Eur. J. Biochem. 198, 59-65 (1991) 0FEBS 1991 0014295691003092
The monoclonal antibody 2G8 is carbohydrate-specific and distinguishes between different forms of vertebrate cholinesterases Jian LIAO ', Hardld HEIDER', Man-Chi SUN', Susi STIEGER' and Urs BRODBECK Institute of Biochemistry and Molecular Biology, University of Bern, Switzerland Institute of Pharmacology and Toxicology, Beijing, China (Received December 34, 1990)
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EJB 90 1488
The monoclonal antibody (mAb) 2G8 (subclass IgG2,) raised against acetylcholinesterase (AChE, EC 3.1.1.7) from electric organ of Torpedo nacline timilei crossreacted with AChE from Torpedo marmorata, electric eel (Electrophorus electricus), flounder (Platichthys Jlesus) body muscle, rat brain, bovine brain, and human brain, this suggests that the epitope to which mAb 2G8 bound had been highly conserved during evolution. N o crossreaction was found with AChE from human and bovine erythrocytes, nor with butyrylcholinesterase (BtChE, EC 3.1.13)from human serum. Binding of mAb 2G8 to the globular G z form of AChE from T. marmorata strongly decreased enzyme activity, while no significant inhibition was found with either collagen-tailed, asymmetric forms, or with the enzymes from flounder body muscle or mammalian sources. The possibility that mAb 2G8 bound to anionic sites of AChE could be excluded since neither edrophonium chloride nor decamethonium bromide influenced the binding of 2G8 to the enzymes. Enzyme-linked immunosorbent assay and Western blot showed that heat-denatured, diisopropylfluorophosphate-treated, CNBr- and trypsin-digested AChE from T. marmorata still reacted with mAb 2G8; this indicates that the epitope to which 2G8 bound, at least partially, belonged to a continuous determinant. Treatment of cholinesterases with N-glycosidase F abolished crossreaction with 2G8, showing that an essential part of the epitope consisted of N-linked carbohydrates.
A number of monoclonal antibodies (mAb) have been raised against acetylcholinesterase (AChE) of different origins which offer a promising means of gaining insight into structure, function, evolution and biosynthesis of this enzyme [ l 91. Several mAbs against AChE for Narke japonica and Torpedo nacline timilei were shown to inhibit enzyme activity [lo- 131, as well as antibodies raised against AChE from rabbit brain and human erythrocytes [ l , 14- 161. Other mAbs were found to recognize glycosylation sites on AChE. One of them, the IgM mAb Elec-39 raised against AChE from the electric organ of Electrophorus electricus and belonging to the HNK-1 anticarbohydrate family [17, 181, recognized asymmetric AChE from Electrophorus species as well as their lytic globular derivatives but did not inhibit enzyme activity. It did not crossreact with asymmetric forms of AChE from Torpedo but recognized the amphiphilic G z form from that organ. Between different species, most mAbs to mammalian AChE display a relatively low degree of crossreactivity [19]. One mAb, AE-2, was reported to have a relatively wide range of crossreactivity to AChE from several species [2, 7, 151. This mAb inhibited AChE from several sources [14, 16, 201, presumably by binding to one of the anionic sites [14, 151. Correspondence to U. Brodbeck, Institute of Biochemistry and Molecular Biology, University of Bern, Buhlstrasse 28, CH-3012 Bern, Switzerland Abbreviations. AChE, acetykhohnesterdse; BtChE, butyrylcholinesterase; EAIA, enzyme antigen immunoassay; ATCh, acetylthiocholine. Enzymes. Acetylcholinesterase (EC 3.1.1.7); butyrylcholinesterase (EC 3.1.1.8); N-glycosidase F (EC 3.2.2.18); trypsin (EC 3.4.21.4).
Studies on the primary structure and on the kinetic properties of AChE showed that the different forms of AChE share regions of common structural domains, e. g. the catalytic center and the peripheral anionic sites [21-291. Moreover, studies on the crossreactivity of mAb with different forms of AChE isolated from various sources further substantiated the existence of such common structural domains [4, 5, 15, 30, 311. At present, relatively little is known about glycosylation of AChE, in general, or about structural similarities of Nlinked carbohydrates between vertebrate AChE, in particular. From the primary structure of AChE from Torpedo californica [23] and Torpedo marmorata [24], it can be deduced that both asymmetric and globular forms of the enzyme contain four potential N-linked glycosylation sites. G 4 AChE from fetal bovine serum contains five potential sites [28] while butyrylcholinesterase (BtChE) from human serum contains nine [25]. Within one species, composition and structure of oligosaccharide chains may vary between AChE from different tissues. This was suggested by Meflah et al. [32] who reported differences in lectin binding for AChE from bovine lymphocytes, erythrocytes and brain. Interaction with lectins also distinguished between AChE from fetal bovine serum and bovine erythrocytes [7]. Differences in post-translational glycosylation might be a signal for differential association of the enzyme with the extracellular matrix or the cell membrane P31. In this paper, we present an example of an mAb, i.e. mAb 2G8, raised against AChE from the electric organ of T . nacline timilei which displayed a wide range of crossreactivity with
60 AChE from different vertebrates. The essential part to which 2G8 bound is an N-linked carbohydrate epitope which appears to be highly conserved during evolution.
Enzyme antigen immunoassay (EAIA)
The AChE activity was measured at room temperature using the method of Ellman et al. [34] with 1 mM acetylthiocholine (ATCh) iodide and 0.25 mM 5,5'-dithiobis(2-nitrobenzoic acid) in 100 mM sodium phosphate pH 7.4, containing 0.1 o/o Triton X-100. The same procedure was used to assay BtChE activity except that butyrylthiocholine (BtCh) iodide was used as substrate. In the cases of EAIA, AChE activity was measured in microplates according to the methods of Rasmussen et al. [8] and Stieger et al. [35].
EAIA was conducted based on the procedures of Rasmussen et al. [42] as follows. mAbs were first diluted in coating buffer (10 mM phosphate pH 8.0 containing 144 mM NaCl, 0.02% NaN3). Into each well of the polystyrene microplate (Immuno Nunc plate I, Denmark) 100 p1 mAb, blank or control was pipetted and incubated at 4°C for 24 h. The plates were then washed twice with buffer (10 mM sodium phosphate pH 7.2 containing 0.05% Tween-20). Blocking was carried out with coating buffer containing 1YObovine serum albumin (Sigma), 200 pl/well, and by incubation at 37°C for 1 h. The plates were then washed as in the second step. Samples (100 pl/well) of vertebrate AChE, diluted in 10 mM sodium phosphate pH 7.4 containing 144 mM NaCl and 0.1 YO Triton X-100, were pipetted into the wells and incubated at room temperature for 150 min on a slow shaker. The plates were then washed five times with washing buffer and developed by the addition of 100 pl/well assay buffer [l mM acetylthiocholine iodide and 0.25 mM 5,5'-dithiobis(2nitrobenzoic acid) in 100 mM sodium phosphate pH 7.4 containing 0.1% Triton X-1001. Absorbance was measured at 405 nm using the Molecular Devices Vm,, kinetic microplate reader.
Protein assay
Solid-phase immunoassay
Protein concentration was determined in polystyrenemicroplates (from Nunc, Denmark) according to Serrensen and Brodbeck [36] using the Pierce BCA kit with bovine serum albumin (Pierce Chemicals) as protein standard.
Ascitic fluid was kindly provided by G. Z. Yan and Y. B. Xin (Institute of Microbiology and Epidemiology, Beijing 100850, China). It contained mAb 2G8 raised against AChE from electric organ of T. nacline timilei [9] which was purified according to Wang et al. [13] by protein-A-Sepharose CL4B chromatography (Pharmacia Fine Chemicals, Uppsala, Sweden) or purified by chromatography on DEAE - Affi-gel blue (Bio-Rad) according to Bruck et al. [37]. mAb 4F19, raised against human erythrocyte AChE, was obtained as published previously [S]. Monoclonal anti-human IgM clone MB-11 was from Miles-Yeda Ltd (Israel).
Dot-blot immunoassays were carried out based on the procedures of S~rrensenet al. [14]. Aliquots (2 p1) of vertebrate AChE, diluted with 10 mM sodium phosphate pH 7.4 containing 0.14 M NaCl and 0.1% Triton X-100, were spotted directly onto strips of nitrocellulose membrane (Bio-Rad) using a Hamilton syringe. The strips were incubated at 37°C for 1 h with 20 mM Tris/HCl pH 7.4, containing 0.14 M NaCl and 3% bovine serum albumin (buffer A) and then rinsed once with buffer A devoid of albumin (buffer B); mAb (ascitic fluid), diluted 1 : 200 with buffer A containing 0.05% Tween20, was added. Incubation was carried out at 4°C overnight. The strip was rinsed four times with buffer B and incubated at room temperature for 2 h with horseradish-peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) diluted I : 1000 with buffer A containing 0.05% Tween-20. The strip was washed five times with buffer B and developed with a solution of 0.5 mg/ml of diaminobenzidine in buffer B containing 1 pl/ml of 30% H202.
Ch olinesterases
Enzyme-linked immunosorbent-assay (ELISA)
AChE was purified by affinity chromatography using two consecutive steps on the same resin. Unless otherwise stated, purifications were carried out on a resin containing trimethylammonium m-phenylenediamine as affinity ligand [38] as follows: AChE from bovine and human erythrocyte membranes according to Brodbeck et al. [38]; AChE from rat brain, bovine brain, and human brain according to Smensen et al. [39,40]; AChE from electric organ of T . marmorata according to Stieger and Brodbeck [41]. AChE from electric eel was purified from Sigma product (no. C-2879) by the procedures reported for the Torpedo enzymes [41]. The enzyme from flounder (Platichthys flesus) body muscle was prepared according to Stieger et al. [35] using edrophonium - Sepharose as high-affinity matrix. BtChE from human serum was kindly provided by Dr Oksana Lockridge (Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68105-1065, USA).
ELISA was performed as follows. Samples were first diluted with coating buffer (10 mM phosphate pH 8.0 containing 144 mM NaCl and 0.02% NaN3). The wells were coated with 100 p1 of the diluted specimens at 4°C for 24 h and then rinsed twice with washing buffer (10 mM sodium phosphate pH 7.2 containing 0.05% Tween-20). Postcoating was carried out at 37 "C for 1 h by the addition of 200 pl/well of the coating buffer containing 1YO bovine serum albumin washing three times, 100 pl/well of mAb diluted in washing buffer was added and incubated at 37°C for 1 h. After washing five times, 100 pl/well of second antibody solution (horseradish-peroxidase-conjugated goat anti-mouse IgG, diluted 1 : 1000 in washing buffer) was added to the plate and incubated at 37°C for 1 h. After washing five times, 100 ,ul/well of a solution of 0.1 YO o-phenylenediamine dihydrochloride (Sigma) containing 0.03% H 2 0 2in substrate buffer (35 mM citric acid, 65 mM Na2HP04 pH 5.0) was added to the wells. The reaction was
MATERIALS AND METHODS Unless otherwise stated, all chemicals were analytical grade and either from Sigma (St Louis, USA), Merck (Darmstadt, FRG), Fluka (Buchs, Switzerland), Bio-Rad (Richmond, USA) or Boehringer (Mannheim, FRG). AChE assay
Antibodies
61 allowed to proceed at room temperature for 15 min and stopped by adding 100 pl/well of 2 M H2S04then absorbance at 490 nm was measured on the microplate reader. SDS/polyacrylamide gel electrophoresis
SDSjPAGE was performed essentially according to the method described by Laemmli [43] using 5 - 3 5% polyacrylamide gradient gels. Protein was stained with Coomassie brilliant blue R-250. Electroblotting and Western blot on nitrocellulose and [email protected] membranes
After electrophoresis, proteins from SDSjPAGE and peptides from Tricine SDSjPAGE were electrophoretically transferred to nitrocellulose paper or to polyvinyldifluoride membranes in a semi-dry-blot system (Ancos, Denmark) according to Heider et al. [29] and Ploug et al. [44], respectively. After the transfer, nitrocellulose and polyvinyldifluoride membranes were rinsed three times with 50 m M Tris/HC1 p H 7.4 containing 0.14 M NaCl. Then, 20 m M TrisjHCl pH 7.4 containing 0.14 M NaCl and 3% bovine serum albumin was added, and blocking was carried out at room temperature for 2 h. Thereafter, the procedure described for the solid-phase immunoassay was followed. N-Glycosidase F treatment
AChE and BtChE were first dialysed against 20 m M sodium phoshate pH 7.2 containing 20 mM EDTA and 0.1YOnoctyl glucoside. Then, 50 IU AChE or 5 IU BtChE were treated in 50 p120 m M sodium phosphate p H 7.2, containing 20 m M EDTA and 1YOn-octyl glucoside, 0.1% SDS and 1% mercaptoethanol. To each sample, 1 unit N-glycosidase F (Boehringer Mannheim) was added and incubated at 37 "C for 16 h. RESULTS As shown previously [9, 131, mAb 2G8 raised against AChE from T. nacline timilei strongly inhibited that enzyme when acetylcholine was used as substrate. On the other hand, with indophenylacetate only a modest inhibition was seen. In the present study, the properties of 2G8 toward dimeric and tetrameric AChE from several vertebrates were evaluated. The dimeric G 2 form of AChE from T. marmorata was inhibited by 2G8. The extent of inhibition varied with time and concentration of antibody. When antigen and antibody were present in equal amounts, little inhibition was seen. It increased with increasing amounts and reached 75y0 at a fivefold molar excess of antibody. The formation of the complex was slow as it took 24 h to achieve maximal inhibition. Longer incubation did not significantly increase the extent of inhibition. Since the inhibition was seen with ATCh only, it was of interest to determine if positively charged inhibitors of AChE would influence the binding of 2G8 to the G2 form of AChE from T. rnarmorata. Neither high concentrations of edrophonium, which occupies the anionic site of the catalytic center of AChE, nor decamethonium, which binds to both the central and the peripheral anionic sites, significantly decreased the rate of binding of 2G8 to AChE (results not shown). Since the active site of AChE has been highly conserved during evolution, we expected that the antibody 2G8 would
not only inhibit AChE from T. murmorata but also AChE from various other sources. This was, however, not the case as the inhibition by 2G8 was specifically seen only with the G2 form of AChE from T. marmorata. The asymmetric form of AChE from T. marmorata was inhibited only to 7%, even at a fivefold molar excess of antibody. Even less inhibition was seen with the G2 form of AChE from flounder body muscle, and practically no inhibition was observed with the other forms of AChE tested, i. e. G4 forms of AChE from rat, bovine and human brain, and the G2 form of AChE from bovine and human red cell membranes. Further, the antibody did not inhibit the G4 form of BtChE from human serum, the active site of which also shows a high degree of similarity to AChE (results not shown). Since 2G8 significantly inhibited the globular form of AChE from T. marmorata only but not the asymmetric form from this source or from other vertebrate AChE, it was of interest to us to determine whether this antibody recognized the latter forms at all. Crossreactivity was assessed using the enzyme-antigen immunoassay for AChE. When increasing amounts of AChE were added, an increased signal at 405 nm resulted, showing increased binding of AChE by 2G8 (Fig. 1 A). From the initial slopes of the binding curves, the relative extent of crossreactivity may be assessed. The G2 form of AChE from T. rnarmorata gave the steepest initial slope, showing the highest degree of crossreactivity. Decreasing crossreactivities were seen with the G4 form of AChE from bovine and rat brain, with the G2 form from flounder muscle, and with the G4 form of AChE from human brain. No crossreactivity a t all was determined with the G2 form of AChE from bovine and human red cell membranes or with the G4 form of BtChE from human serum. In this assay, the asymmetric form of AChE from T. marmorata showed a high degree of self-adsorption to the wells of micrplates yielding a background too high for reliable measurements. The crossreactivity of the asymmetric form of AChE from T. marmorata could thus not be determined with this assay. When plates were coated with increasing amounts of antibody to which a fixed amount of antigen was added, the dose/ response curves shown in Fig. 1 B were obtained. Again, no crossreaction was seen with the G2form of AChE from bovine and human red cell membranes as or with the G, form of BtChE from human serum. The crossreactivity of 2G8 with several forms of AChE was further ascertained using a solid-phase immunoassay which was independent of the catalytic activity of the antigen. In this method, the antigens were applied as dots on nitrocellulose sheets followed by incubation with antibody. Complexes formed between antigens and antibody were visualized by incubation with goat anti-mouse IgG conjugated to horseradish peroxidase and developed with diaminobenzidine. As shown in Fig. 2, both globular and asymmetric forms of AChE from T. marmorata gave a strong signal in the dot-blot while AChE from the electric organ of E. electricus gave a weaker one. Interestingly, a strong signal was obtained with the enzyme from flounder body muscle which shows a substrate specificity intermediate between a true AChE and a BtChE [35]. On the other hand, weaker signals were obtained with the G 4 form of AChE from mammalian brains. As shown previously [39, 451, AChE in brain consists mainly of a membrane-bound amphiphilic G4 form while 15 - 20% of all extractable activity is attributed to hydrophilic tetramers. It is of interest to note that in the dot-blot, there appear to be differences between these two forms of AChE as the amphiphilic form of the enzyme from bovine brain gave a stronger
62 0.12 I
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Protein applied (pg)
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Antibody (pg/wellj
Fig. 2. Dot-blot imrnunoassay f o r the crossreactivity of rnAb 2G8 with AChE and BtChE. The indicated amount of different vertebrate cholinesterases was applied in 2 p1 10 mM phosphate pH 7.4, containing 0.14 M NaCI, to a sheet of nitrocellulose paper. The sheet was incubated with the mAb 2G8 followed by incubation with horseradishperoxidase-conjugated goat anti-mouse IgG and developed with diaminobenzidine. (1) Gz form of AChE from T. marmorata; (2) asymmetric forms of AChE from T. rnarmorata; (3) AChE from electric eel; (4) G 2 form of cholinesterase from flounder (P.flesus)body muscle; ( 5 ) G4 form of AChE from rat brain; (6) G4 form of AChE from bovine brain; (7) G4 hydrophilic form of AChE from bovine brain; (8) G 2 form of AChE from bovine erythrocytes; (9) G4 form of AChE from human brain; (10) G2 form AChE from human erythrocytes; (1 1) BtChE from human serum
Fig. 1. Crossreactivity ojrnAb 2G8 with AChE and BtChEfrorn dgferent origins. Crossreactivity was assessed by the enzyme antigen immunoassay as described in Materials and Methods. (A) The response obtained with increasing amounts of antigen added to the microtitre wells coated with 0.5 pg purified mAb 2G8 per well. (B) The dose/response curves obtained after coating the wells with increasing amounts of antibody 2G8 to which 1 IU/well of the different enzyme and boiled for 3 min in presence of SDS. Since this treatment samples were added ( 0 )G2 form of AChE from T. marrnorata; (0) fully denatured the enzyme, it appeared that the reactivity of G2 form of cholinesterase from flounder (P.,flesus)body muscle; ( A ) our particular monoclonal antibody towards AChE was most G4 form of AChE from rat brain; ( W ) G4 form of AChE from bovine likely due to the recognition of a continuous rather than a brain; (A)G4 form of AChE from human brain; No crossreaction conformational epitope. In order to substantiate this assump( 0 )was seen with G z forms of AChE from bovine and human tion, the G2 form of the enzyme from T. m a r m o r a t a was erythrocytes, nor with BtChE from human serum
signal than the hydrophilic form from the same source (Fig. 2). In the dot-blot, no reaction was seen with the G 2 form of AChE from bovine and human red cell membranes or with the G4 form of BtChE from human serum. As shown in Fig. 3A, 2G8 reacted on a Western blot with tetrameric AChE from mammalian sources whereas no reaction was seen with either the mammalian G2 form of AChE or with BtChE from human serum. The band pattern obtained with AChE from E. electricus showed that this commercially available enzyme preparation was partially degraded, a n observation which is in line with that made by Small [46]. The native subunit of this enzyme migrated in the SDS gel to a position of 75 kDa while the fragments were seen at approximately 50 and 25 kDa. Despite this partial proteolysis, both the native subunit at 75 kDa as well as the breakdown products at 25 kDa and 50 kDa reacted with 2G8 (results not shown). The crossreactivity on Western blots was assessed on AChE samples which had been reduced by mercaptoethanol
inactivated in several ways. As shown in Table 1, neither heat denaturation nor inactivation by diisopropylfluorophosphate abolished the reactivity towards 2G8 either in the ELISA or on the Western blot. Cleavage of the enzyme by CNBr preserved crossreactivity, albeit at a somewhat lower level, while degradation of AChE by trypsin reduced the croessreactivity to a weak signal both in the ELISA and on the Western blot. These results showed that the binding of 2G8 to AChE occurred largely independently of a n intact tertiary structure of the enzyme, indicating that the epitope to which 2G8 bound is at least partially continuous. Another important feature of 2G8 pertains to the difference in reactivity towards tetrameric and dimeric AChE from mammalian sources. In view of the fact that the amino acid sequences of the two forms are identical or highly similar within the same species, one would expect similar crossreactivities if the antibody bound to a continuous epitope consisting of an identical or highly similar peptide sequence. Our results presented thus far clearly showed that this was not the case. Alternatively, the marked difference in crossreactivity could be explained in terms of different glycosylation patterns of dimeric and tetrameric forms of mammalian AChE if 2G8
63
A
I 2 3 4 5 6 7 8 9 1 0 11 12 13 14 15 16
kDa
43 30 20 14
--
B
,
Table 1. Reaction of G 2form of AChE from T. marmorata with 2G8 after different treatments After treatment indicated, equal amounts of the G 2 form of AChE from T. marmoratu were used for coating the plate in ELISA. Ascitic fluid (1 :200 dilution) was used for incubation followed by goat antimouse IgG conjugated to horseradish peroxidase and development with o-phenylenediamine dihydrochloride. Absorbance was measured at 490 nm after stopping the reaction with 100 ~ 1 M2 H 2 S 0 4per well. Results are given as the response compared to that of the native enzyme. Trypsin and CNBr treatment were carried out according to [29]. For Western blots, equal amounts of treated samples were used (details as described in Materials and Methods). n. d, not determined; iPr2P-F, diisopropylfluorophosphate AChE
ELISA
Western blot
%
1 2 3 4 5 6 7 8 9 10 1 1 1213141516
Native enzyme Heat-denatured iPr&F-treated CNBr-treated Trypsin-treated
100 104 92 52 16
n. d
++++ ++++ +++ +
kDa
94 67 43 30 20 14
---
i
c - -
Fig. 3. SDSjPAGE and Westernblot of different cholinesteruses before and ajter treatment with N-glycosidase F. Electrophoresis was carried out in reducing condition on a 5 - 15% gradient polyacrylamide gel. (A) Proteins stained with Coomassie brilliant blue R-250; (B) proteins revealed by Western blotting. Lanes 1 and 2, G 2 form of AChE from T. marmorata; lanes 3 and 4, asymmetric forms of AChE from T. marmouata;lanes 5 and 6. Gz form of cholinesterase from flounder ( P . flesus) body muscle; lanes 7 and 8, G4 form of AChE from bovine brain; lanes 9 and 10, G4form of AChE from human brain; lanes 11 and 12, Gz form of AChE from human erythrocytes; lanes 13 and 14, G z form of AChE from bovine erythrocytes; lanes 15 and 16, BtChE from human serum. Odd numbers, before, and even numbers, after, treatment with N-glycosidase F. The gels were calibrated with phosphorylase h (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa), and lactalbumin (14 kDa)
recognized carbohydrate residues attached to AChE. To answer this question, the different forms of AChE were treated with N-glycosidase F and the crossreactivity with 2G8 was assessed before and after deglycosylation. As further seen in Fig. 3A, the mass of the native subunits of the different forms of AChE investigated ranged between 65 - 81 kDa, whereas BtChE appeared at 87 kDa. Treatment of AChE with Nglycosidase F decreased the subunit mass of all forms of AChE, most of them migrating to a position on the gel corresponding to an apparent molecular mass of 59-60 kDa. A significant difference was seen between the G2 and the G4 forms of AChE from T. marrnorata. After deglycosylation, the former was at 56 kDa while the latter moved to 60 kDa on the gel. Fig. 3A further showed that AChE from bovine
sources had a higher subunit mass than the corresponding human forms, However, after deglycosylation, all forms moved to the same position on the SDS gel. This result indicated that bovine AChE bears more carbohydrate residues than the human enzyme. In addition, the native G2 form from mammals show a higher subunit mass than the G4 form the same species. After treatment with N-glycosidase F, both G2 and the G4 forms had the same subunit mass, indicating that within one species the G2 form shows a higher degree of glycosylation than the G4 form. The largest difference in subunit mass was seen in BtChE, showing that this enzyme bears even more carbohydrate chains. Western blots carried out with 2G8 before treatment with N-glycosidase F showed that all forms of AChE, except the enzyme from red cell membranes and BtChE, crossreacted with 2G8. However, after deglycosylation with N-glycosidase F, no crossreaction could be seen on the Western blot with any of the AChE forms (Fig. 3 B). This result clearly showed that an essential part of the epitope recognized by 2G8 consisted of N-linked carbohydrate chains.
DISCUSSION
In the present study, we investigated the crossreactivity of mAb 2G8 with dimeric and tetrameric AChE from several species. This mAb was originally raised against the enzyme from T. nacline timilei; our present results show that it crossreacted with all forms of AChE tested except the G2 form from human and bovine erythrocytes. mAb 2G8 was shown to inhibit the enzyme from T. nacline timilei [9, 131 and it is of interest to note that it also inhibited the G2 form of the enzyme from T. marmorata, while virtually no inhibition was seen with the asymmetric form. The fact that a fivefold molar excess of antibody was necessary to reach maximal inhibition suggests the presence of multiple binding sites on AChE for 2G8. AChE from other vertebrates was not inhibited by 2G8. This could have meant that 2G8 did not recognize AChE from other vertebrates at all, or that the antibody crossreacted in a non-inhibitory manner. From the results presented in Fig. 1, it is seen that the latter was the case except for the G2 forms from mammalian erythrocytes which did not crossreact at all. This differential reactivity is of interest as the G 2 form from
64 non-mammalian sources, i. e. from T. marmorata and flounder body muscle, readily crossreacted with 2G8. The crossreactivity of 2G8 with AChE from vertebrates was therefore also assessed in dot-blot experiments which showed that 2G8 is able to recognize the asymmetric form of AChE from T. marmorata as well as the enzyme from flounder and mammalian G 4 forms. This form of the enzyme is mainly found in brain where it exists as a membrane-bound form (80%) as well as a soluble form (20%). In the dot-blot the enzyme from flounder body muscle gave a stronger response with mAb 2G8 than that observed in the EAIA. This apparent discrepancy is explained by the difference in substrate specificity of the flounder enzyme which in evolution stands between AChE and BtChE [35]. The K , value for ATCh of the flounder enzyme is about 12 times higher than that of the other forms of AChE tested [35]. Western blotting of AChE showed that mAb 2G8 also reacted with enzyme that had been denatured by treatment with SDS and heat. mAb 2G8 thus recognized a continuous and not a conformational epitope. The band pattern seen for AChE from E. electricus is of interest as it revealed degradation of the commercially available enzyme to two immunoreactive fragments of 50 and 25 kDa. This further supports the notion that 2G8 binds to more than one epitope and that these epitopes are far apart on the primary structure of eel AChE. CNBr cleavage of AChE from T. marmorata yielded a number of peptides of which at least three gave a positive reaction with 2G8 on a Western blot (unpublished result). Since 2G8 could dinstinguish between G 2and G4 forms of mammalianAChE, it is reasonable to assume that the epitope recognized by 2G8 consisted of carbohydrate residues. This notion was substantiated by the results obtained from treatment with N-glycosidase F, an enzyme which specifically cleaves N-linked oligosaccharide chains. This enzyme reduced the molecular mass of all samples of AChE as well as of BtChE, showing that all forms carried N-linked carbohydrates. Except for the G 2 form of T. marmorata, all forms, after deglycosylation, moved on the gel with an apparent molecular mass of 59-60 kDa. Since this molecular mass corresponds to that calculated from available amino acid sequences, it is reasonable to assume that the proteins were completely deglycosylated and that they did not carry any 0linked carbohydrates. As shown previously [29], AChE from bovine brain and erythrocytes does not contain Nacetylgalactosamine, providing further evidence that these forms of mammalian AChE do not contain 0-linked carbohydrates. On the other hand, AChE from chromaffin cells appears to contain both N- and 0-linked carbohydrate chains [47]. After deglycosylation by N-glycosidase F, none of the forms of AChE investigated by us reacted with 2G8 on the Western blot. This result clearly demonstrated that the epitope to which 2G8 bound consists of N-linked carbohydrates. As suggested by the groups of Taylor [23] and MassouliC [24], both asymmetric and globular forms of AChE from Torpedo contain four potential N-glycosylation sites. The subunits of the asymmetric forms of T. marmorata consist of 575 amino acid residues, whereas the dimeric form has 535 residues [24]. As seen from Fig. 3, the subunit of the asymmetric form is about 4 kDa heavier than that of the globular form. Treatment with N-glycosidase F decreased the subunit mass by about 9 kDa in both forms, indicating that both forms are equally glycosylated. At present, however, it is not possible to assign definitely the number of oligosaccharide chains and their site of attachment. In view of the conclusion that the epitope to which 2G8 bound consists of N-linked carbohydrates, our
finding that 2G8 inhibited only the globular form of AChE from T. marmorata is of special interest. Both forms are essentially identical in their amino acid sequence [24] bearing identical potential glycosylation sites. The difference in inhibition seen with 2G8 thus suggests that there must be at least one difference in the sites at which N-linked carbohydrate chains are attached. Our results further suggest that in G z AChE from T. marmorata the one glycosylation site through which inhibition is conveyed is closer to the active site than in the asymmetric form. Our results show further that bovine AChE (both dimeric and tetrameric) is more heavily glycosylated than the corresponding human enzyme. Further, dimeric AChE from red cell membranes bears more carbohydrate residues than the tetrameric brain enzyme. Despite this, 2G8 did not react with dimeric AChE from either human or bovine red cell membranes, showing that a set of N-linked carbohydrates must exist on that form which is definitively different from that of the tetrameric brain form. The finding that BtChE is more heavily glycosylated than AChE (Fig. 3) is in line with the proposal by Lockridge et al. [25] that BtChE contains nine potential glycosylation sites. The structure of the N-linked carbohydrates of tetrameric BtChE, however, must definitely be different from that of tetrameric AChE from brain as 2G8 did not react with BtChE at all. We thank Dr Oksana Lockridge for comments and discussion. We are grateful to Miss Monika Boschung for expert technical assistance and to Mr Han Zhang for purifying the mAb by protein-ASepharose C L 4 B . We are also indebted to Susi Jungi for her excellent photographs. This research was supported by Swiss National Science Foundation grant (31-9119.87) as well as by contributions of the Wander Stlftung Bern and, in part, by the Chinese natural science foundation.
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65 16. Olson, C. E., Chhajlani,V., August, J. T. & Schmell, E. D. (1990) Arch. Biochem. Biophys. 277, 361 - 367. 17. Musset, F., Frobert, Y., Grassi, J., Vigny, M., Boulla, G., Bon, S. & Massoulie, J. (1987) Biochimie 6Y, 147-1556, 18. Bon, S.. Meflah. K., Musset, F., Grassi, J. & Massoulie, J. (1987) J . Neurochem. 49, 1720- 1731. 19. Rakonczay, 2.& Brimijoin, S. (1988) in Subcellular Biochemistry (Harris, J. R., ed.) vol. 12, pp. 335-378, Plenum Publishing Corp., New York. 20. Wolfe, A. D. (1989) Biochim. Biophys. Actu 997, 232-235. 21. Brimijoin, S. (1983) Prog. Neurohiol. 21, 291 -322. 22. MacPhee-Quigley, K., Taylor, T. & Taylor, S. S. (1985) J . Biol. C%PHI. 260, 12 185- 12 1 89. 23. Schumachcr. M., Camp, S., Maulet, Y., Newton, M., MacPheeQuigley, K., Tylor, S. S., Friedmann, T. & Taylor, P. (1986) Noture Sly, 407-409. 24. Sikorav, J. L., Krejei, E. & Massoulie, J. (1987) E M B O J . 6, 1865- 1873. 25. Lockridge, O., Bartels, C. F., Vaughan, T. A,, Wong, C. K., Norton, S. E. &Johnson, L. L. (1 987) J . B i d . Chem. 262.549 557. 26. Prody, C. A., Zevin-Sonkin, D., Genatt, A., Goldberg, 0. & Soreq, H. (1987) Proc. Natl Acad. Sci. U S A 84, 3555-3559. 27. Soreq, H. & Prody, C. A. (1989) Prog. Clin. Biol. Res. 28Y, 347359. 28. Doctor, B. P., Chapman, T. C., Christner, C. E., Deal, C. D., De La Hoz, D. M., Gentry, M. K., Ogert, R . A,, Rush, R. S., Smyth, K. K . & Wolfe, A.D. (1990) FEBS Lett. 266, 123-127. 29. Heider, H., Litynski, P., Stieger, S. & Brodbeck, U. (1991) Cell. Mol. Neurohiol. 11, 105- 1 18. 30. Doctor, B. P., Camp, S.. Gentry, M.-K., Taylor, S. S. & Taylor, P. (1983) Proc. Natl Acud. Sci. U S A 80, 5157- 5771.
31. Rakonczay, Z. & Brimijoin, S. (1985) Biochim. Biophys. Actu 832, 127 - 134. 32. Meflah. K., Bernard, S. & Massoulie, J. (1984) Biochimie66, 5969. 33. Abramson, S. N., Ellisman, M. H., Deerinic, T. J., Maulet, Y., Gentry, M. K., Doctor, B. P. & Tylor, P. (1989) J . Cell. B i d . 108,2301-2311. 34. Ellman, G . L., Courtney, D. K., Andres, V. & Featherstone. R. M. (1961) Biochem. Pharmacol. 7, 88-95. 35. Stieger, S., Gentinetta, R. & Brodbeck, U. (1989) Eur. J . Biochem. 181,633- 642. 36. Serrensen, K . & Brodbeck, U. (1986) Experientia 42, 161- 162. 37. Bruck, C., Portetelle, D., Glineur, C. & Bollen, A. (1982) J . Immunol. Methods 53, 313- 319. 38. Brodbeck, U., Gentinetta, R. & Ott, P. (1981) in Membrane proteins: a lahoratory manual (Azzi, A., Brodbeck, U. & Zahler, P., eds) pp. 85 -96, Springer-Verlag, Berlin. 39. Sorensen, K., Gentinetta, R. & Brodbeck, U. (1982) J. Neurochern. 39, 1050 - 1060. 40. Snrensen, K., Brodbeck, U., Landauer, P. & Lieflinder, M. (1985) Comp. Biochem. Physiol. SOC, 263 -268. 41. Stieger, S. & Brodbeck, U (1985) J . Neurochem. 44,48-56. 42. Rasmussen, A. G., Arends, J. & Larsen, S. 0. (1989) Scnnd. J . Clin. Luh. Invest. 49, 503 - 512. 43. Laemmli, U. K. (1970) Nature 227, 680-685. 44. Ploug, M., Jensen, A. L. & Barkholt, V. (1989) Anal. Biochem. 181.33-39. 45. Gennari, K . & Brodbeck, U (1985) J . Neurochem. 44, 697704. 46. Small. D. H. (1 990) Trends Biochem. Sci. 6 , 21 3 - 21 5. 47. Bon, S., Bader, M.-F., Aunis, D., Massoulit, J. & Henry, J.-P. (1990) Eur. J . Biochem. 190, 221 -232.
The monoclonal antibody 2G8 is carbohydrate-specific and distinguishes between different forms of vertebrate cholinesterases Jian LIAO ', Hardld HEIDER', Man-Chi SUN', Susi STIEGER' and Urs BRODBECK Institute of Biochemistry and Molecular Biology, University of Bern, Switzerland Institute of Pharmacology and Toxicology, Beijing, China (Received December 34, 1990)
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EJB 90 1488
The monoclonal antibody (mAb) 2G8 (subclass IgG2,) raised against acetylcholinesterase (AChE, EC 3.1.1.7) from electric organ of Torpedo nacline timilei crossreacted with AChE from Torpedo marmorata, electric eel (Electrophorus electricus), flounder (Platichthys Jlesus) body muscle, rat brain, bovine brain, and human brain, this suggests that the epitope to which mAb 2G8 bound had been highly conserved during evolution. N o crossreaction was found with AChE from human and bovine erythrocytes, nor with butyrylcholinesterase (BtChE, EC 3.1.13)from human serum. Binding of mAb 2G8 to the globular G z form of AChE from T. marmorata strongly decreased enzyme activity, while no significant inhibition was found with either collagen-tailed, asymmetric forms, or with the enzymes from flounder body muscle or mammalian sources. The possibility that mAb 2G8 bound to anionic sites of AChE could be excluded since neither edrophonium chloride nor decamethonium bromide influenced the binding of 2G8 to the enzymes. Enzyme-linked immunosorbent assay and Western blot showed that heat-denatured, diisopropylfluorophosphate-treated, CNBr- and trypsin-digested AChE from T. marmorata still reacted with mAb 2G8; this indicates that the epitope to which 2G8 bound, at least partially, belonged to a continuous determinant. Treatment of cholinesterases with N-glycosidase F abolished crossreaction with 2G8, showing that an essential part of the epitope consisted of N-linked carbohydrates.
A number of monoclonal antibodies (mAb) have been raised against acetylcholinesterase (AChE) of different origins which offer a promising means of gaining insight into structure, function, evolution and biosynthesis of this enzyme [ l 91. Several mAbs against AChE for Narke japonica and Torpedo nacline timilei were shown to inhibit enzyme activity [lo- 131, as well as antibodies raised against AChE from rabbit brain and human erythrocytes [ l , 14- 161. Other mAbs were found to recognize glycosylation sites on AChE. One of them, the IgM mAb Elec-39 raised against AChE from the electric organ of Electrophorus electricus and belonging to the HNK-1 anticarbohydrate family [17, 181, recognized asymmetric AChE from Electrophorus species as well as their lytic globular derivatives but did not inhibit enzyme activity. It did not crossreact with asymmetric forms of AChE from Torpedo but recognized the amphiphilic G z form from that organ. Between different species, most mAbs to mammalian AChE display a relatively low degree of crossreactivity [19]. One mAb, AE-2, was reported to have a relatively wide range of crossreactivity to AChE from several species [2, 7, 151. This mAb inhibited AChE from several sources [14, 16, 201, presumably by binding to one of the anionic sites [14, 151. Correspondence to U. Brodbeck, Institute of Biochemistry and Molecular Biology, University of Bern, Buhlstrasse 28, CH-3012 Bern, Switzerland Abbreviations. AChE, acetykhohnesterdse; BtChE, butyrylcholinesterase; EAIA, enzyme antigen immunoassay; ATCh, acetylthiocholine. Enzymes. Acetylcholinesterase (EC 3.1.1.7); butyrylcholinesterase (EC 3.1.1.8); N-glycosidase F (EC 3.2.2.18); trypsin (EC 3.4.21.4).
Studies on the primary structure and on the kinetic properties of AChE showed that the different forms of AChE share regions of common structural domains, e. g. the catalytic center and the peripheral anionic sites [21-291. Moreover, studies on the crossreactivity of mAb with different forms of AChE isolated from various sources further substantiated the existence of such common structural domains [4, 5, 15, 30, 311. At present, relatively little is known about glycosylation of AChE, in general, or about structural similarities of Nlinked carbohydrates between vertebrate AChE, in particular. From the primary structure of AChE from Torpedo californica [23] and Torpedo marmorata [24], it can be deduced that both asymmetric and globular forms of the enzyme contain four potential N-linked glycosylation sites. G 4 AChE from fetal bovine serum contains five potential sites [28] while butyrylcholinesterase (BtChE) from human serum contains nine [25]. Within one species, composition and structure of oligosaccharide chains may vary between AChE from different tissues. This was suggested by Meflah et al. [32] who reported differences in lectin binding for AChE from bovine lymphocytes, erythrocytes and brain. Interaction with lectins also distinguished between AChE from fetal bovine serum and bovine erythrocytes [7]. Differences in post-translational glycosylation might be a signal for differential association of the enzyme with the extracellular matrix or the cell membrane P31. In this paper, we present an example of an mAb, i.e. mAb 2G8, raised against AChE from the electric organ of T . nacline timilei which displayed a wide range of crossreactivity with
60 AChE from different vertebrates. The essential part to which 2G8 bound is an N-linked carbohydrate epitope which appears to be highly conserved during evolution.
Enzyme antigen immunoassay (EAIA)
The AChE activity was measured at room temperature using the method of Ellman et al. [34] with 1 mM acetylthiocholine (ATCh) iodide and 0.25 mM 5,5'-dithiobis(2-nitrobenzoic acid) in 100 mM sodium phosphate pH 7.4, containing 0.1 o/o Triton X-100. The same procedure was used to assay BtChE activity except that butyrylthiocholine (BtCh) iodide was used as substrate. In the cases of EAIA, AChE activity was measured in microplates according to the methods of Rasmussen et al. [8] and Stieger et al. [35].
EAIA was conducted based on the procedures of Rasmussen et al. [42] as follows. mAbs were first diluted in coating buffer (10 mM phosphate pH 8.0 containing 144 mM NaCl, 0.02% NaN3). Into each well of the polystyrene microplate (Immuno Nunc plate I, Denmark) 100 p1 mAb, blank or control was pipetted and incubated at 4°C for 24 h. The plates were then washed twice with buffer (10 mM sodium phosphate pH 7.2 containing 0.05% Tween-20). Blocking was carried out with coating buffer containing 1YObovine serum albumin (Sigma), 200 pl/well, and by incubation at 37°C for 1 h. The plates were then washed as in the second step. Samples (100 pl/well) of vertebrate AChE, diluted in 10 mM sodium phosphate pH 7.4 containing 144 mM NaCl and 0.1 YO Triton X-100, were pipetted into the wells and incubated at room temperature for 150 min on a slow shaker. The plates were then washed five times with washing buffer and developed by the addition of 100 pl/well assay buffer [l mM acetylthiocholine iodide and 0.25 mM 5,5'-dithiobis(2nitrobenzoic acid) in 100 mM sodium phosphate pH 7.4 containing 0.1% Triton X-1001. Absorbance was measured at 405 nm using the Molecular Devices Vm,, kinetic microplate reader.
Protein assay
Solid-phase immunoassay
Protein concentration was determined in polystyrenemicroplates (from Nunc, Denmark) according to Serrensen and Brodbeck [36] using the Pierce BCA kit with bovine serum albumin (Pierce Chemicals) as protein standard.
Ascitic fluid was kindly provided by G. Z. Yan and Y. B. Xin (Institute of Microbiology and Epidemiology, Beijing 100850, China). It contained mAb 2G8 raised against AChE from electric organ of T. nacline timilei [9] which was purified according to Wang et al. [13] by protein-A-Sepharose CL4B chromatography (Pharmacia Fine Chemicals, Uppsala, Sweden) or purified by chromatography on DEAE - Affi-gel blue (Bio-Rad) according to Bruck et al. [37]. mAb 4F19, raised against human erythrocyte AChE, was obtained as published previously [S]. Monoclonal anti-human IgM clone MB-11 was from Miles-Yeda Ltd (Israel).
Dot-blot immunoassays were carried out based on the procedures of S~rrensenet al. [14]. Aliquots (2 p1) of vertebrate AChE, diluted with 10 mM sodium phosphate pH 7.4 containing 0.14 M NaCl and 0.1% Triton X-100, were spotted directly onto strips of nitrocellulose membrane (Bio-Rad) using a Hamilton syringe. The strips were incubated at 37°C for 1 h with 20 mM Tris/HCl pH 7.4, containing 0.14 M NaCl and 3% bovine serum albumin (buffer A) and then rinsed once with buffer A devoid of albumin (buffer B); mAb (ascitic fluid), diluted 1 : 200 with buffer A containing 0.05% Tween20, was added. Incubation was carried out at 4°C overnight. The strip was rinsed four times with buffer B and incubated at room temperature for 2 h with horseradish-peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) diluted I : 1000 with buffer A containing 0.05% Tween-20. The strip was washed five times with buffer B and developed with a solution of 0.5 mg/ml of diaminobenzidine in buffer B containing 1 pl/ml of 30% H202.
Ch olinesterases
Enzyme-linked immunosorbent-assay (ELISA)
AChE was purified by affinity chromatography using two consecutive steps on the same resin. Unless otherwise stated, purifications were carried out on a resin containing trimethylammonium m-phenylenediamine as affinity ligand [38] as follows: AChE from bovine and human erythrocyte membranes according to Brodbeck et al. [38]; AChE from rat brain, bovine brain, and human brain according to Smensen et al. [39,40]; AChE from electric organ of T . marmorata according to Stieger and Brodbeck [41]. AChE from electric eel was purified from Sigma product (no. C-2879) by the procedures reported for the Torpedo enzymes [41]. The enzyme from flounder (Platichthys flesus) body muscle was prepared according to Stieger et al. [35] using edrophonium - Sepharose as high-affinity matrix. BtChE from human serum was kindly provided by Dr Oksana Lockridge (Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68105-1065, USA).
ELISA was performed as follows. Samples were first diluted with coating buffer (10 mM phosphate pH 8.0 containing 144 mM NaCl and 0.02% NaN3). The wells were coated with 100 p1 of the diluted specimens at 4°C for 24 h and then rinsed twice with washing buffer (10 mM sodium phosphate pH 7.2 containing 0.05% Tween-20). Postcoating was carried out at 37 "C for 1 h by the addition of 200 pl/well of the coating buffer containing 1YO bovine serum albumin washing three times, 100 pl/well of mAb diluted in washing buffer was added and incubated at 37°C for 1 h. After washing five times, 100 pl/well of second antibody solution (horseradish-peroxidase-conjugated goat anti-mouse IgG, diluted 1 : 1000 in washing buffer) was added to the plate and incubated at 37°C for 1 h. After washing five times, 100 ,ul/well of a solution of 0.1 YO o-phenylenediamine dihydrochloride (Sigma) containing 0.03% H 2 0 2in substrate buffer (35 mM citric acid, 65 mM Na2HP04 pH 5.0) was added to the wells. The reaction was
MATERIALS AND METHODS Unless otherwise stated, all chemicals were analytical grade and either from Sigma (St Louis, USA), Merck (Darmstadt, FRG), Fluka (Buchs, Switzerland), Bio-Rad (Richmond, USA) or Boehringer (Mannheim, FRG). AChE assay
Antibodies
61 allowed to proceed at room temperature for 15 min and stopped by adding 100 pl/well of 2 M H2S04then absorbance at 490 nm was measured on the microplate reader. SDS/polyacrylamide gel electrophoresis
SDSjPAGE was performed essentially according to the method described by Laemmli [43] using 5 - 3 5% polyacrylamide gradient gels. Protein was stained with Coomassie brilliant blue R-250. Electroblotting and Western blot on nitrocellulose and [email protected] membranes
After electrophoresis, proteins from SDSjPAGE and peptides from Tricine SDSjPAGE were electrophoretically transferred to nitrocellulose paper or to polyvinyldifluoride membranes in a semi-dry-blot system (Ancos, Denmark) according to Heider et al. [29] and Ploug et al. [44], respectively. After the transfer, nitrocellulose and polyvinyldifluoride membranes were rinsed three times with 50 m M Tris/HC1 p H 7.4 containing 0.14 M NaCl. Then, 20 m M TrisjHCl pH 7.4 containing 0.14 M NaCl and 3% bovine serum albumin was added, and blocking was carried out at room temperature for 2 h. Thereafter, the procedure described for the solid-phase immunoassay was followed. N-Glycosidase F treatment
AChE and BtChE were first dialysed against 20 m M sodium phoshate pH 7.2 containing 20 mM EDTA and 0.1YOnoctyl glucoside. Then, 50 IU AChE or 5 IU BtChE were treated in 50 p120 m M sodium phosphate p H 7.2, containing 20 m M EDTA and 1YOn-octyl glucoside, 0.1% SDS and 1% mercaptoethanol. To each sample, 1 unit N-glycosidase F (Boehringer Mannheim) was added and incubated at 37 "C for 16 h. RESULTS As shown previously [9, 131, mAb 2G8 raised against AChE from T. nacline timilei strongly inhibited that enzyme when acetylcholine was used as substrate. On the other hand, with indophenylacetate only a modest inhibition was seen. In the present study, the properties of 2G8 toward dimeric and tetrameric AChE from several vertebrates were evaluated. The dimeric G 2 form of AChE from T. marmorata was inhibited by 2G8. The extent of inhibition varied with time and concentration of antibody. When antigen and antibody were present in equal amounts, little inhibition was seen. It increased with increasing amounts and reached 75y0 at a fivefold molar excess of antibody. The formation of the complex was slow as it took 24 h to achieve maximal inhibition. Longer incubation did not significantly increase the extent of inhibition. Since the inhibition was seen with ATCh only, it was of interest to determine if positively charged inhibitors of AChE would influence the binding of 2G8 to the G2 form of AChE from T. rnarmorata. Neither high concentrations of edrophonium, which occupies the anionic site of the catalytic center of AChE, nor decamethonium, which binds to both the central and the peripheral anionic sites, significantly decreased the rate of binding of 2G8 to AChE (results not shown). Since the active site of AChE has been highly conserved during evolution, we expected that the antibody 2G8 would
not only inhibit AChE from T. murmorata but also AChE from various other sources. This was, however, not the case as the inhibition by 2G8 was specifically seen only with the G2 form of AChE from T. marmorata. The asymmetric form of AChE from T. marmorata was inhibited only to 7%, even at a fivefold molar excess of antibody. Even less inhibition was seen with the G2 form of AChE from flounder body muscle, and practically no inhibition was observed with the other forms of AChE tested, i. e. G4 forms of AChE from rat, bovine and human brain, and the G2 form of AChE from bovine and human red cell membranes. Further, the antibody did not inhibit the G4 form of BtChE from human serum, the active site of which also shows a high degree of similarity to AChE (results not shown). Since 2G8 significantly inhibited the globular form of AChE from T. marmorata only but not the asymmetric form from this source or from other vertebrate AChE, it was of interest to us to determine whether this antibody recognized the latter forms at all. Crossreactivity was assessed using the enzyme-antigen immunoassay for AChE. When increasing amounts of AChE were added, an increased signal at 405 nm resulted, showing increased binding of AChE by 2G8 (Fig. 1 A). From the initial slopes of the binding curves, the relative extent of crossreactivity may be assessed. The G2 form of AChE from T. rnarmorata gave the steepest initial slope, showing the highest degree of crossreactivity. Decreasing crossreactivities were seen with the G4 form of AChE from bovine and rat brain, with the G2 form from flounder muscle, and with the G4 form of AChE from human brain. No crossreactivity a t all was determined with the G2 form of AChE from bovine and human red cell membranes or with the G4 form of BtChE from human serum. In this assay, the asymmetric form of AChE from T. marmorata showed a high degree of self-adsorption to the wells of micrplates yielding a background too high for reliable measurements. The crossreactivity of the asymmetric form of AChE from T. marmorata could thus not be determined with this assay. When plates were coated with increasing amounts of antibody to which a fixed amount of antigen was added, the dose/ response curves shown in Fig. 1 B were obtained. Again, no crossreaction was seen with the G2form of AChE from bovine and human red cell membranes as or with the G, form of BtChE from human serum. The crossreactivity of 2G8 with several forms of AChE was further ascertained using a solid-phase immunoassay which was independent of the catalytic activity of the antigen. In this method, the antigens were applied as dots on nitrocellulose sheets followed by incubation with antibody. Complexes formed between antigens and antibody were visualized by incubation with goat anti-mouse IgG conjugated to horseradish peroxidase and developed with diaminobenzidine. As shown in Fig. 2, both globular and asymmetric forms of AChE from T. marmorata gave a strong signal in the dot-blot while AChE from the electric organ of E. electricus gave a weaker one. Interestingly, a strong signal was obtained with the enzyme from flounder body muscle which shows a substrate specificity intermediate between a true AChE and a BtChE [35]. On the other hand, weaker signals were obtained with the G 4 form of AChE from mammalian brains. As shown previously [39, 451, AChE in brain consists mainly of a membrane-bound amphiphilic G4 form while 15 - 20% of all extractable activity is attributed to hydrophilic tetramers. It is of interest to note that in the dot-blot, there appear to be differences between these two forms of AChE as the amphiphilic form of the enzyme from bovine brain gave a stronger
62 0.12 I
I
.-E E
v
c v)
2 Q
0.00
0.05
0.10
8 9
Enzymes (IUiwellj
a
*
.
10 0.25
-
B
11
0.01 0.05 0.20 0.40 1.00 0.20
Protein applied (pg)
.-E c
v
0.15
0.10 d
0.05
0.00 0.00
0.10
0.20
0.30
0.40
0.50
Antibody (pg/wellj
Fig. 2. Dot-blot imrnunoassay f o r the crossreactivity of rnAb 2G8 with AChE and BtChE. The indicated amount of different vertebrate cholinesterases was applied in 2 p1 10 mM phosphate pH 7.4, containing 0.14 M NaCI, to a sheet of nitrocellulose paper. The sheet was incubated with the mAb 2G8 followed by incubation with horseradishperoxidase-conjugated goat anti-mouse IgG and developed with diaminobenzidine. (1) Gz form of AChE from T. marmorata; (2) asymmetric forms of AChE from T. rnarmorata; (3) AChE from electric eel; (4) G 2 form of cholinesterase from flounder (P.flesus)body muscle; ( 5 ) G4 form of AChE from rat brain; (6) G4 form of AChE from bovine brain; (7) G4 hydrophilic form of AChE from bovine brain; (8) G 2 form of AChE from bovine erythrocytes; (9) G4 form of AChE from human brain; (10) G2 form AChE from human erythrocytes; (1 1) BtChE from human serum
Fig. 1. Crossreactivity ojrnAb 2G8 with AChE and BtChEfrorn dgferent origins. Crossreactivity was assessed by the enzyme antigen immunoassay as described in Materials and Methods. (A) The response obtained with increasing amounts of antigen added to the microtitre wells coated with 0.5 pg purified mAb 2G8 per well. (B) The dose/response curves obtained after coating the wells with increasing amounts of antibody 2G8 to which 1 IU/well of the different enzyme and boiled for 3 min in presence of SDS. Since this treatment samples were added ( 0 )G2 form of AChE from T. marrnorata; (0) fully denatured the enzyme, it appeared that the reactivity of G2 form of cholinesterase from flounder (P.,flesus)body muscle; ( A ) our particular monoclonal antibody towards AChE was most G4 form of AChE from rat brain; ( W ) G4 form of AChE from bovine likely due to the recognition of a continuous rather than a brain; (A)G4 form of AChE from human brain; No crossreaction conformational epitope. In order to substantiate this assump( 0 )was seen with G z forms of AChE from bovine and human tion, the G2 form of the enzyme from T. m a r m o r a t a was erythrocytes, nor with BtChE from human serum
signal than the hydrophilic form from the same source (Fig. 2). In the dot-blot, no reaction was seen with the G 2 form of AChE from bovine and human red cell membranes or with the G4 form of BtChE from human serum. As shown in Fig. 3A, 2G8 reacted on a Western blot with tetrameric AChE from mammalian sources whereas no reaction was seen with either the mammalian G2 form of AChE or with BtChE from human serum. The band pattern obtained with AChE from E. electricus showed that this commercially available enzyme preparation was partially degraded, a n observation which is in line with that made by Small [46]. The native subunit of this enzyme migrated in the SDS gel to a position of 75 kDa while the fragments were seen at approximately 50 and 25 kDa. Despite this partial proteolysis, both the native subunit at 75 kDa as well as the breakdown products at 25 kDa and 50 kDa reacted with 2G8 (results not shown). The crossreactivity on Western blots was assessed on AChE samples which had been reduced by mercaptoethanol
inactivated in several ways. As shown in Table 1, neither heat denaturation nor inactivation by diisopropylfluorophosphate abolished the reactivity towards 2G8 either in the ELISA or on the Western blot. Cleavage of the enzyme by CNBr preserved crossreactivity, albeit at a somewhat lower level, while degradation of AChE by trypsin reduced the croessreactivity to a weak signal both in the ELISA and on the Western blot. These results showed that the binding of 2G8 to AChE occurred largely independently of a n intact tertiary structure of the enzyme, indicating that the epitope to which 2G8 bound is at least partially continuous. Another important feature of 2G8 pertains to the difference in reactivity towards tetrameric and dimeric AChE from mammalian sources. In view of the fact that the amino acid sequences of the two forms are identical or highly similar within the same species, one would expect similar crossreactivities if the antibody bound to a continuous epitope consisting of an identical or highly similar peptide sequence. Our results presented thus far clearly showed that this was not the case. Alternatively, the marked difference in crossreactivity could be explained in terms of different glycosylation patterns of dimeric and tetrameric forms of mammalian AChE if 2G8
63
A
I 2 3 4 5 6 7 8 9 1 0 11 12 13 14 15 16
kDa
43 30 20 14
--
B
,
Table 1. Reaction of G 2form of AChE from T. marmorata with 2G8 after different treatments After treatment indicated, equal amounts of the G 2 form of AChE from T. marmoratu were used for coating the plate in ELISA. Ascitic fluid (1 :200 dilution) was used for incubation followed by goat antimouse IgG conjugated to horseradish peroxidase and development with o-phenylenediamine dihydrochloride. Absorbance was measured at 490 nm after stopping the reaction with 100 ~ 1 M2 H 2 S 0 4per well. Results are given as the response compared to that of the native enzyme. Trypsin and CNBr treatment were carried out according to [29]. For Western blots, equal amounts of treated samples were used (details as described in Materials and Methods). n. d, not determined; iPr2P-F, diisopropylfluorophosphate AChE
ELISA
Western blot
%
1 2 3 4 5 6 7 8 9 10 1 1 1213141516
Native enzyme Heat-denatured iPr&F-treated CNBr-treated Trypsin-treated
100 104 92 52 16
n. d
++++ ++++ +++ +
kDa
94 67 43 30 20 14
---
i
c - -
Fig. 3. SDSjPAGE and Westernblot of different cholinesteruses before and ajter treatment with N-glycosidase F. Electrophoresis was carried out in reducing condition on a 5 - 15% gradient polyacrylamide gel. (A) Proteins stained with Coomassie brilliant blue R-250; (B) proteins revealed by Western blotting. Lanes 1 and 2, G 2 form of AChE from T. marmorata; lanes 3 and 4, asymmetric forms of AChE from T. marmouata;lanes 5 and 6. Gz form of cholinesterase from flounder ( P . flesus) body muscle; lanes 7 and 8, G4 form of AChE from bovine brain; lanes 9 and 10, G4form of AChE from human brain; lanes 11 and 12, Gz form of AChE from human erythrocytes; lanes 13 and 14, G z form of AChE from bovine erythrocytes; lanes 15 and 16, BtChE from human serum. Odd numbers, before, and even numbers, after, treatment with N-glycosidase F. The gels were calibrated with phosphorylase h (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa), and lactalbumin (14 kDa)
recognized carbohydrate residues attached to AChE. To answer this question, the different forms of AChE were treated with N-glycosidase F and the crossreactivity with 2G8 was assessed before and after deglycosylation. As further seen in Fig. 3A, the mass of the native subunits of the different forms of AChE investigated ranged between 65 - 81 kDa, whereas BtChE appeared at 87 kDa. Treatment of AChE with Nglycosidase F decreased the subunit mass of all forms of AChE, most of them migrating to a position on the gel corresponding to an apparent molecular mass of 59-60 kDa. A significant difference was seen between the G2 and the G4 forms of AChE from T. marrnorata. After deglycosylation, the former was at 56 kDa while the latter moved to 60 kDa on the gel. Fig. 3A further showed that AChE from bovine
sources had a higher subunit mass than the corresponding human forms, However, after deglycosylation, all forms moved to the same position on the SDS gel. This result indicated that bovine AChE bears more carbohydrate residues than the human enzyme. In addition, the native G2 form from mammals show a higher subunit mass than the G4 form the same species. After treatment with N-glycosidase F, both G2 and the G4 forms had the same subunit mass, indicating that within one species the G2 form shows a higher degree of glycosylation than the G4 form. The largest difference in subunit mass was seen in BtChE, showing that this enzyme bears even more carbohydrate chains. Western blots carried out with 2G8 before treatment with N-glycosidase F showed that all forms of AChE, except the enzyme from red cell membranes and BtChE, crossreacted with 2G8. However, after deglycosylation with N-glycosidase F, no crossreaction could be seen on the Western blot with any of the AChE forms (Fig. 3 B). This result clearly showed that an essential part of the epitope recognized by 2G8 consisted of N-linked carbohydrate chains.
DISCUSSION
In the present study, we investigated the crossreactivity of mAb 2G8 with dimeric and tetrameric AChE from several species. This mAb was originally raised against the enzyme from T. nacline timilei; our present results show that it crossreacted with all forms of AChE tested except the G2 form from human and bovine erythrocytes. mAb 2G8 was shown to inhibit the enzyme from T. nacline timilei [9, 131 and it is of interest to note that it also inhibited the G2 form of the enzyme from T. marmorata, while virtually no inhibition was seen with the asymmetric form. The fact that a fivefold molar excess of antibody was necessary to reach maximal inhibition suggests the presence of multiple binding sites on AChE for 2G8. AChE from other vertebrates was not inhibited by 2G8. This could have meant that 2G8 did not recognize AChE from other vertebrates at all, or that the antibody crossreacted in a non-inhibitory manner. From the results presented in Fig. 1, it is seen that the latter was the case except for the G2 forms from mammalian erythrocytes which did not crossreact at all. This differential reactivity is of interest as the G 2 form from
64 non-mammalian sources, i. e. from T. marmorata and flounder body muscle, readily crossreacted with 2G8. The crossreactivity of 2G8 with AChE from vertebrates was therefore also assessed in dot-blot experiments which showed that 2G8 is able to recognize the asymmetric form of AChE from T. marmorata as well as the enzyme from flounder and mammalian G 4 forms. This form of the enzyme is mainly found in brain where it exists as a membrane-bound form (80%) as well as a soluble form (20%). In the dot-blot the enzyme from flounder body muscle gave a stronger response with mAb 2G8 than that observed in the EAIA. This apparent discrepancy is explained by the difference in substrate specificity of the flounder enzyme which in evolution stands between AChE and BtChE [35]. The K , value for ATCh of the flounder enzyme is about 12 times higher than that of the other forms of AChE tested [35]. Western blotting of AChE showed that mAb 2G8 also reacted with enzyme that had been denatured by treatment with SDS and heat. mAb 2G8 thus recognized a continuous and not a conformational epitope. The band pattern seen for AChE from E. electricus is of interest as it revealed degradation of the commercially available enzyme to two immunoreactive fragments of 50 and 25 kDa. This further supports the notion that 2G8 binds to more than one epitope and that these epitopes are far apart on the primary structure of eel AChE. CNBr cleavage of AChE from T. marmorata yielded a number of peptides of which at least three gave a positive reaction with 2G8 on a Western blot (unpublished result). Since 2G8 could dinstinguish between G 2and G4 forms of mammalianAChE, it is reasonable to assume that the epitope recognized by 2G8 consisted of carbohydrate residues. This notion was substantiated by the results obtained from treatment with N-glycosidase F, an enzyme which specifically cleaves N-linked oligosaccharide chains. This enzyme reduced the molecular mass of all samples of AChE as well as of BtChE, showing that all forms carried N-linked carbohydrates. Except for the G 2 form of T. marmorata, all forms, after deglycosylation, moved on the gel with an apparent molecular mass of 59-60 kDa. Since this molecular mass corresponds to that calculated from available amino acid sequences, it is reasonable to assume that the proteins were completely deglycosylated and that they did not carry any 0linked carbohydrates. As shown previously [29], AChE from bovine brain and erythrocytes does not contain Nacetylgalactosamine, providing further evidence that these forms of mammalian AChE do not contain 0-linked carbohydrates. On the other hand, AChE from chromaffin cells appears to contain both N- and 0-linked carbohydrate chains [47]. After deglycosylation by N-glycosidase F, none of the forms of AChE investigated by us reacted with 2G8 on the Western blot. This result clearly demonstrated that the epitope to which 2G8 bound consists of N-linked carbohydrates. As suggested by the groups of Taylor [23] and MassouliC [24], both asymmetric and globular forms of AChE from Torpedo contain four potential N-glycosylation sites. The subunits of the asymmetric forms of T. marmorata consist of 575 amino acid residues, whereas the dimeric form has 535 residues [24]. As seen from Fig. 3, the subunit of the asymmetric form is about 4 kDa heavier than that of the globular form. Treatment with N-glycosidase F decreased the subunit mass by about 9 kDa in both forms, indicating that both forms are equally glycosylated. At present, however, it is not possible to assign definitely the number of oligosaccharide chains and their site of attachment. In view of the conclusion that the epitope to which 2G8 bound consists of N-linked carbohydrates, our
finding that 2G8 inhibited only the globular form of AChE from T. marmorata is of special interest. Both forms are essentially identical in their amino acid sequence [24] bearing identical potential glycosylation sites. The difference in inhibition seen with 2G8 thus suggests that there must be at least one difference in the sites at which N-linked carbohydrate chains are attached. Our results further suggest that in G z AChE from T. marmorata the one glycosylation site through which inhibition is conveyed is closer to the active site than in the asymmetric form. Our results show further that bovine AChE (both dimeric and tetrameric) is more heavily glycosylated than the corresponding human enzyme. Further, dimeric AChE from red cell membranes bears more carbohydrate residues than the tetrameric brain enzyme. Despite this, 2G8 did not react with dimeric AChE from either human or bovine red cell membranes, showing that a set of N-linked carbohydrates must exist on that form which is definitively different from that of the tetrameric brain form. The finding that BtChE is more heavily glycosylated than AChE (Fig. 3) is in line with the proposal by Lockridge et al. [25] that BtChE contains nine potential glycosylation sites. The structure of the N-linked carbohydrates of tetrameric BtChE, however, must definitely be different from that of tetrameric AChE from brain as 2G8 did not react with BtChE at all. We thank Dr Oksana Lockridge for comments and discussion. We are grateful to Miss Monika Boschung for expert technical assistance and to Mr Han Zhang for purifying the mAb by protein-ASepharose C L 4 B . We are also indebted to Susi Jungi for her excellent photographs. This research was supported by Swiss National Science Foundation grant (31-9119.87) as well as by contributions of the Wander Stlftung Bern and, in part, by the Chinese natural science foundation.
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