Journal of Neurochemistry Raven Press, Ltd., New York 0 I990 International Society for Neurochemistry

Studies on the Topography of the Catalytic Site of Acetylcholinesterase Using Polyclonal and Monoclonal Antibodies Robert A. Ogert, Mary K. Gentry, Earl C. Richardson, Carolyn D. Deal, *Stewart N. Abramson, Carl R. Alving, *Palmer Taylor, and B. P. Doctor Division of Biochemistry, Walter Reed Army Institute of Research, Washington, D.C.; and *Department of Pharmacology, University of California at San Diego, School of Medicine, La Jolla, California, U.S.A.

Abstract: Polyclonal and monoclonal antibodies were generated against a synthetic peptide (25 amino acid residues) corresponding to the amino acid sequence surrounding the active site serine of Torpedo californica acetylcholinesterase (AChE). Prior to immunization, the peptide was either coupled to bovine serum albumin or encapsulated into liposomes containing lipid A as an adjuvant. To determine whether this region of AChE is located on the surface of the enzyme and thus accessible for binding to antibodies, or located in a pocket and thus not accessible to antibodies, the immunoreactivity of the antibodies was determined using enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, Western blots, and competition ELISA. The polyclonal antibody and several ofthe monoclonal antibodies failed to react with either Torpedo or fetal bovine serum AChE in their native conformations, but showed significant cross-reactivity with the denatured enzymes. Human serum butyrylcholinester, which has a high degree of amino acid sequence homology with

these AChEs, failed to react with the same antibodies in either native form or denatured form. Chymotrypsin also failed to react with the monoclonal antibodies in either form. Eighteen octapeptides spanning the entire sequence of this region were synthesized on polyethylene pins, and epitopes of representative monoclonal antibodies were determined by ELISA. The reactivity of peptides suggests that a portion of the 25 mer peptide in AChE containing the active site serine is the primary epitope. It is not exposed on the surface of the enzyme and is most likely sequestered in a pocket-like conformation in the native enzyme. Key Words: Acetylcholinesterase-Monoclonal antibodies-Active site-TopographyConformation-Fetal bovine serum-Epitope-Synthetic peptide. Ogert R. A. et al. Studies on the topography of the catalytic site of acetylcholinesterase using polyclonal and monoclonal antibodies, J. Neurochem. 55, 756763 ( 1 990).

Acetylcholinesterase (AChE; acetylcholine acetylhydrolase, EC 3.1.1.7) catalyzes the hydrolysis of the neurotransmitter acetylcholine and is synthesized in cells forming cholinergic synapses. A mechanism of catalysis has been described for the serine hydrolases of known structure, such as trypsin, chymotrypsin, and subtilisin (Blow et al., 1969; Chambers and Stroud, 1977). In the case of cholinesterases, a similar, if not identical, mechanism of catalysis has been proposed on the basis of kinetic behavior and inhibitor specificity (Froede and Wilson, 1971 ;Aldridge and Reiner, 1972). Briefly, the nucleophilic serine in the catalytic site of

the enzyme attacks the carbonyl carbon of the substrate. A transition state enzyme-substrate complex is formed, and the tetrahedral transition-state intermediate is hydrolyzed to yield an acyl enzyme, which is subsequently hydrolyzed to yield acetate and free enzyme. For serine proteases, such as chymotrypsin, trypsin, and subtilisin, a mechanism generally known as a “charge relay” is envisioned by which the peptide bond is hydrolyzed. The amino acids Asp’’*, His”, and Ser195 are those implicated in proton transfer in the case of chymotrypsin. The folding pattern of this protein as

Received August 22, 1989; revised January 23, 1990 accepted January 24, 1990. Address correspondence and reprint requests to Dr. B. P. Doctor at Division of Biochemistry, Walter Reed Army Institute of Research, Washington, DC 20307-5 100, U.S.A. Abbreviations used. AChE, acetylcholinester; BSA, bovine serum albumin; BuChE, butyrylcholinesterase; LXP, dicetyl phosphate;

DFP, diisopropyl fluorophosphate; DMPC, dimynstoyl phosphatidylcholine; ELISA, enzyme-linkedimmunosorbent assay; FBS, fetal bovine serum; IgA, IgG, and IgM, immunoglobulins A, G, and M, respectively; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis;PBS, phosphate-bufferedsaline; SDS, sodium dodecyl sulfate.

756

TOPOGRAPHY OF AChE ACTIVE SITE

deduced by x-ray diffraction studies of single crystals showed that the catalytic site formed by these three amino acids is located in a pocket (Blow et al., 1969). Although located at different positions in the linear sequence for subtilisin, these same three amino acids have been implicated in a charge-relay system for this enzyme, and the protein folding pattern allows for the formation of the same dimensions of the catalytic site pocket (Chambers and Stroud, 1977). The primary structures of several cholinesterases have been deduced recently either from cloned cDNA sequences (Hall and Spierer, 1986; Schumacher et al., 1986; McTiernan et al., 1987; Prody et al., 1987) or by direct amino acid sequence determination (MacPhee-Quigley et al., 1986; Lockridge et al., 1987; Doctor et al., 1988). These reports locate the active site serine and show substantial amino acid sequence homology with other serine hydrolases immediately around the active site serine. It should be noted, however, that the location of the histidine and the putative aspartic acid are not in the same relative order within the sequence compared with serine proteases, and the overall amino acid sequence homology between serine proteases and cholinesterases is far below that expected if these enzymes were to belong to the same family (Schumacher et al., 1986). The two histidine residues (425, 440) conserved in all cholinesterases are located in the third disulfide loop. The highest degree ofamino acid sequence homology is in the proximity of histidine 440. Also, seven or eight aspartic acid residues are located in the same position in all cholinesterases. Two or three of these are preferred candidates for participating in the charge-relay system (Doctor et al., 1988). In subtilisin, the folding pattern of the protein in proximity to the catalytic site creates a domain similar to the catalytic site domain in chymotrypsin, in spite of the fact that the three amino acids involved in the charge relay are located at different positions within the primary sequence (Chambers and Stroud, 1977). Therefore, it is of interest to determine if the catalytic site serine region in the cholinesterases is located in a pocket similar to the serine proteases. Such information can be obtained by x-ray diffraction studies of single crystals (Sussman et al., 1988). In the absence of high-resolution crystal structure, we have generated polyclonal antibodies and monoclonal antibodies (mAbs) against a synthetic peptide corresponding to the amino acid sequence surrounding the active site serine of TorpedocalifornicaAChE and employed these antibodies to determine whether this region is located on the surface of the enzyme or within a pocket of restricted access. If this region is located on the surface of the enzyme, in all likelihood the antibodies would be able to bind to the enzyme in its native conformation. If, on the other hand, this region is located in a pocket, as in the serine proteases, the antibodies would bind to the denatured enzyme and not to the native conformation. The results presented in this article show that some of the mAbs and the

75 7

polyclonal antibodies generated against a peptide corresponding to the active site serine-containing region of Torpedo AChE failed to bind to the enzyme in its native state, but recognized the enzyme after it was reduced and denatured. These results and competition studies with truncated peptides in this region suggest that the active site serine in AChE is indeed located in a pocket-like conformation. MATERIALS AND METHODS Enzymes Fetal bovine serum AChE (FBS-AChE) was purified as previously described (De La Hoz et al., 1986). The purification of 11s catalytic subunits originating from the asymmetric form of Torpedo californica AChE has been described (Lee and Taylor, 1982; Lee et al., 1982). Human serum butyrylcholinesterase (BuChE) was purified by the two-step procedure of Lockridge and La Du (1978). Chymotrypsin and TPCK-treated trypsin were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

Immunochemical reagents Biotinylated goat anti-mouse antibody, streptavidin-conjugated horseradish peroxidase, goat anti-mouse antibody (H L chain), goat anti-mouse immunoglobulin M (IgM) conjugated to microspheres, enzyme-linked immunosorbent assay (ELISA) substrates, and all other immunological reagents were purchased from Kirkegaard and Perry Laboratories (Gaithersburg, MD, U.S.A.).

+

Other chemicals and reagents [3H]Diisopropylfluorophosphate (DFP, 3.5 Ci/mM) was obtained from New England Nuclear Corp. (Boston, MA, U.S.A.). Lipids for the preparation of liposomes were obtained from the following sources: dimynstoyl phosphatidylcholine (DMPC), Sigma Chemical Co.; cholesterol, Calbiochem (La Jolla, CA, U.S.A.); dicetyl phosphate (DCP), K & K Laboratories (Plainview, NY, U.S.A.). Lipid A (E. coli 01 1 1 lipopolysaccharide, lot no. 798957) produced by the Westphal method (Difco Labs, Detroit, MI, U.S.A.), was prepared by acetic acid hydrolysis as described by Chang and Nowotny (1975). It was made soluble in chloroform by treatment with EDTA to remove heavy metals and polycationic contaminants and extracted by the Bligh-Dyer procedure (Dancey et al., 1977).

Active site peptide A peptide identical to the active site serine-containing region of AChE isolated from T. californica (KTVTIFGESAGGASVGMHILSPGSR,Ly~''*-Arg*'~;peptide D-9 1) was synthesized in the laboratory of Dr. Russell Doolittle (University of California at San Diego) by the Memfield solidphase method (Memfield, 1986). The authenticity of the amino acid sequences was determined by gas-phase sequence analysis (Applied Biosystems Protein Sequencermodel 470A). Eighteen overlapping octapeptidescorrespondingto restricted sequences of peptide D-9 1 were synthesized on polyethylene pins using the procedure described by Geysen et al. ( I 984).

Preparation of liposomes containing synthetic peptide The D-91 peptide was dissolved in ammonium formate and diluted in 0.15 A4 NaCI. The detailed procedure for the preparation of liposomes has been described previously (AlJ. Neurmhem., Vol. 55, No. 3, 1990

758

R. A . OGERT ET AL.

ving et al., 1984). The following modification was adapted for encapsulation of synthetic peptide. Lipid A (40 nmol of lipid A phosphate/pmol of phosphatidylcholine) was added to a pear-shaped flask and the contents dried in vacuo. The lipids DMPC/cholesterol/DCP (molar ratio 2.0: 1.5:0.22), solubilized in chloroform, were added to the flask, dried by rotary evaporation, and dispersed by shaking with a 5 mg/ ml solution of peptide. The liposomes thus formed were washed twice with 5 volumes of 0.15 iZI NaCl by centrifugation for 10 min at 10,000 g and were finally suspended in 0.15 M NaC1.

50 p1 of [3H]DFP-labeled enzyme were incubated overnight at room temperature. Two milligrams of microspheres (Igh4conjugated) were added to each reaction vial and allowed to incubate for 2 h with intermittent shaking. Microspheres were sedimented from the reaction mixture at 2,500 g for 10 min. The microspheres were washed three times with 2 ml of PBS/ 0.05% Tween 20. Radioactivity in all the supernatant washes was determined after pooling the samples (free radioactivity). Radioactivity in sedimented microspheres was also measured (bound radioactivity). Percent immunoprecipitation was calculated based on the ratio of bound to total counts.

Immunization and production of mAb against the synthetic peptide encapsulated in liposomes

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots

Female Balb/C mice (Jackson Laboratories) were immunized by intraperitoneal injection with approximately 100 pg of peptide encapsulated in liposomes. They were reimmunized by injection with the same amounts of freshly prepared liposome-encapsulated peptide on days 12,22, 33, and 37. The spleens were removed on day 40. Spleen cells were fused using polyethylene glycol loo0 with the P3 X 63Ag8.653 mouse myeloma line as described elsewhere (Gentry et al., 1982). Antibody-secreting cell lines were identified by a solidphase microtiter plate radioimmunoassay (Zollinger et al., 1976). This assay utilized 5 pg of active site peptide/well for detection of reactive mAbs. The secondary antibody used in this procedure was a goat anti-mouse (IgG, IgA, IgM) radiolabeled with '1 by the chloramine-T method (Hunter, 1973). The hybridomas were cloned using a semisolid agarose technique (Gentry, 1985). Determination of the isotype of each mAb was made using tissue culture supernatants in a solidphase radioimmunoassay technique (Rothman et al., 1988). mAbs were amplified by growing hybridoma cells in the peritoneal cavities of pristane-primed Balb/C mice to produce antibody-containing ascitic fluid (Rener, 1985).

Electrophoresis of proteins was camed out using the Mini Protean I1 slab gel apparatus (Bio-Rad, Richmond, CA, U.S.A.). Equal volumes of protein and sample buffer containing 0.5 M Tris-HCI, pH 6.8, 5% glycerol, 10% (wt/vol) SDS, 5% 2-~-mercaptoethanol,O.O5%bromophenol blue, and 0.05 mg/ml pyronin Y were heated in boiling water for 3 min. Proteins were separated by discontinuous SDS-PAGE at 160 V using 1.5-mm slab gels containing 8% acrylamide/ 0.8% N,N'-methylene-bis-acrylamide. The stacking gel was a 4% gel in 0.125 M Tris, pH 6.8. Proteins were detected by staining with 0.1% Coomassie Blue R-250 in fixative containing 40% methanol and 10% acetic acid. Electrophoretic transfer of proteins from gels to nitrocellulose was performed (TE50 Transphor unit, Hoefer Scientific, San Francisco, CA, U.S.A.) at 100 V and 4°C for 75 min, using a transfer buffer consisting of 20 mM Tris, 150 mMglycine, with 20% methanol. Blotted proteins were detected with amido black (0.25 mg/ml in 10%methanol, 5% acetic acid). Immunodetection of blotted proteins was performed using biotin-labeled goat anti-mouse secondary antibody and peroxidase or phosphatase-coupled avidin. 4Chloro- I -naphthol and hydrogen peroxide were used as substrates with the peroxidase conjugate, and 5-bromo-4chloro-3-indoyl phosphate and nitroblue tetrazolium with the phosphatase conjugate. mAb was incubated overnight with nitrocellulose blotted strips at room temperature. Secondary antibody conjugate was allowed to react for 2 h. Development of background signals was blocked with caseinBSA-PBS buffer for 1 h.

ELISA This assay utilized either 0.5 pg of enzyme/well or 5 pg of active site peptide/well. Antigen was diluted in phosphatebuffered saline (PBS; 50 m M phosphate, 150 m M NaCl, pH 7.4) and allowed to attach to ELISA plates (Immulon 4, Dynatech, Springfield, VA, U.S.A.) overnight at 4°C or, in the case of the synthetic peptide, to evaporate to dryness at room temperature. Casein (I%) and bovine serum albumin (BSA; 1%) in PBS were used as a blocking buffer to reduce nonspecific background binding. Casein (I%) in PBS was used in the ELISA with the polyclonal antibodies. The ELISA plates were blocked for I h at room temperature. All assays utilized ascitic fluids as a source of mAbs. The secondary antibody used was goat anti-mouse (IgG, IgM, H L chain) conjugated to horseradish peroxidase. mAb was allowed to react with antigen for 2 h before being washed with PBS/ 0.05% Tween 20. Secondary antibody incubation was for I h. Microtiter plates were read 30 min after the addition of substrate at 405 nm using an EL3 10 microplate reader (Biotek Instruments, Winooski. VT, U.S.A.). For mapping the epitopes of mAbs, I8 overlapping octapeptides on polyethylene pins were used. Instead of drying the synthetic peptide antigen on microtiter plate wells, the reactions were camed out on the tips of the pins. All reagents, antibodies, and spectrophotometric determinations were the same as described above.

+

Immunoprecipitation mAb from ascitic fluid was diluted in casein-BSA-PBS buffer at selected dilutions; 50 p1 of antibody dilution and

J. Neiiroclietn., Vol. 55, No. 3, 1990

Radiolabeling of active site serine of AChE with 13H]DFP Chymotrypsin, Torpedo AChE, human BuChE, and FBSAChE were radiolabeled with [3H]DFPby the following procedure. A 2X molar ratio of ['HIDFP (mixture of labeled and nonlabeled) to subunit molecular weight of enzyme was allowed to incubate with the selected enzyme for 24 h at room temperature. The aged enzyme was separated from free [3H]DFP on a 1.5 X 20 cm Biorad P6 gel column using 50 mM phosphate buffer, pH 8.0.

Reduction, denaturation, and alkylation of enzymes (RDA) All enzymes, either [3H]DFPaged or native, were reduced in the presence of 10 mM dithiothreitol for 1 h at 37°C. NMethylmaleimide (40 mM) was used for a period of I h at 37°C to alkylate the reduced enzyme. Denaturation was carried out in 6 M guanidine-HC1 for 90 min at 55°C. The reduced, alkylated, and denatured enzyme was dialyzed against three changes of 50 m M PBS.

TOPOGRAPHY OF AChE ACTIVE SITE

759

arose 4B). This antibody preparation was used for the determination of its reactivity with FBS-AChE. D-

\ 0

I

1

RESULTS

2

10 1 102 103 Fold Dilution o f Antibody

104

FIG. 1. Cross-reactivityof polyclonal antibodies raised against an active center peptide (D-91) with Torpedo 11S AChE. [3H]DFPlabeled Torpedo 11s AChE was incubated with rabbit polyclonal

antibodies.The ability of the antibodies to precipitate AChE relative to an acetone-precipitatedcontrol (percent of control precipitate) is plotted versus the dilution of antibody. 0 , native Torpedo 1I S AChE; a, denatured Torpedo 11S AChE. Inset: Torpedo 11S AChE (10 pg/lane) was separated on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with the polyclonal antibodies at 100-fold dilution. Reactivity with control antibodies (lane 1) and antibodies incubated with the active site peptide (1 X M) for 2 h (lane 2) is shown. Monomers (M) and dimers (D) of catatytic subunits are indicated.

Determination of AChE activity Enzymatic activity was determined by the method of Ellman et al. (1961) as described by Main et al. (1974). One unit of enzyme activity caused hydrolysis of 1 mM acetylthiocholine/min at 25°C in 0. I M phosphate buffer, pH 8.0.

Protein determination Protein concentrations were typically determined by absorbance at 280 nm. More specific determinations were made using the method of Lowry et al. ( 1951).

Polyclonal antibodies Rabbit polyclonal antibodies were generated against the active site peptide (D-9 1) coupled to BSA and were assayed for their ability to precipitate [3H]DFP-labeled Torpedo 1 IS AChE (Fig. 1). Although the antibodies were able to recognize and precipitate the denatured enzyme, they were unable to recognize the enzyme in its native conformation. The antibodies were also able to react with the denatured enzyme on Western blots, and this reactivity was blocked by prior incubation of the antibodies with the active site peptide (Fig. 1, inset). The polyclonal antibodies were able to recognize denatured FBS-AChE and Torpedo 1 IS AChE, as well as the active site peptide D-9 1 using ELISA. However, the polyclonal antibodies were not able to recognize the enzymes in their native conformation (Fig. 2). No reactivity with either native or denatured human serum BuChE was observed (data not shown). It was necessary to remove anti-BSA antibodies from the rabbit polyclonal antibodies by absorption on Sepharose 4B-BSA affinity gel prior to use in ELISA, because the presence of trace amounts of BSA in FBS-AChE gave false positive results. For comparison, the amino acid sequences of active site regions of Torpedo 1 IS AChE (Schumacher et al., 1986), FBS-AChE (Doctor et a]., 1988), and human serum BuChE (Lockridge et a]., 1987) are given in Fig. 3. mAbs When culture supernatants from all fusion wells were tested for mAbs specific for the active site peptide, over 100 were reactive with peptide D-9 1. ELISAs were per-

Competition ELISA The specificities of the mAbs were determined by a competition ELISA. The microtiter plate wells were coated with antigen, either reduced, denatured, and alkylated FBS-AChE or Torpedo 1 IS AChE. The amount of antigen employed was such that antigen would be in excess over antibody. The plates were allowed to sit overnight at 4°C with casein-BSAPBS blocker. The wells were aspirated, and equal volumes of serially diluted synthetic peptide ( I nmol/ml of stock solution) and selected concentrations (pretitrated) of antibodies were added to appropriate wells. The mixture of antibody and peptide was allowed to incubate in the plate at room temperature for 2 h. The plates were washed with 0.05% Tween-20 in PBS, and the remainder of the ELISA procedure was followed as described above.

Polyclonal antibodies Polyclonal antibodies were generated against the D-9 I peptide by coupling the peptide to BSA and using this conjugate as an antigen in rabbits (Abramson et al., 1989). Immunoprecipitation and Western blot assays using the polyclonal antibodies were also previously described (Abramson et al., 1989). Anti-BSA antibodies present in the immunized rabbit plasma were removed by passing the plasma through an affinity column (BSA attached to CNBr-activated Seph-

10

100

1000

ANTIBODY

10000

DILUTION

Cross-reactivityof polyclonal antibodies raised against an active center peptide (D-91) with Torpedo 11s AChE and FBSAChE. Anti-BSA antibodies from immune rabbit serum were removed prior to the ELISA. A. synthetic peptide D-91; 0, Torpedo 11S AChE; a, Torpedo 11S AChE (reduced and denatured); 0, FBS-AChE; A, FBS-AChE (reduced and denatured). FIG. 2.

J h ' e u r ~ ~ ~. ~I lof~ a55, t ~No. 3. I990

R. A . OGERT ET AL.

760 5

10

15

20

25

TORPEDO AChE FBS AChE HUM BUChE

FIG. 3. Amino acid sequences of active site regions of cholinesterases. The active site serine is shown in a block. HUM, human serum.

formed on the tissue culture supernatants to examine the level of cross-reactivity with native and denatured Torpedo I 1s AChE. Based on the magnitude of reactivity with either native or denatured enzyme, 10 hybridomas were selected for cloning and further characterization. All 10 of the selected mAbs were of the IgM isotype. These mAbs were tested for their cross-reactivity with Torpedo 11s AChE (parent protein), FBS-AChE, human serum BuChE, and chymotrypsin in native conformation, as well as in reduced, alkylated, and denatured form. Assays were carried out using an ELISA with fixed amounts of antigen and varying dilutions of ascitic fluid. None of the 10 mAbs tested showed reactivity with either native or denatured human serum BuChE or chymotrypsin (data not shown). Based on the cross-reactivity of these mAbs with native or denatured FBS-AChE and Torpedo 1 1S AChE, they were divided into three groups (Table 1). The first group was characterized by reactivity with denatured Torpedo 1 1S AChE and FBS-AChE, but not with the native conformation of either enzyme. Four of the 10 cloned hybridomas exhibited this pattern of reactivity (Table 1, mAbs 1-4). The second group showed the same reactivity toward the denatured enzymes as the first group; however, the mAbs in this

group also showed weak cross-reactivity with native Torpedo 1 1s AChE, but not with the native FBS-AChE (Table 1, mAbs 5-7). This group required high concentration of antigen and also failed to immunoprecipitate. The third group showed the same pattern of recognition as the second group, but additionally these antibodies were reactive with the native FBS-AChE. Figure 4 shows the results of the ELISA for one mAb selected from each of the three groups. The ELISA was modified to measure the antigen concentration dependence of antibody binding by using a fixed concentration of each mAb and serial dilution of antigens. The results for two of the three mAbs tested are shown in Fig. 5. The same pattern ofcross-reactivity and specificity of binding for each mAb was observed. The extent of cross-reactivity of one of these three mAbs was also investigated by immunoprecipitation (Fig. 6). The results in Figs. 4-6 are in essential agreement with the ELISA results in Table 1. 1.2

0.8

I

h

B

TABLE I . Cross-reactivity of anti-active site peptide mAbs with FBS-AChE and Torpedo 11sAChE AChE Torpedo 1 IS mAb I. 2. 3. 4.

PI-5G06-12 PI - I B03-05 PI-5W6-32 PI-12BI-04

5. PI-ID1 1-12 6. PI-4A02-05 7. PI-5W6-21

8. P 1-6806-03 9. PI-16HI-08 10. PI-14A7-09

Native -

+ + + ++ + ++

Denatured

+++ + + + +++ ++ +++ ++ + ++

FBS Native

Denatured

-

-

+ ++ ++ +++ +++ ++ +++

+ + +++

+++ ++ +++

-

-

The cross-reactivity of the mAbs was determined by ELISA. None ofthese mAbscross-reacted with either chymotrypsin or human serum BuChE in native or denatured form. The details of the ELISA are given under Materials and Methods. A lack of detectable reactivity is indicated by -, and the relative degree of reactivity (three times the absorbance at 405 nm over blank) is indicated by +.

J. Neiirochc.m.. V d 55. No. 3, 1990

1 /Ascitic Fluid Dilution FIG. 4. Cross-reactivity of anti-peptide (D-91) mAbs with various

AChEs. The cross-reactivity of three representative mAbs, one from each group (Table I), with native and denatured forms of various cholinesterases. was determined using ELISA. A mAb P15606-12. B mAb P1-1Dll-12. C: mAb P1-14A7-09. The serial dilution of ascitic fluid was used as a source of mAb. Details of the procedure are described under Materials and Methods. The results of antigens which showed no cross-reactivity are not included. A, synthetic peptide 0 9 1 ; 0, Torpedo 1 1 S AChE; D, Torpedo 11S AChE (reduced and denatured);0,FBS-AChE; A,FBSAChE (reduced and denatured).

~~:

TOPOGRAPHY OF AChE ACTIVE SITE

7

A z

0 4

0.4

2 !

L

E C

In

W

0.2

K

a

0

+-

ai

I-

W 2

0.0

0 K

a Y

76 1

t

10

00

10

0.8

ANTIBODY DILUTION

0.4

0.0 0.025

0.100

1.000

1/Antigen Concentration (pmoles) FIG. 5. Cross-reactivity of anti-peptide mAbs with cholinesterases as determined by ELISA using varying concentration of antigens. A mAb P1-5G06-12. B: mAb P1-lDl1-12. A single dilution of each Torpedo 11S mAb (1:160) was used. A, synthetic peptide D-91; 0, AChE; A, Torpedo 11S AChE (reduced and denatured); 0 , FBSAChE; m, FBS-AChE (reduced and denatured); 0, chymotrypsin (reduced and denatured).

Each of the 10 mAbs was tested for immunoreactivity with Torpedo 1 IS AChE and FBS-AChE which had been separated by SDS-PAGE and electrophoretically blotted onto nitrocellulose membranes. The antibodies showed the same levels of recognition for blotted enzyme as they did for the denatured enzymes using other types of assays. To determine whether the epitope for binding of these mAbs to denatured 1 IS Torpedo AChE was located within the amino acid sequence of the peptide, the D-91 peptide was employed to compete with denatured Torpedo 1 IS AChE for binding to one of the mAbs (PI-ID1 1-12). As seen in Fig. 7, the synthetic peptide fully competed with the parent protein for binding to antibody. Elucidation of which region within the active site peptide constitutes the epitopes for two representative mAbs may reveal whether the active site serine was involved in binding to antibody. The epitopes for mAbs P1-1Dll-I2 and PI-14A7-09 were mapped using 18 overlapping octapeptides synthesized on polyethylene pins (Geysen et al., 1984). The results in Fig. 8 show that for mAb PI - 1D 1 1 - 12 significant binding occurred with octapeptides 2-6 (TVTIFGESAGGA), and the greatest binding occurred with octapeptides 4 and 5 (TIFGESAGG). This finding demonstrates that the active site senne region of AChE appears to be part of an epitope for this mAb. On the other hand, very weak binding of mAb P 1- 14A7-09 occurred with octapep-

FIG. 6. lmmunoprecipitation of AChEs and chymotrypsin by antipeptide mAb (Pl-5D06-21). Details of the immunoprecipitation are given under Materials and Methods. 0.Torpedo 11s AChE; A, Torpedo 11S AChE (reduced and denatured); 0, FBS-AChE; B, FBS-AChE (reduced and denatured); +, chymotrypsin (reduced and denatured).

tides 4-6 and with octapeptides 13- 18. Binding with a lower affinity to several octapeptides in two different regions of peptide D-9 I by mAb P 1- 14A7-09 may explain why it binds to both denatured and native AChE. mAb P 1- 1D 1 1- 12 shows relatively significant and specific binding to one region (octapeptides 2-6), which may explain its strong binding to denatured AChE. This region contains the active site senne.

DISCUSSION Although several attempts have been made to generate mAbs which alter the catalytic activity of AChEs

0

100

200

300

pMOLE PEPTIDE PER WELL

FIG. 7. Competition with Torpedo 11S AChE (reduced, denatured, alkylated) by peptide D-91 for binding to mAb P1-1Dll-12. Twenty picomoles of denatured Torpedo 11S AChE were added to each well. The peptide D-91 varied from 0 to 1 nmol/well.

J. Neurochem.. Vol. 55, No. 3%1990

R. A . OGERT ET AL.

762

t

I

1487-9

0 60

0 20 I

2

3

4

5

6

7

8

9 10 I I 12 13 14 15 16 17 I8

FIG. 8. Epitope mapping of mAbs P1-1D11-12 and P1-14A7-09. Eighteen overlapping octapeptides were synthesized on polyethylene pins according to Geysen et al. (1984) and used in ELISA. The sequences of peptides are given at the bottom.

(see Brimijoin, 1986),to date only a few mAbs inhibit the catalytic activity ofAChEs (Abe et al., 1983; Brimijoin et al., 1985; Sakai et al., 1985; Sorenson et al., 1987; Doctor et al., 1988; Ashani et al., 1990).Further characterization of these antibodies has revealed that their epitopes appear to be located in regions other than the active site (Brimijoin et al., 1985; Ashani et al., 1990). Therefore, to obtain mAbs which recognize an epitope including the active site serine, a peptide was prepared which corresponds to the sequence of the tryptic fragment of Torpedo AChE containing the active site serine (MacPhee-Quigleyet al., 1986),and the peptide was used to generate polyclonal antibodies and mAbs. Generation of antibodies, either polyclonal or monoclonal, against synthetic peptides of reasonable length usually requires covalent attachment of the peptides to camer proteins, such as serum albumin or keyhole limpet hemocyanin. In order to generate mouse mAbs, the D-91 peptide was encapsulated in liposomes which contained lipid A as adjuvant. Over 100 antibody-positive hybridomas were found to be reactive with the synthetic peptide. The results in Table 1 demonstrate that not only did the encapsulation of peptide in liposomes allow the generation of mAbs that recognized the antigen peptide specifically,but that they also cross-reacted with the protein from which the peptide sequence was derived. Furthermore, several of these mAbs and polyclonal antibodies displayed a high degree of specificity in that they recognized the parent J. Nrurochrm , Vol 55. No. 3. 1990

protein in its denatured form, but not in the native conformation, perhaps because in native form this region of AChE is not available for binding to antibodies. All 10 of the cloned mAbs were assayed by ELISA. The antibodies also exhibited similar specificities and binding properties when tested by other immunochemical methods. Each mAb could be classified into one of three groups based on reactivity with the AChE enzymes. The first group exhibited reactivity only with Torpedo 1 IS AChE and FBS-AChE in the denatured form (similar to polyclonal antibodies). The second group of mAbs showed weak binding to native Torpedo 1 IS AChE, in addition to the denatured form of both AChEs. The third group showed no preference for denatured over native conformation, but instead was reactive with both AChEs in the native, as well as denatured, form. The folding pattern of chymotrypsin, as deduced by x-ray diffraction studies of single crystals, shows that the catalytic site formed by Asp102,His5’, and Ser’” is located in a pocket (Blow et al., 1969). In subtilisin, the same three amino acids (Asp32,His64,and Se?2’; Kraut, 1971) are implicated in formation of a chargerelay system, although they are located in different positions within the linear sequence from those in chymotrypsin (Chambers and Stroud, 1977). The protein folding pattern of subtilisin allows the formation of a similar type and dimension of the active site pocket as found in chymotrypsin. It would be interesting to determine if cholinesterases have a similar pocket-like conformation in which the catalytic site is located, because their mechanism of catalysis appears to be similar. Obviously, the elucidation of the detailed structure of the AChE catalytic site pocket will have to await xray diffraction data on single crystals; however, the results presented here provide a first glimpse of the catalytic site in terms of suspected topography. The results presented here show that the polyclonal antibodies and one group of the mAbs generated against the synthetic peptide corresponding to the amino acid sequence of a region of Torpedo AChE which contains active site serine showed binding to the denatured form of the enzyme, but not to the native form. These antibodies showed similar specificity with FBS-AChE, but had no reactivity with human serum BuChE, chymotrypsin, or nonrelated peptides. The epitope for one of this group of mAbs consists of a truncated region within this peptide which contains the active site serine. Based on the results presented here, it appears that in native form the active site serinecontaining regions of both Torpedo AChE and FBSAChE are located in a pocket, and thus are not accessible for binding to antibodies generated against a peptide corresponding to the amino acid sequence of this region. Binding of native forms of Torpedo AChE and FBS-AChE to the mAbs described under the third group indicates that (a) liposome-encapsulated peptide D-9 1 may have assumed some conformation which is recognized by these mAbs, and/or (b) a portion of this

TOPOGRAPHY OF AChE ACTIVE SITE region is located on the surface of the enzyme in native form, thus constituting a partial epitope for these mAbs. Acknowledgment: A portion of these d a t a was submitted t o T h e Catholic University o f America, Washington, D.C., by Robert A. Ogert as partial fulfillment o f t h e requirements for a n M. S. degree i n Cell Biology. T h e authors wish t o t h a n k Dr. J. M. Mullins of Catholic University for his guidance a n d advice t o Mr. Ogert during his studies. This work was supported i n part by USPHS grant GM 18360 and D A M R D C contract D A M D 17-87-C-7109.

REFERENCES Abe T., Sakai M., and Saisu H. (1983) A monoclonal antibody against catalytic subunits of acetylcholinesterase in the electric organ of an electric ray, Narke japonica. Neurosci. Lett. 38, 6 1-66. Abramson S. N., Ellisman M., Deerinck T., Maulet Y., Doctor B. P., Gentry M. K., and Taylor P. (1989) Differences in structure and distribution of the molecular forms of acetylcholinesterase. J. Cell Biol. 108, 230 1-23 1 1. Aldridge W. N. and Reiner E. ( I 972) Enzyme Inhibitors as Substrates, p. 37. North-Holland Publishing Co., Amsterdam. Alving C. R., Shichijo J., and Maltsby-Baltzer I. (1984) Preparation and use of liposomes in immunological studies, in Liposome Technology, L’ol. II (Gregoriadis G., ed), pp. 157-175. CRC Press, Boca Raton. Ashani Y., Gentry M. K., and Doctor B. P. (1990) Differences in conformational stability between native and phosphorylated acetylcholinesterase as evidenced by a monoclonal antibody. Biochemisiry 29, 2456-2463. Blow D. M., Birktoft J. J., and Hartley B. S. (1969) Role of a buried aspartic acid group in the mechanism of action of chymotrypsin. Nature 221, 337-340. Brimijoin S. (1986) Immunology and molecular biology of the cholinesterases: current results and prospects. Int. Rev. Neurobiol. 28, 363-410. Brimijoin S., Mintz K. P., and Alley M. C. (1985) Production and characterization of separate monoclonal antibodies to human acetylcholinesterase and butyrylcholinesterase. Mol. Pharmacol. 24,5 13-520. Chambers J. L. and Stroud R. M. (1977) Difference Fourier refinement of the structure of diisopropylphosphoryl trypsin at 1.5 angstrom with a minicomputer technique. Aciu Crysi. 33, 18241837. Chang C. and Nowotny A. (1975) Relation of structure to function in bacterial 0 antigens. VII. Endotoxicity of “lipid A,” Immunochemisiry 12, 19. Dancey G. F., Yasuda T., and Kinsky S. C. (1977) Enhancement of liposomal model membrane immunogenicity by incorporation of lipid A. J. Immunol. 119, 1868. De La Hoz D., Doctor B. P., Ralston J. S., Rush R. S., and Wolf A. D. (1986) A simplified procedure for the purification of large quantities of fetal bovine serum acetylcholinesterase. Life Sci. 39, 195-199. Doctor B. P., Smyth K. K., Gentry M. K., Ashani Y., Christner C. E., Ogert R. A., and Smith S. W. (1988) Structural and immunochemical properties of fetal bovine serum acetylcholinesterase, in Prog. Clin. Bid. Rex 289, 305-316. Ellman G. L., Courtney K. D.. Andes V. Jr., and Featherstone R. M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. Froede H. C. and Wilson 1. B. (1971) Acetylcholinesterase, in The Enzymes, 3rd ed. (Boyer P. D., ed), p. 87. Academic Press, New York. Gentry M. K. (1985) Cloning of hybridomas on semisolid agarose. J. Tiss. Cult. Meihods 19, 179- I8 1. Gentry M. K., Henchal E. A,, McGown J., Brandt W. E., and Dalrymple J. M. (1982) Identification of distinct antigenic determinants on Dengue-2 virus using monoclonal antibodies. Am. J. Trop. Med. Hyg. 31,548-555.

763

Geysen H. M., Meloen R. H., and Barteling S. J. (1984) Use ofpeptide synthesis to probe viral antigens for epitope to a resolution of a single amino acid. Proc. Nail. Acad. Sci. USA 81, 3998-4002. Hall L. M. C. and Spierer P. (1986) The Ace locus of Drosophila me/anogaster: structural gene for acetylcholinesterase with an unusual 5‘ leader. EMBO J. 5,2949-2954. Hunter R. M. (1973) Radioimmunoassay, in Handhook o/EsperimentalImmunologj~(Weir D. M., ed), pp. 17.1-17.36. Blackwell Scientific, Oxford. Kraut J. (197 I ) Subtilisin: x-ray structure, in The Enzymc>s,Vo/. 3 (Boyle P. D., ed), pp. 547-560. Academic Press, New York. Lee S. L. and Taylor P. (1982) Structural characterization of the asymmetric (17 + 13) S species of acetylcholinesterase from Torpedo. 11. Component peptides obtained by selective proteolysis and disulfide bond reduction. J. Biol. Chem. 257, 1229212301. Lee S. L., Heinemann S., and Taylor P. (1982) Structural characterization of the asymmetric ( 1 7 + 13) S forms of acetylcholinesterase from Torpedo. 1. Analysis of subunit composition. J Bid. Chem. 257, 12283-12291. Lockridge 0. and La Du B. N. (1978) Comparison of atypical and usual human serum cholinesterase: purification, number of active sites, substrate affinity, and turnover number. J. Bid. Chem 253,361-366. Lockridge O., Bartels C. F., Vaughan T. A., Wong C. K., Norton S. E., and Johnson L. L. ( 1 987) Complete amino acid sequence of human serum cholinesterase. J. Biol. Chem. 262, 549-557. Lowry 0. H., Rosebrough N. J., F a r A. L., and Randall R. J. (195 1) Protein measurement with the Folin phenol reagent. J . Biol. Chem. 193,265-275. MacPhee-Quigley K., Vedick T. S., Taylor P., and Taylor S. S. (1986) Profile of disulfide bonds in acetylcholinesterase. J . Biol. Chem. 261, 13565-13570. Main A. R., Soucie W. G., Buxton I. L., and Anne E. (1974) The purification of cholinesterase from horse serum. Biochem. J. 143, 733-744. McTiernan C., Adkins S., Chatonnet A., Vaughan T. A,, Bartels C. F., Kott M., Rosenberry T. L., La Du B. N., and Lockridge 0. (1987) Brain cDNA clone for human cholinesterase. Pruc. Nail. Acad. Sci. USA 84,6682-6686. Memfield B. (1986) Solid phase synthesis. Science 232, 341-347. Prody C. A., Zevin-Sonkin D., Genatt A., Goldberg O., and Oreg H. (1987) Isolation and characterization of full length cDNA clones coding for cholinesterase from fetal human tissue. Proc. ,Vatl. Acad. Sci. USA 84,3555-3559. Rener J. (1985) Monoclonal antibody production in ascites fluid. J. Tiss. Cult. Methods 9, 187-189. Rothman S. W., Gentry M. K.. Brown J. E., Foret D. A,, Stone M. J., and Strickler M. P. (1988) Immunochemical and structural similarities in toxin A and toxin B of Clostridiirrndificileshown by binding to monoclonal antibodies. Toxicon 26, 583-597. Sakai M., Saisu H., Koshigoe N., and Aka T. (1985) Detergent soluble form of acetylcholinesterase in the electric organ of electric rays. Its isolation, characterization and monoclonal antibodies. Eur. J. Biochem. 148, 197-206. Schumacher M., Camp S., Maulet Y., Newton M., MacPhee-Quigley K., Taylor S., Friedmann T., and Taylor P. (1986) Primary structure of Torpedo callfornica acetylcholinesterase deduced from its cDNA sequence. Nature 319, 407-409. Sorenson K., Brcdbeck V., Rasmussen A. G., and Norgaard-Pedenen B. (1987) An inhibitory monoclonal antibody to human acetylcholinesterases. Biochim. Biophys. Acia 912, 56-62. Sussman J. L., Hare1 M., Frolow F., Varon L., Toker L.. Futerman A. H., and Silman I. (1988) Purification and crystallization of a dimeric form of acetylcholinesterase from Torpedo californica subsequent to solubilization with phosphatidylinositol-specific phospholipase C. J. Mol. Biol. 203, 800-803. Zollinger W. D., Dalrymple J. M., and Artenstein M. S. (1976) Analysis of parameters affecting the solid phase radioimmunoassay quantitation of antibody to meningococcal antigens. J. Immunol. 117, 1788- 1798.

J Neitrnchem , 1’01 55. No 3. 1990