Addition of a Glycophosphatidylinositol to Acetylcholinesterase

Coussen, Françoise; Ayon, Annick; Goff, Anne Le; Leroy, Jacqueline; Massoulié, Jean; Bon, Suzanne

doi:10.1074/jbc.m010817200

Cited by 25 publications

(27 citation statements)

References 46 publications

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“…The cellular extracts and the medium also contained amphiphilic uncleaved molecules and nonamphiphilic lytic molecules (15). The level of activity was considerably lower in the case of the A370N/V372T mutant, as discussed below, and this mutant produced an unusual nonamphiphilic species.…”

Section: Effects Of Fhb Mutations On Molecular Formsmentioning

confidence: 91%

“…For PI-PLC digestion, samples of detergent extracts (50 l) were incubated in TMg buffer for 1 h at 30°C with a 1% volume (0.025 IU) of PI-PLC from Bacillus thuringiensis (Immunotech (Marseille, France or Glyko Europe, Upper Heyford, UK)). Electrophoretic analyses in nondenaturing conditions and evaluation of PI-PLC-sensitive and -resistant components were performed as previously described (15).…”

Section: ϫ5mentioning

confidence: 99%

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Acetylcholinesterase H and T Dimers Are Associated through the Same Contact

Morel¹,

Leroy

Ayon

et al. 2001

Journal of Biological Chemistry

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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...

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Section: Effects Of Fhb Mutations On Molecular Formsmentioning

confidence: 91%

Section: ϫ5mentioning

confidence: 99%

Acetylcholinesterase H and T Dimers Are Associated through the Same Contact

Morel¹,

Leroy

Ayon

et al. 2001

Journal of Biological Chemistry

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“…The C2 mutant was derived by deletion of most of the C-terminal peptide from the H variant of AChE, leaving a cysteine at position 6 downstream of the common catalytic domain (23). Plasmids were transfected in COS cells with the DEAE-dextran method, as described previously (24), usually with 4 g of DNA/100-mm dish.…”

Section: Methodsmentioning

confidence: 99%

Determinants of the t Peptide Involved in Folding, Degradation, and Secretion of Acetylcholinesterase

Falasca¹,

Perrier

Massoulié

et al. 2005

Journal of Biological Chemistry

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The C-terminal 40-residue t peptide of acetylcholinesterase (AChE) forms an amphiphilic ␣ 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 ␣ 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.

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“…These signals comprise a stop codon at the C-terminal end and, proceeding upstream, a short stretch of hydrophobic residues (8-21 amino acids), a hydrophilic/neutral spacer region (usually 5-12 residues) and, finally, the cleavage/ attachment site of the protein to the GPI anchor, denoted the ω-site (Chen et al, 2001;Coyne et al, 1993). The ω and ω+2 residues are both critical for anchor addition and are usually residues with small side chains; ω -1 residues have also been observed to affect the efficiency of glypiation (Coussen et al, 2001). …”

Section: Sequence Analysismentioning

confidence: 99%

Minimal mutations are required to effect a radical change in function in CEA family members of the Ig superfamily

Naghibalhossaini

Stanners

2004

Journal of Cell Science

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GPI anchorage in the CEA family results in the acquisition of radically different functions relative to TM anchorage, including inhibition of differentiation and anoikis, disruption of tissue architecture and promotion of tumorigenicity. CEA GPI anchors, as determined by the carboxy-terminal exon of CEA, demonstrate biological specificity in their ability to confer these functional changes. CEA family GPI anchorage appears to have evolved twice independently during the primate radiation, in a manner suggestive of evolution from more primitive TM-anchored CEACAM1. We show here that very few mutations in the TM exon of present-day human CEACAM1 are required to give efficient GPI anchorage and the biological specificity of CEA GPI anchors, i.e., to give the differentiation-blocking function of GPI-anchored CEA. Such a change in anchorage could therefore represent a relatively facile means for producing radical change in molecular function of Ig superfamily members during evolution.

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Addition of a Glycophosphatidylinositol to Acetylcholinesterase

Cited by 25 publications

References 46 publications

Acetylcholinesterase H and T Dimers Are Associated through the Same Contact

Acetylcholinesterase H and T Dimers Are Associated through the Same Contact

Determinants of the t Peptide Involved in Folding, Degradation, and Secretion of Acetylcholinesterase

Minimal mutations are required to effect a radical change in function in CEA family members of the Ig superfamily

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