Old and new questions about cholinesterases.

Cholinesterases have been intensively studied for a long time, but still offer many fascinating and fundamental questions regarding their evolution, activity, biosynthesis, folding, post-translational modifications, association with structural proteins (ColQ, PRiMA and maybe others), export or degradation. They constitute an excellent model to study these processes, particularly because of the sensitivity and specificity of enzymic assays. In addition, a number of provocative ideas concerning their proposed non-conventional, or non-catalytic functions deserve to be further documented.

Readthrough acetylcholinesterase expression remains minor after stress or exposure to inhibitors.

The gene of mammalian acetylcholinesterase (AChE) generates multiple molecular forms, by alternative splicing of its transcripts and association of the tailed variant (AChET) with structural proteins. In the mammalian brain, the major AChE species consists of AChET tetramers anchored to the cell membrane of neurons by the PRiMA protein (Perrier et al., 2002). Stress and anticholinesterase inhibitors have been reported to induce rapid and long-lasting expression of the readthrough variant (AChER) in the mouse brain (Kaufer et al., 1998). In the readthrough transcript, there is no splicing after the last exon encoding the catalytic domain, so that the entire alternatively spliced 3' region is maintained. It encodes a C-terminal peptide with no specific interaction properties: COS cells transfected with AChER produce a soluble, nonamphiphilic monomeric form. We quantified AChER and total AChE expression in the mouse brain after an immobilization stress and after heat shock in neuroblastoma cells, and compared the observed effects with those induced by irreversible AChE inhibition (Perrier et al., 2005).

The C-terminal peptides of acetylcholinesterase: cellular trafficking, oligomerization and functional anchoring.

In vertebrates, the catalytic domain of acetylcholinesterase (AChE) may be associated with several C-terminal peptides generated by alternative splicing in the 3' region of transcripts. The "readthrough" (R) variant results from a lack of splicing after the last exon encoding the catalytic domain. Such a variant has been observed in Torpedo and in mammals; its C-terminal r peptide, also called "AChE Related Peptide" (ARP), is poorly conserved between rodents and humans. In rodents, it is significantly expressed in embryonic tissues and at a very low level in the brain of adult mice; it may be increased under various stress conditions, but remains very low. The "hydrophobic" (H) variant generates glycolipid (GPI)-anchored dimers, which are expressed in muscles of Torpedo, and in blood cells of mammals; H variants exist in Torpedo and in mammals, but apparently not in other vertebrate classes, suggesting that they were lost during evolution of early vertebrates and re-appeared independently in mammals. The "tailed" (T) variant exists in all vertebrate cholinesterases and their C-terminal t peptides are strongly conserved; in mammals, AChE(T) subunits represent the major type of acetylcholinesterase in cholinergic tissues. They produce a wide variety of oligomeric forms, ranging from monomers to heteromeric assemblies containing the anchoring proteins ColQ (collagen-tailed forms) and PRiMA (membrane-bound tetramers), which constitute the major functional enzyme species in mammalian muscles and brain, respectively. The oligomerization of AChE(T) subunits depends largely on the properties of their C-terminal t peptide. These peptides contain seven conserved aromatic residues, including three tryptophans, and are organized in an amphiphilic alpha helix in which these residues form a hydrophobic cluster. The presence of a cysteine is required for dimerization, while aromatic residues are necessary for tetramerization. In the collagen-tailed molecules, four t peptides form a coiled coil around a proline-rich motif (PRAD) located in the N-terminal region of ColQ. The t peptide also strongly influences the folding and cellular trafficking of AChE(T) subunits: the presence of hydrophobic residues induces partial misfolding leading to inactive protein, while aromatic residues, organized or not in an amphiphilic helix, induce intracellular degradation through the "Endoplasmic Reticulum Associated Degradation" (ERAD) pathway, rather than secretion. It has been proposed that the r and t C-terminal peptides, or fragments of these peptides, may exert independent, non cholinergic biological functions: this interesting possibility still needs to be documented, especially in view of their various degrees of evolutionary conservation.

The readthrough variant of acetylcholinesterase remains very minor after heat shock, organophosphate inhibition and stress, in cell culture and in vivo.

Acetylcholinesterase (AChE) exists in various molecular forms, depending on alternative splicing of its transcripts and association with structural proteins. Tetramers of the 'tailed' variant (AChE(T)), which are anchored in the cell membrane of neurons by the PRiMA (Proline Rich Membrane Anchor) protein, constitute the main form of AChE in the mammalian brain. In the mouse brain, stress and anticholinesterase inhibitors have been reported to induce expression of the unspliced 'readthrough' variant (AChE(R)) mRNA which produces a monomeric form. To generalize this observation, we attempted to quantify AChE(R) and AChE(T) after organophosphate intoxication in the mouse brain and compared the observed effects with those of stress induced by swimming or immobilization; we also analyzed the effects of heat shock and AChE inhibition on neuroblastoma cells. Active AChE molecular forms were characterized by sedimentation and non-denaturing electrophoresis, and AChE transcripts were quantified by real-time PCR. We observed a moderate increase of the AChE(R) transcript in some cases, both in the mouse brain and in neuroblastoma cultures, but we did not detect any increase of the corresponding active enzyme.

Determinants of the t peptide involved in folding, degradation, and secretion of acetylcholinesterase.

The C-terminal 40-residue t peptide of acetylcholinesterase (AChE) forms an amphiphilic alpha helix with a cluster of seven aromatic residues. It allows oligomerization and induces a partial degradation of AChE subunits through the endoplasmic reticulum-associated degradation pathway. We show that the t peptide induces the misfolding of a fraction of AChE subunits, even when mutations disorganized the cluster of aromatic residues or when these residues were replaced by leucines, indicating that this effect is due to hydrophobic residues. Mutations in the aromatic-rich region affected the cellular fate of AChE in a similar manner, with or without mutations that prevented dimerization. Degradation was decreased and secretion was increased when aromatic residues were replaced by leucines, and the opposite occurred when the amphiphilic alpha helix was disorganized. The last two residues (Asp-Leu) somewhat resembled an endoplasmic reticulum retention signal and caused a partial retention but only in mutants possessing aromatic residues in their t peptide. Our results suggested that several "signals" in the catalytic domain and in the t peptide act cooperatively for AChE quality control.

Expression of PRiMA in the mouse brain: membrane anchoring and accumulation of 'tailed' acetylcholinesterase.

We analysed the expression of PRiMA (proline-rich membrane anchor), the membrane anchor of acetylcholinesterase (AChE), by in situ hybridization in the mouse brain. We compared the pattern of PRiMA transcripts with that of AChE transcripts, as well as those of choline acetyltransferase and M1 muscarinic receptors which are considered pre- and postsynaptic cholinergic markers. We also analysed cholinesterase activity and its molecular forms in several brain structures. The results suggest that PRiMA expression is predominantly or exclusively related to the cholinergic system and that anchoring of cholinesterases to cell membranes by PRiMA represents a limiting factor for production of the AChE tailed splice variant (AChET)-PRiMA complex, which represents the major AChE component in the brain. This enzyme species is mostly associated with cholinergic neurons because the pattern of PRiMA mRNA expression largely coincides with that of ChAT. We also show that, in both mouse and human, PRiMA proteins exist as two alternative splice variants which differ in their cytoplasmic regions.