The membrane anchor of mammalian brain acetylcholinesterase consists of a single glycosylated protein of 22 kDa

Boschetti, Nicola; Brodbeck, Urs

doi:10.1016/0014-5793(96)00041-5

Cited by 23 publications

(15 citation statements)

References 23 publications

(16 reference statements)

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“…The banding patterns in lanes 1 and 2 of Fig. 2B are identical to those in autoradiographs obtained after labeling bovine brain AChE with the hydrophobic photoactivated reagent 125 I-labeled TID (2,3,32). Since this reagent essentially labels only subunit P (2,3,32), these patterns provide strong evidence that G13T recognizes subunit P. Fig.…”

Section: Determination Of Peptide Sequences Of Small Proteins Copurisupporting

confidence: 60%

“…2B are identical to those in autoradiographs obtained after labeling bovine brain AChE with the hydrophobic photoactivated reagent 125 I-labeled TID (2,3,32). Since this reagent essentially labels only subunit P (2,3,32), these patterns provide strong evidence that G13T recognizes subunit P. Fig. 2C shows that the C17V antiserum, directed against mCutA, recognized a low molecular mass band of around 10 kDa, which was observed in nonreduced as well as in reduced samples and therefore was not disulfide-linked to AChE.…”

Section: Determination Of Peptide Sequences Of Small Proteins Copurimentioning

confidence: 69%

“…This reagent selectively labeled a 20-kDa protein that was disulfide-linked to catalytic subunits and released by disulfide reduction (2, 3, 10, 32); it was designated subunit P (4). Boschetti and Brodbeck (32) reported that the released TID-labeled subunit P corresponded to a doublet of 20-kDa bands that differed only in their extent of glycosylation. Sequencing of these bands revealed the same N-terminal sequence, EPQKSCSKYTD.…”

Section: Determination Of Peptide Sequences Of Small Proteins Copurimentioning

confidence: 99%

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Two Distinct Proteins Are Associated with Tetrameric Acetylcholinesterase on the Cell Surface

Perrier¹,

Cousin²,

Boschetti³

et al. 2000

Journal of Biological Chemistry

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In mammalian brain, acetylcholinesterase (AChE) exists mostly as a tetramer of 70-kDa catalytic subunits that are linked through disulfide bonds to a hydrophobic subunit P of approximately 20 kDa. To characterize P, we reduced the disulfide bonds in purified bovine brain AChE and sequenced tryptic fragments from bands in the 20-kDa region. We obtained sequences belonging to at least two distinct proteins: the P protein and another protein that was not disulfide-linked to catalytic subunits. Both proteins were recognized in Western blots by antisera raised against specific peptides. We cloned cDNA encoding the second protein in a cDNA library from bovine substantia nigra and obtained rat and human homologs. We call this protein mCutA because of its homology to a bacterial protein (CutA). We could not demonstrate a direct interaction between mCutA and AChE in vitro in transfected cells. However, in a mouse neuroblastoma cell line that produced membrane-bound AChE as an amphiphilic tetramer, the expression of mCutA antisense mRNA eliminated cell surface AChE and decreased the level of amphiphilic tetramer in cell extracts. mCutA therefore appears necessary for the localization of AChE at the cell surface; it may be part of a multicomponent complex that anchors AChE in membranes, together with the hydrophobic P protein.

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Section: Determination Of Peptide Sequences Of Small Proteins Copurisupporting

confidence: 60%

Section: Determination Of Peptide Sequences Of Small Proteins Copurimentioning

confidence: 69%

Section: Determination Of Peptide Sequences Of Small Proteins Copurimentioning

confidence: 99%

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Two Distinct Proteins Are Associated with Tetrameric Acetylcholinesterase on the Cell Surface

Perrier¹,

Cousin²,

Boschetti³

et al. 2000

Journal of Biological Chemistry

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“…AChE is located on the surface of erythrocytes, with the active site oriented towards the outside of the cell, whereas in the lipid bilayer it is anchored via a carboxy-terminal binding domain. 54) The activities of the tested compounds were investigated by determining the rate of hydrolysis of acetylthiocholine using the method of Ellman et al 41) Native enzymatic activity of AChE from erythrocytes was inhibited by indoloquinolones in a concentration (0.005, 0.06 mM) within which this amphiphilic agent has been demonstrated to be nonlytic for intact erythrocytes. As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Membrane Perturbations Induced by New Analogs of Neocryptolepine

Jaromin

Korycińska

Pįętka-Ottlik

et al. 2012

Biological & Pharmaceutical Bulletin

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antihyperglycemic, 8,9) anti-inflammatory 10,11) and antimalarial activities.12-15) Phyto-Riker (Gihoc) Pharmaceuticals Ltd. (Accra, Ghana) has developed an herbal preparation (Phyto-laria ® ) from the dried root of C. sanguinolenta for the treatment of malaria. According to clinical studies, this tea-bag formulation of the root powder (2.5 g) has been shown to be highly effective in the treatment of acute uncomplicated malaria, with an overall cure rate of 93.5%. Moreover, the laboratory findings did not suggest any toxicity. 16)However, indoloquinoline alkaloids are quite a rare group of natural products. This fact caused significant interest for the synthesis of indoloquinoline alkaloid derivatives, because of their potential biomedical and pharmaceutical value. 1,[17][18][19][20][21][22][23][24][25][26][27] Previous studies have indicated that all indolo[2,3-b]-quinolines belonging to the 5H-series possess interesting biological activities, including inhibition of the growth of Gram-positive bacteria and pathogenic fungi, cytotoxicity against KB cells and stimulation of the formation of calf thymus topoisomerase II mediated DNA cleavage. 17) 5,11-Dimethyl-5H-indolo [2,3-b] quinoline (DIMIQ), a benzoiso-α-carboline derivative, is one of the most interesting and active representatives of this group (Fig. 1). DIMIQ displayed strong antibacterial, antimycotic and cytotoxic activity in vitro as well as significant antitumor properties in vivo. 17,28,29) The influence of analogs of indolo [2,3-b] quinolines on cell cycle effects and their cytotoxicity have been extensively studied in vitro against several cell lines of different origin. Additionally, their ability to inhibit topoisomerase II activity was also evaluated. 18,[30][31][32][33] Osiadacz et al. have performed energy calculations for different DIMIQ orientations inside the DNA duplex. The studies have shown that the most preferred intercalation position, characterized by the lowest complex energy, was the parallel orientation of the long DIMIQ axis and the neighbor base-pair axes. 34)Since therapeutic and toxic effects of drugs are strongly influenced by their lipid affinity, the study of interactions with membranes was necessary. It was also of major importance to Regular Article

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“…In addition, PRiMA contains a putative N-glycosylation site between the polyproline stretches and the transmembrane domain (17). Alternative splicing produces two PRiMA variants, which differ in their C-terminal domains as follows: the intracellular domains of the major variant (PRiMA I) and of the minor variant (PRiMA II) contain 40 and 11 residues, respectively (18).…”

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confidence: 99%

Assembly of Acetylcholinesterase Tetramers by Peptidic Motifs from the Proline-rich Membrane Anchor, PRiMA

Noureddine

Schmitt

Liu

et al. 2007

Journal of Biological Chemistry

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The membrane-bound form of acetylcholinesterase (AChE) constitutes the major component of this enzyme in the mammalian brain. These molecules are hetero-oligomers, composed of four AChE catalytic subunits of type T (AChE T ), associated with a transmembrane protein of type 1, called PRiMA (proline-rich membrane anchor). PRiMA consists of a signal peptide, an extracellular domain that contains a proline-rich motif (14 prolines with an intervening leucine, P 4 LP 10 ), a transmembrane domain, and a cytoplasmic domain. Expression of AChE T subunits in transfected COS cells with a truncated PRiMA, without its transmembrane and cytoplasmic domains (P stp54 mutant), produced secreted heteromeric complexes (T 4 -P stp54 ), instead of membrane-bound tetramers. In this study, we used a series of deletions and point mutations to analyze the interaction between the extracellular domain of PRiMA and AChE T subunits. We confirmed the importance of the polyproline stretches and defined a peptidic motif (RP 4 LP 10 RL), which induces the assembly and secretion of a heteromeric complex with four AChE T subunits, nearly as efficiently as the entire extracellular domain of PRiMA. It is noteworthy that deletion of the N-terminal segment preceding the prolines had little effect. Interestingly, short PRiMA mutants, truncated within the proline-rich motif, reduced both cellular and secreted AChE activity, suggesting that their interaction with AChE T subunits induces their intracellular degradation.In the nervous tissue and muscles of mammals, acetylcholinesterase (AChE, 2 EC 3.1.1.7) controls cholinergic transmission by rapidly hydrolyzing the neurotransmitter acetylcholine after its release from nerve terminals. The functional localization of AChE depends on the association of its T splice variant with structural proteins (1-4). Thus, the physiologically active AChE species correspond essentially to the collagen-tailed forms at the neuromuscular junctions and to membrane-bound tetramers in the brain.The AChE T splice variant is characterized by its 40-residue C-terminal peptide (t peptide), which contains a C-terminal cysteine and seven aromatic residues, including three evenly spaced tryptophans, and can be organized as an amphiphilic ␣-helix (5). This peptide behaves as an autonomous interaction domain (the tryptophan (W) amphiphilic tetramerization domain (WAT)) (6); it allows oligomerization of AChE T subunits into homomeric dimers (T 2 ) and tetramers (T 4 ), as well as heteromeric associations of tetramers with anchoring proteins (7-9).In the collagen-tailed forms, AChE T tetramers are associated with a specific collagen, called ColQ (7, 10). This interaction has been extensively studied; it is based on a tight interaction between four t peptides (6) and a proline-rich motif, called PRAD ("proline-rich attachment domain"), located in the N-terminal noncollagenous region of ColQ (11). Synthetic t and PRAD peptides (40 and 15 residues, respectively) spontaneously form a complex, the structure of which has been determined by cry...

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The membrane anchor of mammalian brain acetylcholinesterase consists of a single glycosylated protein of 22 kDa

Cited by 23 publications

References 23 publications

Two Distinct Proteins Are Associated with Tetrameric Acetylcholinesterase on the Cell Surface

Two Distinct Proteins Are Associated with Tetrameric Acetylcholinesterase on the Cell Surface

Membrane Perturbations Induced by New Analogs of Neocryptolepine

Assembly of Acetylcholinesterase Tetramers by Peptidic Motifs from the Proline-rich Membrane Anchor, PRiMA

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