Monoclonal antibodies against chicken brain ... - FEBS Press

Eur. J. Biochem. 164,95-102 (1987) 0 FEBS 1987

Monoclonal antibodies against chicken brain acetylcholinesterase Their use in immunopurification and immunochemistry to demonstrate allelic variants of the enzyme William R. RANDALL, Karl W. K. TSIM, Josephine LA1 and Eric A. BARNARD Medical Research Council Molecular Neurobiology Unit, Medical Research Council Centre, Cambridge (Received June 19/December 1, 1986) - EJB 86 0632

Acetylcholinesterase (AChE) from 1-day chicken brain was enriched over 2000-fold by affinity chromatography using N-methylacridinium-Sepharose. This preparation was used to prepare monoclonal antibodies (mAb) directed against AChE, of which two were extensively characterised for further application. Both mAbs bound to the enzyme from the chicken with high affinity (Kd w 8 x lo-'' M) and one mAb, in addition, recognised AChE from quail brain and muscle. Neither mAb cross-reacted with mammalian or fish AChE. Both mAbs recognised AChE in the endplate region of adult chicken skeletal muscle and bound with equal affinity to the three major oligomeric forms found in early ambryonic muscle. One mAb was used to immunopurify chicken brain AChE to homogeneity (over 12000-fold enrichment), with nearly complete recovery of the enzyme and without detectable proteolytic breakdown. The other mAb recognised AChE after immunoblotting and was used to screen crude brain extracts from individual chickens for allelic variations. Evidence is presented to show that two allelic forms occur, represented in SDS-PAGE by a doublet polypeptide of MI w 110000, this pattern is maintained after deglycosylation of the N-linked oligosaccharides. This variation was found throughout development and in both the brain and the muscle of individuals. We conclude that the gene encoding the catalytic subunit of chicken AChE is polymorphic with either one or two equally active alleles being expressed.

Acetylcholinesterase (AChE) is an oligomeric enzyme of importance in nerve and muscle, which is found to exist in three general classes : (a) predominantly hydrophilic, globular forms, soluble in aqueous solutions of low salt concentration; (b) asymmetric forms requiring high salt for extraction from their tissues; (c) globular, predominantly hydrophobic forms requiring detergent for their extraction (for references see review [l]). Classes (a) and (b) are the major forms in skeletal muscles, while in the brains of mammals and birds the AChE is principally in the membrane-bound hydrophobic forms, associated into tetramers [2-41. The brain form of AChE has been purified from several mammals [3, 5-81 and the monomer has been found to be in each species a single polypeptide of MI z 70000. The corresponding avian form is also of interest, since much work on the biology of AChE has used it, especially from the chick owing to the greater accessibility of developmental stages in the avian embryo. There is a need in such studies for monoclonal antibodies directed against the avian AChE, and we have embarked upon their production. The avian AChE is clearly fairly different from the mammalian, since the molecular mass of each form Correspondence to W. R. Randall, MRC Molecular Neurobiology Unit, University of Cambridge Medical School, Hills Road, Cambridge, England CB2 2QH Abbreviations. mAb, monoclonal antibody; AChE, acetylcholinesterase; Dip-F, diisopropylfluorophosphate; TAPA-Sepharose, N,N,N-trimethyl-(m-aminophenyl)ammonium-Sepharose; isoOMPA, tetraisopropylpyrophosphoramide; PLD, posterior latissimus dorsi. Enzyme. Acetylcholinesterase (EC 3.1.1.7).

is about 40% greater than the corresponding mammalian form [9 - 111, and monoclonal or polyclonal antibodies against mammalian [12- 141or fish [15,16] AChE have shown little cross-reactivity to avian AChE. This emphasises the need for monoclonal antibodies (mAb) directed specifically against avian AChE. Satisfaction of that need should also lead to an immunoaffinity method for the complete purification of the avian AChE forms of all classes. In this report we present a method for enrichment of chicken brain AChE to a degree sufficient for the production of mAbs against its monomeric subunit. We describe two such mAbs and detail the characteristics of their binding to the antigen. We have used one of these, immobilised on a Sepharose matrix, to accomplish affinity purification of the brain AChE to homogeneity. The use of the other monoclonal antibody to identify the AChE subunit within individual birds yielded evidence that the gene encoding the catalytic subunit of AChE is polymorphic, with one or two alleles being expressed. While this work was in progress an interesting report appeared by Rotundo [17] on a different affinity purification method for chicken brain AChE and briefly described a monoclonal antibody, 1A2, prepared against it. Two polypeptide chains, a of M , 105000 and p of Mr 100000, were found and it was suggested that the tetrameric AChE molecule is composed of two heterodimers, i.e. having the structure a2 pz. The speculation was made that this heterogeneity is of functional significance. The evidence presented here clarifies this situation and shows that the structure of the tetrameric form of the brain AChE is a4.

96 The protein was eluted using the glycine buffer at pH 11.0 and neutralised immediately with 1 M Tris/HCl (pH 7.5). The Materials material (immunoaffinity eluate) was pumped onto a column Ovomucoid inhibitor (type 11) was from Sigma, free or containing N,N,N-trimethyl-(m-aminopheny1)ammoniumconjugated immunoglobulins from DAKO, concanavalin- Sepharose (TAPA-Sepharose), washed with 0.5 M NaC1/ A -Agarose from BRL, [3H]acetylcholine chloride (60 Ci 0.05% Triton X-100/10 mM Hepes (pH 7.5) and eluted with mmol - ') and [ 3H]diisopropylfluorophosphate (Dip-F; 50 Ci 0.1 M phenyltrimethylammonium bromide in the same medimmol- ') from Amersham. The affinity resins used were either um. Fractions containing the peak AChE activity were pooled N-methylacridinium-Sepharose,a gift from Professor J. (approximately 10 ml), dialysed against 0.05% Triton X-1001 Massoulie, having been prepared as described by Dudai and 10mM Hepes (pH 7.5) and further concentrated (purified Silman [18], or N,N,N-trimethyl-(m-aminopheny1)ammo- concentrate), as previously described, when necessary. nium-Sepharoqe a gift from Dr R. Raba [19]. Materials and methods not specified were as given elsewhere [9] or were from Enzyme assay standard sources AChE activity was assayed radiometrically [21] using a substrate concentration of 0.788 mM and sufficient [3H]aceEnrichment of'AC'hE,for immunisation tylcholine chloride to produce 100000 cpm when totally Brain from one-day chickens (approximately 60 g, freshly hydrolysed. The [3H]acetylcholine chloride used was reremoved) was homegenised (Polytron) in 10 vol. 400mM lyophilised from 5% acetic acid to remove the contaminant [3H]aceticacid, stored in aliquots under liquid N2 and thawed NaC1/12.5 mM Napi (pH 7.5)/5 mM EGTAjl mg ml-' bacitracin, followed by centrifugation (30 min, 30000 x g). All only once. Fractions eluting from the columns were diluted reactions were at 4"C. The pellets were rehomogenised in as needed to obtain measurable hydrolysis rates within the 8 vol. 50 mM Napi (pH 7.5)/0.5 M NaCl/0.5% Triton X-1001 incubation time of the assay. Although the decamethonium 1 mg ml-' bacitracin and centrifuged (1 h, 100000 xg). The present inhibited >95% of the enzyme activity there was supernatants were pooled and pumped onto a concanavalin- sufficient activity remaining to determine the elution peak. A- Agarose column (50 ml) overnight. The column was All assays were performed after incubation for 30 min with washed with 8 column volumes of 10mM Napi (pH 7.5)/ 0.1 mM tetraisopropylpyrophosphoramide (iso-OMPA), to 0.5 M NaCl/O.S'% Triton X-100 and the enzyme was eluted confine the activity to AChE [9]. 1 unit of AChE activity is with 5 column volumes of the same medium containing 0.5 M defined 1 pmol acetylcholine chloride hydrolysed minMolecular forms of AChE were separated using sucrose a-methylmannoside. The eluate was dialysed against two changes of 4 1 I 0 mM Napi (pH 7.5)/0.5?4 Triton X-100 (buf- density gradients as previously described [9], with similar fer 1) for 16 h and then pumped (60 ml h-') onto a DEAE- radiometric assay of the fractions. Sephacel column (40 ml) pre-equilibrated in buffer 1. Elution was performed with a 0 -250 mM NaCl gradient in 180 ml Deglycosy Lation buffer 1. Fractions containing the AChE activity were pooled Endoglycosidase F [22] was used. Samples (30 pl contain( z 50 - 180 mM NaCl region) and dialysed overnight. The enzyme was pumped through an affinity column (see above; ing 0.02 unit purified brain AChE) were incubated for 4 h at 20 ml column volume), pre-equilibrated with buffer 1, and the 37°C with or without endoglycosidase F (20 units ml-' column was then washed with 10 mM Napi (pH 7.5)/0.050/, diluted in 50 mM Nap, pH 6.1/0.1YO2-mercaptoethanol/0.1% Triton X- 100 (buffer 2) containing also 10 mM NaC1. Elution sodium dodecyl sulfate/50 mM EDTA/O.I M NaCl), then was with the same medium containing also 1 0 m M deca- denatured by adding SDS-PAGE sample buffer (defined methonium. Fractions containing the peak AChE activity below) on a heating block at 100"C for 5 min. (approximately 10 ml) were pooled and dialysed against two changes of 4 I buffer 2 at 4'C for 24 h. The dialysate was Screening protocol concentrated to approximately 0.5 ml in an Amicon ultrafiltration cell and either used fresh for immunisations or Hybridoma medium was screened for the presence of stored at - 20 ' C for further analysis. AChE-binding antibodies by a solid-phase immunoadsorption assay. Hybridoma medium (100 p1) was incubated for at least 3 h at room temperature in a 96-well polyethylene Immunopurl~ic.atiori microtitre plate (Microtest 111, Falcon), which had been preMonoclonal antibody ACB-1 was purified from coated with rabbit anti-(mouse immunoglobulin) (30 pg hybridoma medium using protein-A - Sepharose 4B [20]. ml-') and pre-adsorbed with 0.5% bovine serum albumin in Antibody was eluted with 0.1 M citric acidlsodium citrate (pH phosphate-buffered saline (NaCl/Pi). The wells were washed 3.0) and neutraliscd with 1 M Tris/HCl (pH 7.5). The protein three times with NaC1/Pi (200 pl) and an AChE solution was coupled Lo CNBr-activated Sepharose 4B (1 mg protein/ (100 pl) from an extract of one-day chick brain (homogenised ml activated Sepharose) and washed with acid (0.1 M citric 1 : 100 in NaC1/Pi/0.5% Triton X-100) was added and shaken acid, pH 3.0) and then with base (0.1 M glycine, pH 11.0) to for at least 4 h at 4°C. The wells were washed three times with NaC1/Pi/0.05% Nonidet P-40 (200 pl) and the AChE activity remove any uncoupled antibody. Brain (300 g) was homogenised and extracted as above immobilised on the surface was assayed directly in the well. except that 10 mM Hepes (pH 7.5) was used instead of the The reaction was carried out in 100 pl of a solution containing phosphate buffers. The supernatant was pumped onto and 10 mM Hepes pH 7.5/0.5 M NaCl/0.5% Triton X-100, and recirculated overnight (4°C) through a column containing contained the same concentrations of 3H-labelled and 35 ml beads, and washed with 20 volumes of 2 M NaCl/ unlabelled acetylcholine chloride as previously described. The 10mm Hepes (pH 7.5)/0.1% Triton X-100 and then with reaction was stopped by addition of 100 pl 1 M chloroacetic 0.05 M glycine (pH 9.0)/0.05 M NaCl/O.l% Triton X-100. acid/0.5 M NaOH/2 M NaCI. The solution was transferred MATERIALS AND METHODS

'.

97 directly into scintillation vials to which 5 ml scintillation cocktail was added and counted. Typically, non-specific binding was less than 2% and the signal of positive clones was about 70-fold greater than those clones not secreting antibodies against AChE. This solid-phase assay was additionally used for the affinity and competition measurements, with modification as described in the text. Immunisation and cell culture Female BALB/c mice (6-8 weeks) were immunised by intraperitoneal injections with approximately 15 pg purified AChE in complete Freund's adjuvant (DIFCO). Mice were boosted every 2 weeks with a similar amount of antigen in complete Freund's adjuvant. Blood serum taken from the tail vein was measured by the solid-phase immunoassay for the presence of antibodies binding AChE [using rabbit anti(mouse immunoglobulin) as the immobilising antibody] until a positive titre was obtained. These mice were given a final immunisation (15 pg, intraperitoneal) 4 days before hybridisation was performed. Spleen cells were hybridised by a modification [23] of the Kohler-Milstein procedure [24]. The spleen was gently dissociated mechanically in Dulbecco's modified minimum essential medium (DMEM, 2 ml) and the cell suspension was removed, washed in DMEM (18 ml) and pelleted. Exponentially growing myeloma cells (the SP2/OAG34 nonsecreting cell line, 1O7 cells/fusion) were similarly washed, mixed with the spleen cells and pelleted. Fusion was initiated by adding poly(ethyleneglyco1)-1500 (33% in DMEM, 0.3 ml). The suspension was mixed, centrifuged ( 5 min, 700xg) and the pellet was aspirated dry and gently resuspended in selective medium (DMEM/15% fetal calf serum/ 1 pg ml- azaserine/100 pM hypoxanthine/100 units ml- ' each of penicillin and streptomycin, total volume 100 ml). Cells were distributed onto ten 96-well microtitre trays and fed with growth medium (selective medium without azaserine and hypoxanthine, 100 pl/well) after 7 days incubation at 37°C in a humidified atmosphere of 8% COz. Hybridoma supernatants were assayed for immunoreactivity as soon as the cells reached confluence. Hybridomas whose media were positive for AChE binding were expanded and cloned in soft agarose [25] using at least two rounds of recloning for each hybridoma. The monoclonal antibodies obtained were classified by immunoprecipitation of the hybridoma-secreted immunoglobulin using subclass-specific rabbit anti-(mouse immunoglobulins) (Miles Scientific).

'

Gel electrophoresis and immunoblotting Samples were denatured at 100°C, 4 min, in a buffer containing 1YOsodium dodecyl sulfate 'and 1% dithiothreitol and separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) in 10% gels using the methods of Laemmli [26]. Standards used were phosphorylase b (MI 96000), bovine serum albumin (MI 68000), catalase (Mr 60000), ovalbumin ( M ,45000) and lactate dehydrogenase (Mr 36000). Visualisation was by Coomassie blue staining, or after [3H]Dip-F reaction, by fluorography [27] with exposure for 4- 7 days. Proteins from the gel were electroblotted (Bio-Rad Transblot system) onto nitrocellulose paper for 16 h [28]. The blot was adsorbed with hybridoma medium at room temperature (3 h), washed and incubated with a 1 :300 dilution of rabbit anti-(mouse immunoglobulin) antibody conjugated

to horseradish peroxidase. Development was with diaminobenzidine . 4 HCl (0.25 mg ml-')/HzOz (0.06% v/v)/NiS04 (0.3%). Irnrnunochemistry Frozen sections (10 pm) or teased fibres from adult chicken posterior latissimus dorsi (PLD) muslce were incubated with hybridoma medium (2 h). Sections were gently washed three times in NaCI/Pi and incubated (1 h) with either a fluoroscein-conjugated or a peroxidase-conjugated rabbit anti-(mouse immunoglobulin) antibody with development as noted as above. Sections were rinsed five times in NaC1/ Pi, mounted in glycerol/NaCl/Pi jelly and observed under a fluorescence or phase-contrast microscope. RESULTS Enrichment of brain AChE for immunisation More than 90% of the AChE in the one-day chick brain required a non-denaturing detergent for extraction and was presumed to be membrane-bound. Over 90% of the AChE extracted in detergent sedimented as the 12s (tetrameric) form when analysed by sucrose density gradients (data not shown). For the enrichment of AChE by N-methylacridiniumSepharose affinity chromatography, we found it essential first to use ion-exchange chromatography (DEAE-Sephacel; see Materials and Methods) since it removes some proteins which, if present, reduce the efficiency of binding of the AChE to the affinity column. Because the affinity resin also has ionexchange properties [29] we were unable to wash this column with NaCl concentrations greater than 10 mM without displacing some enzyme and other protein. T h s limited the final attained enrichment to a specific activity of approximately 400 pmol ACh hydrolysed min-' mg protein-'. This represented an approximate enrichment of 2000-fold over the initial crude extract and contained approximately 20% AChE by weight. Immunisation This preparation was used to immunise BALB/c mice. Two spleen cell/myeloma fusions were performed and two hybridomas secreting antibodies that bound chick brain AChE were selected to use in the present studies. One, designated ACB-1, is of subclass IgGz,, whereas the other, ACB-2, is of subclass IgG1. Af$nity an epitope competition The affinity of ACB-1 and ACB-2 for AChE from chick brain was measured, using a low concentration of AChE (2 mU ml-l), by serial dilution of the antibody (Fig. 1). Taking into consideration the fact that each IgG molecule contains two recognition sites and that the enzyme is present as a tetramer with one epitope per monomer, we calculated the apparent dissociation constant using the equation: b=

2 [Abl 2 [Ab] %

+

where b is the amount of epitope bound, [Ab] is the concentration of antibody and Kd is the dissociation constant. Since antibody binding to a single epitope of the enzyme tetramer

98 Table 1. mAb reactivity (solidphase assay) AChE activity was measured in the presence of 0.1 mM iso-OMPA for all but the last two samples. Non-specific or pseudocholinesterase (ns-ChE) was measured in the presence of 0.05 mM BW284c51 for these samples. Molecular forms of AChE were separated by sucrose density gradients (see Materials and Methods) and aliquots of the peaks (sedimentation values listed) were used for the assay AChE source

Molecular

ACB-1

ACB-2

+ + + + + +-

+ + + +

-

-

forms I 1

1

100 300 A N T I B O D Y ADDED (ng) lo

j0

1000

Fig. 1. Antigen uffjriify qf anti-AChE antibody. Dilution curves of and ACB-2 ( 0 )were constructed monoclonal antibodies ACB-1 (0) using the solid-phasc assays. AChE (0.2 m u , see Materials and Methods for definition 01' unit) from crude brain extracts of one-day chicks was used; the activity ofthe enzyme bound is expressed as a percentage of the AChE activitl added. The curves were fitted by eye and subsequent calculations of apparent dissociation constant (see text) were performed using the values at 50% binding

is probably sufficient for its immobilisation, the amount of enzyme bound t o the solid phase ( B ) is related to the number of epitopes per molecule ( n ) by

B

=

1 -(1 -b)"

where y1 = 4 for the tetrameric chicken brain form of AChE. The apparent Kd calculated thus from the half-saturation binding values lor IgG is 0.85 nM for ACB-1 and 0.77 nM for ACB-2. ACB-1 and ACB-2 do not compete for the same antigenic site. When AChE was first exposed to saturating amounts of ACB-2 and then assayed for its ability to bind ACB-1 attached to the solid-phase microtitre wells, the ACB-2-AChE complex was found still t o bind to ACB-1 throughout a range of concentrations from 10 ng to 1000 ng (data not shown). The amounts of bound enzyme compared closely with control wells in which the antigen was not initially complexed with ACB-2, showing that the two antibodies recognised different epitopes. Recognition ol'c.lioii~iesterasesfrom different species und tissues

Both ACB-1 and ACB-2 recognised AChE from brain and the three major molecular forms from muscle of the chicken (Table 1). ACB-1 also recognised AChE from quail tissues, albeit at approximately 30% of the binding affinity of the chicken AChE (data not shown). However ACB-2 did not recognise quail AChE nor did either monoclonal antibody recognise AChE from any other animal source tested. In addition, neither or the antibodies bound to non-specific cholinesterase (ns-ChE) from chick plasma or brain, when assayed in the solid-phase system using 0.05 mM 1,5,-bis(4allyldimethylainnroniuinphenyl)-pentan-3-one (BW284c51) to inhibit AChE and omitting iso-OMPA. ACB-1 bound to AChE transferred to nitrocellulose after SDS-PAGE (Fig. 3). ACB-1 recognised the polypeptide of M , I10000 in a sample enriched for chick brain AChE, which was used for the iinmunisations (Fig. 2, lane 1). ACB-1 also reacted with a band of similar M , in brain or muscle crude extracts of newly hatched chicken (lanes 2 and 3) and quail

Chicken brain Chicken musk Quail brain Quail muscle Torpedo electric organ Electrophorus electric organ Rabbit brain Rat brain Guinea-pigbrain Chicken ns-ChE (brain) Chicken ns-ChE (plasma)

7s

12 s 20 s

-

-

-

-

-

-

-

-

-

-

(lanes 4 and 5). ACB-1 did not bind detectably to any component in similar analyses of extracts of the brain of the rabbit, rat or guinea-pig brain or the electric organs of Torpedo or Electrophorus (data not shown). It should be noted that ACB-1 binding to the enriched AChE sample (lane 1) detected a low level of proteolysis of the enzyme, which occurred during this method of preparation. The assignment of the smaller components as breakdown products of the 110000-121,subunit was based on their identification, using SDS-PAGE and autoradiography, after reaction to [3H]Dip-F (0.1 mM) and to their supression when the preparation was first pretreated with eserine (0.1 mM) prior ["]Dip-F treatment (data not shown). Additional support is given to this point by the fact that the breakdown products were suppressed in the crude extracts (lanes 2 and 3), which were prepared in much less time (approximately 20 min) than the AChE-enriched sample and contained an extensive set of protease inhibitors (EGTA, bacitracin, benzamidine, N-ethylmaleimide and ovomucoid ovoinhibitor; see Fig. 2 for concentrations). It should also be noted that two bands are visible in the blots of the quail extracts (lanes 4 and 5), suggesting that there are two forms of the catalytic subunit with different electrophoretic mobilities in the quail. Furthermore, AChE from extracts of quail muscle appeared to migrate slightly more slowly than AChE from quail brain. It was also found that ACB-1 binds to deglycosylated AChE. The purified AChE from chick brain was incubated (4 h, 37°C) with or without endoglycosidase F (20 U/ml; see Materials and Methods for reaction conditions) and transferred to nitrocellulose after SDS-PAGE (Fig. 3). A time series (not shown) confirmed that the maximum change was produced within the period used. The binding of ACB-1 to a protein of approximate M , 96 000 after the endoglycosidase F treatment (Fig. 3, lane 2) indicates that the antibody recognises the polypeptide itself and not its carbohydrate moiety. ACB-2 did not recognise AChE that had been denatured with sodium dodecyl sulfate and transferred to nitrocellulose after SDS-PAGE.

99

1 2 3 4

5

-9 6

-6 8

-4 5

Fig. 2. Antibody recognition of AChE from avian tissues. Aliquots of AChE from the enriched preparation used for immunisation or crude extract samples were separated by SDS-PAGE, blotted onto nitrocellulose and exposed to monoclonal antibody ACB-1 (see Materials and Methods). Crude extracts were prepared with protease inhibitors: EGTA (5 mM), bacitracin (1 mg ml-'), benzamidine (2 mM), N-ethylmaleimide ( 5 mM) and ovomucoid ovoinhibitor (1.2 mg m1-I). The AChE activity, which could be loaded, varied as stated. 1, Preparation enriched for brain AChE (0.1 U). 2, Crude extract from one-day chicken brain (0.02 U). 3, Crude extract from one-day chick muscle (0.004 U). 4, Crude extract from 5-week quail brain (0.015 U). 5, Crude extract from 5-week quail muscle (0.006 U). All of the crude extracts were fully protected from proteolysis (see Materials and Methods). The antibody concentrations were 25 pg/ ml. The numbers shown are the molecular masses, in kDa, of the markers, phosphorylase b (96 kDa), bovine serum albumin (68 kDa) and ovalbumin (45 kDa), used in parallel. Antibodies were localised using peroxidase-labelled rabbit anti-(mouse immunoglobulin)-antibody Cytochemical localisation

Both the antibodies recognise high concentrations of this antigen localised in the endplate regions of adult PLD muscle (Fig. 4). Peroxidase labelling of teased fibers (Fig. 4A) or immunofluorescence of frozen tissue cut in cross-section (Fig. 4B) demonstrated the detailed morphology of the PLD motor endplates. The antibody labelling followed closely the deposition of reaction product using histochemical staining [31] for AChE (data not shown). Immunopurification

AChE from chicken brain was enriched over 10000-fold (Table 2) by a two-step purification using an immunoaffinity column consisting of mAb ACB-2 attached to Sepharose 4B followed by affinity chromatography using TAPA-Sepharose (see Materials and Methods). Since comparable specific activities are found for AChE after the intermediate step (immunoaffinity eluate) and after the final step (purified concentrate), we conclude that the enzyme can be completely purified using the immunoaffinity column alone. Hence the affinity chromatography step was introduced only to concen-

Fig. 3. Anti-AChE monoclonal antibody recognises the deglycosylated protein. Purified chick brain AChE was detected after SDS-PAGE and immunoblotting using ACB-1. 1. Control AChE (0.02 U) incubated without endoglycosidase F for 4 h. 2. AChE incubated in the presence of endoglycosidase F (4 h, 0.5 U, see Materials and Methods for conditions). Antibody localisation was performed as in Fig. 2

trate the sample further while permitting over 95% recovery of the active enzyme. TAPA-Sepharose was used in preference to N-methylacridinium Sepharose or DEAE-Sephacel because it bound the purified enzyme under conditions of higher NaCl concentration and did not require dialysis of the preparation prior to its application to the column. SDS-PAGE analysis of the preparation showed a single band at M , I10000 (Fig. 5, lane A). [3H]Dip-F reacts with this component (lane B), and is displaced when the [3H]DipF reacted sample is further treated for 20 min with 1 mM pyridine-Zaldoxime methiodide (lane C). The binding of [jH]Dip-F is also prevented by preincubating the sample with 0.1 mM eserine (not shown). Since this purification procedure required less than 36 h to complete, proteolytic breakdown was nearly absent, as demonstrated by the lack of Dip-Flabelled lower-molecular-mass bands (Fig. 5 , lane B). Allelic differences

Our enrichment of AChE and subsequent isolation of the mAbs was performed using one-day-old White Leghorn chicks obtained from a local commercial source. The enzyme subunit from this strain, identified by ACB-1 after SDS-PAGE and nitrocellulose blotting, always showed a single band at M , 110000 (Fig. 2, lane 2). However, brain extracts from another chicken strain (line 412 from the University of California, Davis, California, in New Hampshire stock) clearly exhibited two polypeptide bands ( a and a') when revealed by ACB-1 (Fig. 6A-2). In addition, brain (Fig. 6A) or muscle (Fig. 6 B) extracts from individual chickens (newly hatched) of the 412 strain always showed the expression of either a or a' or an equal combination of the two. Individuals showed this variation in banding pattern in the adult chickens (Fig. 6C) as well as in the newly hatched chicks. Furthermore the banding

100 Table 2. Immunopurificution of AChE from chick bruin The immunoaffinity purification started with 100 g, homogenised 1:10. AChE was assayed in the presence of 0.1 mM iso-OMPA using 10GI aliquots, which were diluted when necessary. Steps in the purification are defined in the text. Immunoaffnity eluate = neutralised eluate from the immunoaffinity column; Purified conc. = eluate from the TAPA-Sepharose column after dialysis and conccntration Fraction

Crude extract Immunoaffinity eluate Purified conc.

Volume

Total AChE

Protein

Specific activity

Purification factor

ml

pmol min -

mg

pmol min mg protein-’

1080 34 2.5

150 142 131

4166 0.366 0.360

0.18 2030 2041

%

-

A B

AChE recovery

zoo 11 278 11 312

99 98

C

96-

68

Fig 4 Cytothemit id loc uhation of monoclonal antibody ACB-1 Nonfixed teased fibre\ ( A ) or froien sections (B) from adult chicken PLD muscle were incubated with ACB-1 (approximdtely 10 pg, 3 h), washed dnd detected with either peroxidase-conjugated (A) or fluorescein-conlucg‘ited (B) rdbbit anti-(mouse immunoglobulin) antibody Bar, 20 pm

pattern is maintained when the samples are deglycosylated using endoglycosidase F (no shown). This indicates that two alleles are present within the AChE locus of this strain and that their expression is not dependent upon the source or developmental stage of the tissue.

DISCUSSION

AChE from chicken brain was enriched, using acridinium affinity chromatography, 2000-fold to approximately 20% of the total protein by weight. Monoclonal antibodies raised against this preparation bound to AChE but not to ns-ChE in a solid-phase assay. In immunoblotting experiments they specifically bound to the M,-I 10000 band, which comigrates with purified AChE from chicken brain.

-

Fig. 5. Immunopurification ojchicken brain AC’hE. ACh E was purified chromatographically by using mAb ACB-2-Sepharose and concentrated by using TAPA-Sepharose (see Materials and Methods). Analysis by SDS-PAGE and Coomassie blue R-250 staining showed that the sample migrated as a single polypeptide, M , 110000 (A). The sample reacted (15 min) with 0.1 mM [3H]I)ip-F (lo5 cpm) (B) and the introduced tritium was displaced by a further treatment with pyridine-2-aldoxime methiodide (1 mM) for 30 min (C) after removing the free [3H]Dip-Fby filtration (see Materials and Methods). Visualisation was by fluorography. Molecular markers were as in Fig. 2

The monoclonal antibodies obtained against AChE showed high binding affinity for the enzyme and specificity for avian forms of the enzyme. In their binding characteristics these antibodies closely resemble other mAbs, which have been produced against human [12, 131, rabbit [33],rat I321 and chicken [17] AChE. ACB-1 and ACB-2 are not directed against the same epitope, as is shown by their lack of mutually competitive binding to AChE and by the difference between them in the ability to recognise the SDS-denatured protein. The ability of ACB-2 to bind only to native forms of the enzyme suggests that it recognises a conformation-dependent epitope. Because the two mAbs bind to all three major hydro-

101

1

A 2

3

1

B 2

C 3

96-

Fig. 6. AChE subunits from individual chickens show allelic variations. AChE subunits from crude extracts of brain (0.02 U) or muscle (0.004 U) of individual birds (of line 412) were identified with mAb ACB-1 after SDS-PAGE and immunoblotting (as in Fig. 4). Extracts from brain (A) and muscle (B) from I-day-old chickens (three individuals; 1, 2 and 3); brain extracts (C) from 8-week-old chickens. These illustrates the general finding that two distinct bands CI and CI’ are identified in some individuals (A-2, B-2, C lane l), whereas others show either only the CI band (A-3, B-3, C lane 2) or the CI’ band (A-I, B-1). This individual variation is also seen in AChE from crude muscle extracts (B) of the same birds analysed in (A)

dynamically separable forms of AChE in muscle extracts, we conclude that the specific epitopes are on the catalytic subunit in all cases and are not within regions that are unique to the hydrophobic form of it. This result can be compared to previous findings, which indicate that immunodominant epitopes reside on the common regions of the catalytic subunit of AChE from Torpedo electric organ [15, 341, human erythrocyte membrane [12,13], rabbit brain [33], rat brain [32] and chicken brain [17]. A special problem involving the purification of avian AChE using the presently available affinity ligands is the sensitivity of their binding under moderate salt concentrations. As we and others [17, 291 have noted, acridinium and phenylammonium types of resins can exhibit ion-exchange properties and avian AChE is non-specifically eluted at NaCl concentrations above 10 - 50 mM. This is especially important when considering the purification of asymmetric forms of AChE from muscle tissue, which require high salt conditions to remain in solution. The immunoaffinity purification method used for avian AChE circumvents this problem and allows a washing protocol, which is necessary to remove the large amount of contaminating proteins found in brain and muscle tissues. This purification appears to be complete and can be performed using only the immunoaffinity step while the concentration step allows the reduction of the final sample volume. The results indicate that the brain AChE hydrophobic (tetramer) form contains no polypeptides other than the single catalytic subunit. A recent study has used a similar technique to purify rabbit brain AChE, using a mAb

directed against human erythrocyte AChE [8]. Further, we have been able to use the procedure described here successfully to bind and elute AChE from chicken muscle extracts and are currently developing the complete purification of the enzyme from that tissue source. The ability of ACB-1 to detect small amounts of AChE from brain and muscle extracts allowed us to examine the enzyme subunits from individual birds. The clear variation of subunit size within chickens of the line 412 strain indicates that individuals are either homozygous or heterozygous for alleles at the gene locus for AChE. The evidence (Fig. 6) that the subunit banding pattern was not tissue-specific nor dependent on the age of the bird further supports this conclusion. In following the a and a’ phenotypes in brain extracts from the progeny of a few crosses within this strain (unpublished data) the predicted pattern for simple alleles has been found. It is important to note that in a given individual we find that the major pattern (a, a’ or a d ) is constant in the catalytic unit in all of the major forms, and in muscle (Fig. 6 B) as well as in brain. This indicates that a common catalytic unit occurs in the major multiple forms. As noted in the introduction, Rotundo [17] has also observed two similar polypeptides in chicken brain AChE. These were always present in equal amounts. We now would interpret these not as two distinct chains, always occurring in the same AChE oligomer, but as being due to heterozygous animals used as the source. This result would arise when the tissue from many individuals was pooled if the stock used was randomly heterozygous at this locus. We find that the subunit structure of the tetrameric brain AChE is either (x4 or a& or equal amounts of a and a’; all three types have, so far as overall measurements go, the same level of AChE activity. Since these are a result of the pooling of all allelic subunits from a detergent extract of an individual brain sample, we cannot determine whether the tetrameric molecules are associated as a2a>homodimers or as a mixture of aza>heterodimers or a3a’ and aa; heterotetramers. However, since a population of the White Leghorn chickens was found which has only the CI subunit, we conclude that the function of AChE in the nervous system is adequately served with or without this heterogeneity.

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