Respective roles of the catalytic domains and C‐terminal tail peptides in the oligomerization and secretory trafficking of human acetylcholinesterase and butyrylcholinesterase

Abstract

In vertebrates, butyrylcholinesterase (BChE T ) and the T splice variant of acetylcholinesterase (AChE T ) consist of a catalytic domain of approximately 500 residues, followed by C-terminal tail (t) peptides [1,2]. These peptides of 41 and 40 residues, respectively, contain seven strictly conserved aromatic residues, including three evenly spaced tryptophans, and a cysteine located at position )4 from the C-terminus. The t peptide plays an important role in the biosynthesis of cholinesterases, particularly their folding and export. For example, it has been shown that it induces the misfolding of a significant fraction of newly synthesized AChE polypeptides, and that this effect depends on hydrophobicity since it was maintained when the aromatic residues were replaced by leucines. The t peptide also reduces export, as indicated by the fact that Butyrylcholinesterase (BChE) and the T splice variant of acetylcholinesterase that is predominant in mammalian brain and muscles (AChE T ) possess a characteristic C-terminal tail (t) peptide. This t peptide allows their assembly into tetramers associated with the anchoring proteins ColQ and PRiMA. Although the t peptides of all vertebrate cholinesterases are remarkably similar and, in particular, contain seven strictly conserved aromatic residues, these enzymes differ in some of their oligomerization properties. To explore these differences, we studied human AChE (Aa) and BChE (Bb), and chimeras in which the t peptides (a and b) were exchanged (Ab and Ba). We found that secretion was increased by deletion of the t peptides, and that it was more efficient with a than with b. The patterns of oligomers were similar for Aa and Ab, as well as for Ba and Bb, indicating a predominant influence of the catalytic domains. However, addition of a cysteine within the aromatic-rich segment of the t peptides modified the oligomeric patterns: with a cysteine at position 19, the proportion of tetramers was markedly increased for Aa(S19C) and Ba(S19C), and to a lesser extent for Bb(N19C); the Ab(N19C) mutant produced all oligomeric forms, from monomers to hexamers. These results indicate that both the catalytic domains and the C-terminal t peptides contribute to the capacity of cholinesterases to form and secrete various oligomers. Sequence comparisons show that the differences between the t peptides of AChE and BChE are remarkably conserved among all vertebrates, suggesting that they reflect distinct functional adaptations.Abbreviations AchE T , T splice variant of acetylcholinesterase; BChE, butyrylcholinesterase; DEPQ, 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide; Nbs 2 , 5,5¢-dithiobis(2-nitrobenzoic acid); PRAD, proline-rich attachment domains; t, tail.

The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers

“…The core of this heteromeric association is the formation of a coiled-coil cylinder of four ␣-helical t-peptides around the PRAD, organized as a polyproline II helix (22). Our results show that t-peptides derived from vertebrate AChE T and BChE T subunits are compatible to form mixed coiled-coils associated with a PRAD, despite conserved differences between their sequences (12).…”

“…In contrast, the formation of tetramers depends on the presence of a t-peptide, either for AChE T subunits (12,37) or for BChE T subunits (13). Although subunits of type T can probably form nonamphiphilic homotetramers by themselves, the assembly of tetramers is efficiently induced by a PRAD-containing protein, ColQ or PRiMA (21,25), or by a polyproline peptide in the case of soluble BChE tetramers (44).…”

“…DNA Constructs and Transfection-The cDNAs encoding for human AChE T , BChE T , AChE BChE-T (AChE catalytic subunit with a BChE t-peptide), BChE AChE-T (BChE catalytic subunit with an AChE t-peptide), AChE ⌬T , and BChE ⌬T (catalytic subunits lacking t-peptides) were subcloned in pGS vectors, under a CMV promoter, as described previously (12). The cDNAs encoding full-length rat AChE and the full-length mouse PRiMA I, tagged with an HA epitope (YPYDVPDYA) at the C terminus, were subcloned into pEF-BOS vectors, driven by the EF-1␣ promoter (18,25).…”

“…The amounts of the various AChE or BChE forms were determined by summation of the enzymatic activities corresponding to the peaks of their respective sedimentation profiles. The sedimentation values of the enzymes were calculated from the positions of the markers, alkaline phosphatase and ␤-galactosidase, as described previously (12).…”

“…subunits (12,15) that is associated with ColQ. The BChE T subunits of this 20 S hybrid cholinesterase progressively disappear during muscle development from embryo to adult, ultimately resulting in an homogeneous asymmetric AChE form (24).…”