Acetylcholinesterase H and T Dimers Are Associated through the Same Contact

Abstract

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...

The KDEL Receptor Modulates the Endoplasmic Reticulum Stress Response through Mitogen-activated Protein Kinase Signaling Cascades

“…I-II-III-Myc and -HA were made using the following primers and their complements (5Ј-CCGGAAGTCTGAACAGAA-GTTGATCTCAGAGGAGGACCTATAGACACCTTCTGT-ACGC-3Ј for I-II-III-Myc; 5Ј-CCGGAAGTCTTACCCCTAT-GACGTCCCAGATTATGCATGATCCACACCTTCTGTA-CGC-3Ј for I-II-III-HA). An N-glycosylation site (A2538N, V2540T), which is homologous to that produced in acetylcholinesterase (35), was added to the Tg-CD and ChEL constructs using the following primer and its complement oligonucleotide (5Ј-GGACTCAGATGCCCGCATCCTTGCT-AATGCTACATGGTATTACTCCTTGGAGCACTCC-3Ј). Each construct was confirmed by direct DNA sequencing before expression in 293 cells.…”

“…The presence of such a bond may be exploited in an assay of dimerization potential by nonreducing SDS-PAGE. In 293 cells pulse-labeled with 35 S-labeled amino acids and chased for 3 h, recombinant AChE was specifically immunoprecipitated from the chase medium bathing transfected cells, whereas only background bands were recovered from cells transfected with empty vector (Fig. 4B).…”

“…4A, upward arrow) falls in a peptide extension at the extreme carboxyl terminus of the monomeric protein just downstream from the ChEL homology region that includes one of the dimerization helices (33,35). Crystallographic evidence indicates that this unpaired Cys residue can form an intersubunit disulfide bond, which covalently stabilizes AChE homodimers (40).…”

“…2A. Of the various Tg domains, the carboxyl-terminal ChEL domain (581 residues) has distinct homology to acetylcholinesterase (11,12), which undergoes homodimerization via formation of a four-helix bundle (two helices from each monomer (33)(34)(35)). Using the PSIPRED program for prediction of protein secondary structure (38), we examined the sequence of the Tg ChEL domain.…”

“…Such mutations perturb intradomain structure (30), and interestingly, block homodimerization (31). Acquisition of quaternary structure has long been thought to be required for efficient export from the ER (32) as exemplified by authentic acetylcholinesterase (33,34) in which dimerization enhances protein stability and export (35).…”