Characterization of Monoclonal Antibodies that Strongly Inhibit Electrophorus Electricus Acetylcholinesterase

Abstract

In this study, we describe three different monoclonal antibodies (mAbs Elec-403, Elec-408, and Elec-410) directed against Electrophorus electricus acetylcholinesterase (AChE) which were selected as inhibitors for this enzyme. Two of these antibodies , recognized overlapping but different epitopes, competed with snake venom toxin fasciculin for binding to the enzyme, and thus apparently recognized the peripheral site of AChE. In addition, the binding of Elec-403 was antagonized by 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284C51) and propidium, indicating that the corresponding epitope encompassed the anionic site involved in the binding of these lowmolecular-mass inhibitors. The third mAb (Elec-408), was clearly bound to another site on the AChE molecule, and its inhibitory effect was cumulative with those of Elec-403, Elec-410, and fasciculin. All mAbs bound AChE with high affinity and were as strong inhibitors with an apparent K, value less than 0.1 nM. Elec-403 was particularly efficient with an inhibitory activity similar to that of fasciculin. Inhibition was observed with both charged (acetylthiocholine) and neutral substrates (o-nitrophenyl acetate) and had the characteristics of a non-competitive process. Elec-403 and Elec-410 probably exert their effect by triggering allosteric transitions from the peripheral site to the active site. The epitope recognized by mAb Elec-408 has not been localized, but it may correspond to a new regulatory site on AChE.Keywords. acetylcholinesterase ; inhibitory monoclonal antibodies ; peripheral site ; fasciculin.Acetylcholinesterase (acetylcholine acetylhydrolase, AChE) is widely distributed in the central and peripheral nervous systems where it plays a critical role in quickly hydrolyzing the neurotransmitter acetylcholine. Hydrolysis is the result of nucleophilic attack of the carbonyl carbon by a serine residue in the enzyme active center and involves charge transfer via a histidine residue. In accordance with its biological function, a remarkable feature of AChE is its high catalytic rate in cleaving acetylcholine. AChE works at a rate approaching that of a diffusion-controlled process (Rosenbeny, 1975 ;Quinn, 1987). Even if the enzyme preferentially acts on its natural substrate, it can also efficiently hydrolyze neutral acetic esters including indophenyl acetate, naphthyl acetate, indoxyl acetate, and nitrophenyl acetate (Quinn, 1987). Until recently, it was usually considered that the catalytic center of AChE! was composed of an esteratic and an anionic subsite. The esteratic subsite contains the active serine and is the target for irreversible inhibitors, such as organo-phosphorus and carbamate compounds, which block enzyme activity by forming covalent bonds between phosphoryl or carbamoyl groups and the serine residue. The anionic subsite is implicated in orientating the positively charged substrate in the active center. Many reversible inhibitors that possess quater-

Crystal Structure of Snake Venom Acetylcholinesterase in Complex with Inhibitory Antibody Fragment Fab410 Bound at the Peripheral Site

“…Elec410 was reported to inhibit BfAChE "with an apparent K i in the nM range" (31). Curves for BfAChE inhibition by Elec410 and Fab410 spanning 2 orders of magnitude again ascertained formation of equivalent complexes despite the charge heterogeneity of both partners, and validated the use of Fab410 for the structural study (Fig.…”

“…Protein Purification and Preparation-mAb Elec410 was produced as an IgG1, from murine hybridoma (31). Isolation of Elec410 from the ascitic fluid and preparation and purification of Fab410 and its physical characterization by MALDI-TOF MS and various electrophoretic means were described previously (32).…”

“…As well, the absence of Fab410 interaction with EeAChE Leu 282 , which corresponds to BfAChE Ser 280 , reflects the limited reduction in Fab410 affinity observed upon substitution of this residue by the mammalian His counterpart (45). Hence, this experimentally based model of the Fab410-EeAChE complex largely accounts for the greater affinity of EeAChE for Fab410 and the absence of Elec410 effect on human, bovine, Torpedo, and Naja AChEs (27,31) and mAChE (data not shown).…”

“…However, it could be a vestige of the pancreatic origin of the venom gland (30). These snake venom AChEs are inhibited by small, organic PAS ligands such as propidium, albeit at a lower affinity compared with the other species found in neuronal or neuromuscular tissues, but they differ widely in their sensitivity to larger, peptidic PAS ligands such as Fas2 or mAb Elec410 (see below) (27,31,32). For example, the venom enzymes from Bungarus fasciatus (BfAChE) and Ophiophagus hannah are inhibited by Fas2 and Elec410, whereas those from Naja haje and Hemachatus haemachatus are not (27).…”

“…Elec410, one of the three inhibitory mAbs raised against natural Electrophorus electricus AChE (EeAChE), was initially reported to inhibit BfAChE with an apparent K i or IC 50 value "in the nanomolar range" versus the K d value of ϳ0.04 nM reported for the EeAChE antigen (27,31). This property and availability of the two protein sequences (38,44) were instrumental in delineating the binding site of Elec410 (and those of its Elec403 and Elec408 congeners) at the EeAChE surface using complementary biochemical and mutagenesis approaches (45).…”