Quaternary Associations of Acetylcholinesterase

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We introduced various mutations and modifications in the GPI anchoring signal of rat acetylcholinesterase (AChE). 1) The resulting mutants, expressed in transiently transfected COS cells, were initially produced at the same rate, in an active form, but the fraction of GPI-anchored AChE and the steady state level of AChE activity varied over a wide range. 2) Productive interaction with the GPI addition machinery led to GPI anchoring, secretion of uncleaved protein, and secretion of a cleaved protein, in variable proportions. Unproductive interaction led to degradation; poorly processed molecules were degraded rather than retained intracellularly or secreted. 3) An efficient glypiation appeared necessary but not sufficient for a high level of secretion; the cleaved, secreted protein was possibly generated as a by-product of transamidation. 4) Glypiation was influenced by a wider context than the triplet / ؉ 1/ ؉ 2, particularly ؊ 1. 5) Glypiation was not affected by the closeness of the site to the ␣ 10 helix of the catalytic domain. 6) A cysteine could simultaneously form a disulfide bond and serve as an site; however, there was a mutual interference between glypiation and the formation of an intercatenary disulfide bond, at a short distance upstream of . 7) Glypiation was not affected by the presence of an N-glycosylation site at or in its vicinity or by the addition of a short hydrophilic, highly charged peptide (FLAG; DYKDDDDK) at the C terminus of the hydrophobic region.

Section: Methodsmentioning

confidence: 99%

Addition of a Glycophosphatidylinositol to Acetylcholinesterase

Coussen

Ayon

Goff

et al. 2001

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“…Because CutA has been proposed to participate in the membrane anchoring of AChE by the transmembrane protein PRiMA, we also studied the effect of CutA on the recruitment of AChE T tetramers by PRiMA (19,26) and also by an N-terminal fragment of cholinesterase-associated collagen Q, called Qs (17,27). The tetramers were mostly membrane-bound when associated with PRiMA and secreted when associated with Qs.…”

Section: Effect Of Transfected Cuta On the Production And Secretion Omentioning

confidence: 99%

“…Inhibitors-COS cells were transfected by the DEAE-dextran method, as described previously (17), using up to 2 g of pEF-BOS vector per 60-mm dish. After transfection, COS cells were incubated for 3-4 days at 37°C in a medium containing 10% Nuserum (Inotech, Dottikon, Switzerland), which had been pretreated with 10 Ϫ6 M soman to inactivate serum cholinesterases.…”

Section: Transfection and Culture Of Cos Cells And Treatment With Metmentioning

confidence: 99%

Protein CutA Undergoes an Unusual Transfer into the Secretory Pathway and Affects the Folding, Oligomerization, and Secretion of Acetylcholinesterase

Liang

Nunes-Tavares

Xie

et al. 2009

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The mammalian protein CutA was first discovered in a search for the membrane anchor of mammalian brain acetylcholinesterase (AChE). It was co-purified with AChE, but it is distinct from the real transmembrane anchor protein, PRiMA. CutA is a ubiquitous trimeric protein, homologous to the bacterial CutA1 protein that belongs to an operon involved in resistance to divalent ions ("copper tolerance A"). The function of this protein in plants and animals is unknown, and several hypotheses concerning its subcellular localization have been proposed. We analyzed the expression and the subcellular localization of mouse CutA variants, starting at three in-frame ATG codons, in transfected COS cells. We show that CutA produces 20-kDa (H) and 15-kDa (L) components. The H component is transferred into the secretory pathway and secreted, without cleavage of a signal peptide, whereas the L component is mostly cytosolic. We show that expression of the longer CutA variant reduces the level of AChE, that this effect depends on the AChE C-terminal peptides, and probably results from misfolding. Surprisingly, CutA increased the secretion of a mutant possessing a KDEL motif at its C terminus; it also increased the formation of AChE homotetramers. We found no evidence for a direct interaction between CutA and AChE. The longer CutA variant seems to affect the processing and trafficking of secretory proteins, whereas the shorter one may have a distinct function in the cytoplasm.

“…Tetramers of AChE T subunits also assemble around structural proteins; the hydrophobic P protein anchors the enzyme in cell membranes of the central nervous system (4,5), and collagen ColQ attaches it to the basal lamina of neuromuscular junctions (6,7). Isolated T peptides can associate with a short "proline-rich attachment domain" (PRAD) of the N-terminal region of ColQ (8,9); the C-terminal T peptide therefore behaves as an autonomous interaction domain and has been named the WAT domain (10). This suggests that the catalytic domain of AChE does not play a major part in the assembly of an AChE T tetramer around the PRAD.…”

mentioning

confidence: 99%

Acetylcholinesterase H and T Dimers Are Associated through the Same Contact

Morel¹,

Leroy

Ayon

et al. 2001

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Acetylcholinesterase (AChE) exists as AChE H and AChE T subunits, which differ by their C-terminal H or T peptides, generating glycophosphatidylinositol-anchored dimers and various oligomers, respectively. We introduced mutations in the four-helix bundle interface of glycophosphatidylinositol-anchored dimers, and analyzed their effect on the production and oligomerization of AChE H , of AChE T , and of truncated subunits, AChE C (without H or T peptide). Dimerization was reduced for all types of subunits, showing that they interact through the same contact zone; the formation of amphiphilic tetramers (Torpedo AChE T ) and 13.5 S oligomers (rat AChE T ) was also suppressed. Oligomerization appeared totally blocked by introduction of an N-linked glycan on the surface of helix ␣ 7,8 . Other point mutations did not affect the synthesis or the catalytic properties of AChE but reduced or blocked the secretion of AChE T subunits. Secretion of AChE T was partially restored by coexpression with Q N , a secretable protein containing a proline-rich attachment domain (PRAD); Q N organized PRAD-linked tetramers, except for the N-glycosylated mutants. Thus, the simultaneous presence of an abnormal four-helix bundle zone and an exposed T peptide targeted the enzyme toward degradation, indicating a cross-talk between the catalytic and tetramerization domains.Acetylcholinesterase (AChE) 1 is an essential component of the cholinergic synapse. This enzyme allows a precise control of cholinergic transmission by rapidly hydrolyzing the neurotransmitter, acetylcholine. In vertebrates, AChE is encoded by a single gene. In Torpedo and mammals, alternative splicing produces several types of subunits, mainly AChE H and AChE T , which possess the same catalytic domain but distinct C-terminal peptides (Table I). The H and T (tryptophan amphiphilic tetramerization; WAT) peptides determine different post-transcriptional modifications and quaternary associations (1).The H peptide contains one or two cysteines that are close to the catalytic domain and may form intercatenary disulfide bonds and also contains a signal for cleavage and the addition of a glycophosphatidylinositol (GPI). The AChE H subunits thus generate mature GPI-anchored amphiphilic dimers, which retain only a few residues beyond the catalytic domain. X-ray crystallography studies of Torpedo dimers showed that the contact zone between the two monomers is a "four-helix bundle" (FHB), formed by the ␣ 7,8 and ␣ 10 helices from each catalytic domain (Fig. 1); hydrophobic residues occupy a large proportion of this interface (2). The same contact was observed in crystals of mouse AChE from which the C-terminal T peptide had been deleted and which was monomeric in solution (3).The T (WAT) peptide possesses a free cysteine, which is located near its C terminus, at a distance of 36 residues from the catalytic domain (Table I); this peptide forms an amphiphilic ␣ helix, and its presence allows a variety of quaternary associations (1). The AChE T subunits generate monomers, dimers, and te...