Natural Monomeric Form of Fetal Bovine Serum Acetylcholinesterase Lacks the C-Terminal Tetramerization Domain

Abstract

Acetylcholinesterase isolated from fetal bovine serum (FBS AChE) was previously characterized as a globular tetrameric form. Analysis of purified preparations of FBS AChE by gel permeation chromatography revealed the presence of a stable, catalytically active, monomeric form of this enzyme. The two forms could be distinguished from each other based on their molecular weight, hydrodynamic properties, kinetic properties, thermal stability, and the type of glycans they carry. No differences between the two forms were observed for the binding of classical inhibitors such as edrophonium and propidium or inhibitors that are current or potential drugs for the treatment of Alzheimer's disease such as (-) huperzine A and E2020; tacrine inhibited the monomeric form 2-3-fold more potently than the tetrameric form. Sequencing of peptides obtained from an in-gel tryptic digest of the monomer and tetramer by tandem mass spectrometry indicated that the tetramer consists of 583 amino acid residues corresponding to the mature form of the enzyme, whereas the monomer consists of 543-547 amino acid residues. The subunit molecular weight of the protein component of the monomer (major species) was determined to be 59 414 Da and that of the tetramer as 64 239 Da. The N-terminal of the monomer and the tetramer was Glu, suggesting that the monomer is not a result of truncation at the N-terminal. The only differences detected were at the C-terminus. The tetramer yielded the expected C-terminus, CSDL, whereas the C-terminus of the monomer yielded a mixture of peptides, of which LLSATDTLD was the most abundant. These results suggest that monomeric FBS AChE is trimmed at the C-terminus, and the results are consistent with the involvement of C-terminal amino acids in the assembly of monomers into tetramers.

Excessive Expression of Acetylcholinesterase Impairs Glutamatergic Synaptogenesis in Hippocampal Neurons

“…One day after plating, the plating medium was replaced with maintenance medium made up of Neurobasal medium, 1:50 B27 supplement, and 0.5 mM L-glutamine. Serum was not used in the maintenance medium because serum contains high and variable concentrations of AChE (Saxena et al, 2003). Consistent with a previous report (Brewer, 1995), only a small number of the glial cells existed in the culture under this condition.…”

Selective enhancement of the activity of C‐terminally truncated, but not intact, acetylcholinesterase

“…We present evidence towards a mechanistic link between this functional phenomenon and the strongly promoted hypothesis of the in vivo existence of a C‐terminal peptide of T‐AChE common to development and degeneration (Greenfield and Vaux 2002). While conclusive evidence for its existence is still to be provided (work ongoing in this laboratory), proteolytic cleavage of the C‐terminus has already been suggested for a fetal form of T‐AChE (Saxena et al. 2003), thus further supporting our assumption of the T‐AChE C‐terminus being cleaved off.…”

“…1992). Third, the sequence of the truncated form of AChE used in the study here (T548‐AChE), is highly similar to that of a physiological form of AChE detected in fetal bovine serum that has lost its C‐terminal tail supposedly because of proteolytic cleavage (Saxena et al. 2003).…”

Neuronal AChE splice variants and their non‐hydrolytic functions: redefining a target of AChE inhibitors?

“…Always considering that the above results have been obtained in different in vitro , even test tube model systems, they may, taken together, offer a possible scenario for mechanisms active under developmental/apoptotic conditions (Figure ): AChE‐T is cleaved through proteolysis, as has been suggested previously (Saxena et al ., ), and the C‐terminal peptide, T30, is freed (). The resulting, AChE‐Tt remains enzymatically active (, Zimmermann et al ., ).…”