Biogenesis, assembly and trafficking of acetylcholinesterase

Abstract

Acetylcholinesterase (AChE) is expressed as several homomeric and heterooligomeric forms in a wide variety of tissues such as neurons in the central and peripheral nervous systems and their targets including skeletal muscle, endocrine and exocrine glands. In addition, glycolipid-anchored forms are expressed in erythropoietic and lymphopoietic cells. While transcriptional and post-transcriptional regulation is important for determining which AChE oligomeric forms are expressed in a given tissue, translational and post-translational regulatory mechanisms at the level of protein folding, assembly and sorting play equally important roles in assuring that the AChE molecules reach their intended sites on the cell surface in the appropriate numbers. This brief review will focus on the latter events in the cell with the goal of providing novel therapeutic interventional strategies for the treatment of organophosphate and carbamate pesticide and nerve agent exposure. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Keywords: PRAD-KDEL peptides; XVth International Symposium on Cholinergic Mechanisms; acetylcholine esterase; cholinergic mechanisms.

Figure 1

Major events in the life cycle of acetylcholinesterase

Figure 2. Thermal inactivation of mouse AChE at 45°C and stabilization by the PRiMA subunit

A) COS cells were transfected with a plasmid encoding mouse AChE or co-transfected with a second plasmid encoding the mouse transmembrane anchor PRiMA at a 4:1 ratio to induce formation of the membrane-bound tetrameric enzyme. Three days later the cells were extracted and the cell supernatants divided into three equal aliquots; the first remained on ice and the next two were incubated at 45°C for three or fifteen minutes, then rapidly cooled on ice. The AChE oligomeric forms were then analyzed by velocity sedimentation on sucrose gradients and assay of enzyme activity using the Ellman assay. G4, AChE tetramers anchored by the PRiMA subunit; G1/G2, AChE monomers and dimers; Agg, an aggregate of dimers that forms from the mouse AChE subunits originally described by Massoulié and colleagues. When heated there is a rapid inactivation of the unstable monomeric and dimeric forms, whereas the PRiMA-induced tetrameric forms is relatively stable at this temperature. B) The areas under each of the curves illustrated in Figure 2A expressed as percent total at time 0 are plotted versus time to show the kinetics of thermal inactivation of the monomeric and dimeric AChE forms versus the tetrameric AChE assembled with the membrane anchor PRiMA.

Figure 3. Overexpression ColQ or PDI enhance active AChE expression and assembly of oligomeric forms in avian skeletal muscle

A) Overexpression of the non-catalytic collagen tail subunit ColQ, chicken PDI, or both in quail muscle cells in culture. Plasmids expressing each of the proteins were transfected into myoblasts and allowed to fuse and differentiate into myotubes. A12, A8 and A4, collagen-tailed AChE associated with three, two or one tetramer. G1–G4, globular AChE monomers, dimers and tetramers. B) Total AChE activity expressed after transfection with each or both of the additional proteins. There is an additional synergistic effect of expressing both the non-catalytic subunits together with a molecular chaperone. Each bar is the mean ± SEM of three gradients like those shown in A). This research was originally published in the Journal of Biological Chemistry. Ruiz CA, Rotundo RL. Dissociation of transcription, translation, and assembly of collagen-tailed acetylcholinesterase in skeletal muscle. J Biol Chem. (2009) 284:21488–21495. © the American Society for Biochemistry and Molecular Biology.”

Figure 4. Model illustrating the steps involved in assembling the higher order AChE oligomeric forms

Nascent AChE polypeptides are assisted in their folding through the aid of chaperones leading to the formation of partially folded unstable intermediates, some with catalytic activity. These are then further assembled into higher order forms such as the collagen-tailed form or the PRiMA-linked tetramers, or the glycolipid-anchored AChE dimers. MG, molten globule; G1 globular monomer; G2, globular dimer; G4, globular tetramer; NCS, non-catalytic subunit. ECM, extracellular matrix.