London) 266, 550 (1977). 13 G. Kohler and C. Milstein, Nature (London) 256, 495 (1975).
ANTIBODIES TO PHOSPHOLIPASES A 2
supernatant is carefully aspirated, removing as much fluid as possible, and the pellet is loosened by flicking the tube. One milliliter of a prewarmed (37°) 50% (w/v) polyethylene glycol (PEG) (molecular weight 4000; Merck, Darmstadt, FRG) solution in DMEM is added with gentle mixing to the cell slurry over a 1-min interval. The cells are incubated at 37° during the next minute, and then I0 ml of prewarmed (37°) DMEM is added drop by drop for 3 min to dilute the PEG. The cell suspension is pelleted by centrifugation (4°, 200 g, 5 min) and resuspended in 40 ml of HAT selecting medium consisting of DMEM supplemented with 2 mM glutamine (GIBCO Laboratories, Grand Island, NY), penicillin/streptomycin (I00 U/ml and 100 /xg/ml, respectively; Flow Laboratories, Irvine, Scotland), 1 mM nonessential amino acids (M. A. Bioproducts, Walkersville, MD), 20% fetal calf serum (GIBCO), 5% NCTC 109 (M. A. Bioproducts), 100 mM hypoxanthine (Flow), 0.4 mM aminopterin (Sigma), and 16 mM thymidine (Flow). The cell suspension is dispersed in 200-/zl volumes into two 96well tissue culture plates (Falcon, Lincoln Park, NJ) and incubated in a humid atmosphere of 5% CO2-95% air (v/v). After 1 week, the cells are fed with 50/zl of HAT medium per well. Thereafter the cells are fed every 2-3 days according to the degree of cell growth. Supernatants of confluent wells are screened for the presence of specific antibodies by enzymelinked immunosorbent assay (ELISA) (see below). Positive wells are cloned by limiting dilution. HYbridoma cells are cultured in medium in the presence of BALB/c mouse thymocytes (4- to 6-week-old female) at 1 × l 0 7 cells/ml as a feeder layer. After duplicate limiting dilution, the growing hybridoma clones are expanded and then injected intraperitoneally into BALB/c mice (10 7 cells/head) primed with pristane (0.5 ml/head; Aldrich Chemical Co., Milwaukee, WI). Ascites fluid is collected, and constituent antibodies are purified by 50% ammonium sulfate precipitation followed by affinity chromatography on protein A-Sepharose (Pharmacia) by the method of Ey et al. ~4 ELISA Procedure
The wells of microtiter plates (Immulon 2; Dynatech Laboratories, Alexandria, VA) are coated with 50/zl of purified phospholipase A z in PBS (1/~g/ml) for 2 hr. After blocking with 150/~1 of 1% bovine serum albumin (BSA) (Sigma) in PBS (PBA solution) for 2 hr, the plates are treated sequentially with 50/.d of hybridoma supernatant for 2 hr and 50/xl of horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin (Cappel Laboratories, Dowingtown, PA) in PBA (1 : 1000 dilution) for 2 14 p. L. Ey, S. J. Prowse, and C. R. Jenkin, Immunochemistry 15, 429 (1978).
PHOSPHOLIPASE STRUCTURE-FUNCTION TECHNIQUES
hr, then stained with 100 btl of o-phenylenediamine (100/xg/ml in 0.1 M citrate-phosphate buffer, pH 5.0, containing 0.003% (v/v) H202; Wako, Osaka, Japan). The reaction is stopped by adding 50/xl of 4 N H2SO 4 . The optical density at 492 nm is determined by an ELISA reader. All of the above procedures are performed at room temperature.
Application of Monoclonal Antibodies Western Blotting. Samples (such as tissue or cell homogenates) are resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) using a 15% (w/v) gel by the method of Laemmli 15and transferred to a Millipore GVHP filter (Nihon Millipore Kogyo, K. K., Yonezawa, Japan) for immunoblot analysis [7.5 V/cm for 30 min and then 37.5 V/cm for 2 hr, in a buffer containing 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol]. It should be emphasized that the electrophoresis must be carried out in the absence of 2-mercaptoethanol (2-ME), since treatment of the enzyme with 2-ME results in loss of reactivity with all of the antibodies so far established.7-9 The filter is blocked with PBA solution for 2 hr and then incubated with biotinylated antibody (1/zg/ml) diluted with PBA for 2 hr. The biotinylated antibody is prepared by incubation of the antibody with biotin N-hydroxysuccinimide ester (Behring Diagnostics, La Jolla, CA) as described. 7 The filter is incubated for 1 hr in a solution containing 1 : 1000-diluted HRP-conjugated streptavidin (Zymed Laboratories, San Francisco, CA). After washing the filter with PBS, the color is developed by adding 5 mg of HRP color-developing reagent (2chloronaphthol; Bio-Rad Laboratories, Richmond, CA) in 2.5 ml of methanol, 5/.d of 30% H202, and 10 ml of reaction buffer (20 mM Tris-HCl, pH 7.4, 0.5 M NaC1) and incubated for 5 min on a rocking platform. The reaction is stopped by rinsing the filter with distilled water. All the above procedures are carried out at room temperature. Immunoaffinity Purification of Mammalian Group H Phospholipases A 2 Coupling of antibody to sepharose beads. Four milligrams of antibody is coupled to 2 ml of cyanogen bromide-activated Sepharose (Pharmacia) in 0.1 M citrate buffer (pH 6.8) for 30 min at room temperature. The efficiency of the coupling reaction is usually higher than 90% under these conditions. The beads are blocked by incubation with 1 M ethanolamine (pH 8.0) for 2 hr, packed into an Econo column (1 × 10 cm; Pharmacia), ~s U. K. Laemmli, Nature (London) 227, 680 (1970).
ANTIBODIES TO PHOSPHOLIPASES A 2
and then washed extensively with 10 mM Tris-HC1 (pH 7.4) containing 0.15 M NaCI (TBS). Purification o f rat platelet phospholipase A 2 . Washed rat platelets are sonicated 3 times for 30 sec each time. The lysate is incubated for 12 hr at 4° in I0 mM Tris-HCl (pH 7.4) containing I M KC1 (>80% of enzyme activity is solubilized under these conditions) and centrifuged at I00,000 g for 1 hr, after which the supernatant is applied to an anti-rat platelet phospholipase A 2 antibody MD7.1-Sepharose affinity column. To avoid nonspecific adsorption, a precolumn of Sepharose CL-4B (1 × 10 cm; Pharmacia) is attached before the affinity column. The flow rate is below 10 ml/hr, and the effluent is monitored with a UV monitor (Pharmacia) at 280 nm. The column is washed extensively with TBS, and the bound enzyme is subsequently eluted with 50 mM glycine-HCl buffer (pH 2.3). The eluate at low pH gives a single protein band on SDS-PAGE, the molecular weight of which is estimated to be 14,000, corresponding to phospholipase A2 (Fig. 1). The fractions are neutralized with 1 N NaOH immediately after the elution. The recovery of the enzyme activity is approximately 90%. From platelets of 70 rats in a single preparation, 1.3 mg of phospholipase A2 is obtained. This procedure can be applied to other immuno-cross-reactive phospholipases A 2. For example, rabbit platelet phospholipase A2 has been purified by the same procedure as that for rat platelet enzyme using an antirat platelet phospholipase A 2antibody MD7.1-Sepharose affinity column. 16 Purification o f human rheumatoid synovial fluid phospholipase A 2 . Pooled synovial fluid from patients with rheumatoid arthritis is loaded onto an anti-human synovial fluid phospholipase A 2 antibody HP-1-Sepharose column, with a precolumn of Sepharose CL-4B attached before the antibody column. The flow rate is below 10 ml/hr. All of the phospholipase A2 activity in synovial fluid is adsorbed by the antibody column. The column is washed extensively with 10 mM Tris-HCl (pH 7.4) containing 1 M NaCl and then subsequently eluted with 0.1 M glycine-HC1 buffer (pH 2.3). The eluate at low pH contains one major protein band together with two minor ones on SDS-PAGE. Among them, only the major protein with a molecular weight of approximately 14,000 is reactive with the antihuman synovial phospholipase A 2 antibody when the eluate is examined by Western blotting. The fractions are neutralized with 1 N NaOH. The enzyme is purified approximately 1.6 x 105-fold in a single purification step. It should be noted that the recovery of total activity is 200-300%; thus, some inhibitory factor(s) might be removed during this purification step. J6 H. Mizushima, I. Kudo, K. Horigome, M. Murakami, M. Hayakawa, D,-K. Kim, E. Kondo, M. Tomita, and K. Inoue, J. Biochem. (Tokyo) 105, 520 (1989).
PHOSPHOLIPASE STRUCTURE-FUNCTION TECHNIQUES
-.~ 14 kDa 1 . .=
O ¢0 m
o O C ¢Q