The monoclonal antibody 2G8 is carbohydrate‐specific and distinguishes between different forms of vertebrate cholinesterases

Abstract

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. No crossreaction was found with AChE from human and bovine erythrocytes, nor with butyrylcholinesterase (BtChE, EC 3.1.1 3) 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.

Characterization of Monoclonal Antibodies that Strongly Inhibit Electrophorus Electricus Acetylcholinesterase

“…Detectable cross-reactivity was only All mAbs were unable to detect SDS-denatured AChE when tested in dot-blot experiments, indicating that they are directed against conformational or discontinuous epitopes (data not shown). This makes it very unlikely that these mAbs are directed against a carbohydrate epitope as shown for mAb 2G8 (Liao et al, 1991).…”

“…The following inhibitory mAbs were characterized in detail: mAb AE2 (Fambrough et al, 1982;Sorensen et al, 1987;Wolfe, 1989;Doctor et al, 1989;Wolfe et al, 1993), mAbs 6H9 and 25B1 (Ashani et al, 1988(Ashani et al, , 1990(Ashani et al, , 1991, mAb F3-43 Brimijoin et al, 1985), mAb 2G8 (Liao et al, 1991), and mAb C1B7 (Olson et al, 1990). All these antibodies act as non-competitive inhibitors, modulating enzyme activity by binding to allosteric sites on AChE.…”

The Assembly of Proline-rich Membrane Anchor (PRiMA)-linked Acetylcholinesterase Enzyme

“…Different glycosylation patterns of AChE have been found in different tissues and in different molecular forms. In human and bovine, G 2 AChE from erythrocyte membrane is more heavily glycosylated than G 4 AChE in the brain (10,11). In chicken, AChE from muscle is ϳ5 kDa heavier than the enzyme from brain, possibly due to variation of glycosylation (12).…”

Monoclonal antibodies against brain acetycholinesterases which recognize the subunits bearing the hydrophobic anchor

“…The same procedure was used to assay BtChE activity except that 1 mM butyrylthiocholine iodide was used as substrate. Alternatively, AChE activity was determined in an enzyme antigen immunoassay (EAIA) as described by Liao et al (1991). In the assay of BtChE, samples in the test wells were incubated for 40 min at room temperature with 0.05 mM 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW 284C51, Sigma) before adding 1 mM butyrylthiocholine iodide.…”

“…Most mAb to mammalian AChE were found to crossreact with brain and erythrocyte forms of AChE. More recently, several mAb had been raised against AChE from Torpedo nucline timifei and electric eel, which recognized carbohydrate epitopes, thereby distinguishing mammalian brain G,-AChE from erythrocyte G,-AChE (Bon et al, 1987;Liao et al, 1991 and. However, little has been known about the immunochemical differences between AChE from brain and from erythrocytes, nor about the possible differences in immunochemical properties between the pair of subunits bearing the hydrophobic anchor and the pair of subunits devoid of it.…”