Acetylcholinesterase associates differently with its anchoring proteins ColQ and PRiMA

Acetylcholinesterase tetramers are inserted in the basal lamina of neuromuscular junctions or anchored in cell membranes through the interaction of four C-terminal t peptides with proline-rich attachment domains (PRADs) of cholinesterase-associated collagen Q (ColQ) or of the transmembrane protein PRiMA (proline-rich membrane anchor). ColQ and PRiMA differ in the length of their proline-rich motifs (10 and 15 residues, respectively). ColQ has two cysteines upstream of the PRAD, which are disulfide-linked to two AChE(T) subunits ("heavy" dimer), and the other two subunits are disulfide-linked together ("light" dimer). In contrast, PRiMA has four cysteines upstream of the PRAD. We examined whether these cysteines could be linked to AChE(T) subunits in complexes formed with PRiMA in transfected COS cells and in the mammalian brain. For comparison, we studied complexes formed with N-terminal fragments of ColQ, N-terminal fragments of PRiMA, and chimeras in which the upstream regions containing the cysteines were exchanged. We also compared the effect of mutations in the t peptides on their association with the two PRADs. We report that the two PRADs differ in their interaction with AChE(T) subunits; in complexes formed with the PRAD of PRiMA, we observed light dimers, but very few heavy dimers, even though such dimers were formed with the PQ chimera in which the N-terminal region of PRiMA was associated with the PRAD of ColQ. Complexes with PQ or with PRiMA contained heavy components, which migrated abnormally in SDS-PAGE but probably resulted from disulfide bonding of four AChE(T) subunits with the four upstream cysteines of the associated protein.     

Near-complete adaptation of the PRiMA knockout to the lack of central acetylcholinesterase

J. Neurochem. (2012) 122, 1065-1080. ABSTRACT: Acetylcholinesterase (AChE) rapidly hydrolyzes acetylcholine. At the neuromuscular junction, AChE is mainly anchored in the extracellular matrix by the collagen Q, whereas in the brain, AChE is tethered by the proline-rich membrane anchor (PRiMA). The AChE-deficient mice, in which AChE has been deleted from all tissues, have severe handicaps. Surprisingly, PRiMA KO mice in which AChE is mostly eliminated from the brain show very few deficits. We now report that most of the changes observed in the brain of AChE-deficient mice, and in particular the high levels of ambient extracellular acetylcholine and the massive decrease of muscarinic receptors, are also observed in the brain of PRiMA KO. However, the two groups of mutants differ in their responses to AChE inhibitors. Since PRiMA-KO mice and AChE-deficient mice have similar low AChE concentrations in the brain but differ in the AChE content of the peripheral nervous system, these results suggest that peripheral nervous system AChE is a major target of AChE inhibitors, and that its absence in AChE- deficient mice is the main cause of the slow development and vulnerability of these mice. At the level of the brain, the adaptation to the absence of AChE is nearly complete.     

The assembly of proline-rich membrane anchor (PRiMA)-linked acetylcholinesterase enzyme: glycosylation is required for enzymatic activity but not for oligomerization

Acetylcholinesterase (AChE) anchors onto cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric form in vertebrate brain. The assembly of AChE tetramer with PRiMA requires the C-terminal "t-peptide" in AChE catalytic subunit (AChE(T)). Although mature AChE is well known N-glycosylated, the role of glycosylation in forming the physiologically active PRiMA-linked AChE tetramer has not been studied. Here, several lines of evidence indicate that the N-linked glycosylation of AChE(T) plays a major role for acquisition of AChE full enzymatic activity but does not affect its oligomerization. The expression of the AChE(T) mutant, in which all N-glycosylation sites were deleted, together with PRiMA in HEK293T cells produced a glycan-depleted PRiMA-linked AChE tetramer but with a much higher K(m) value as compared with the wild type. This glycan-depleted enzyme was assembled in endoplasmic reticulum but was not transported to Golgi apparatus or plasma membrane.     

Membrane targeting, shedding and protein interactions of brain acetylcholinesterase

The early stages of Alzheimer's disease are characterized by cholinergic deficits and the preservation of cholinergic function through the use of acetylcholinesterase inhibitors is the basis for current treatments of the disease. Understanding the causes for the loss of basal forebrain cholinergic neurons in neurodegeneration is therefore a key to developing new therapeutics. In this study, we review novel aspects of cholinesterase membrane localization in brain and propose mechanisms for its lipid domain targeting, secretion and protein-protein interactions. In erythrocytes, acetylcholinesterase (AChE) is localized to lipid rafts through a GPI anchor. However, the main splice form of AChE in brain lacks a transmembrane peptide anchor region and is bound to the 'proline-rich membrane anchor', PRiMA, in lipid rafts. Furthermore, AChE is secreted ('shed') from membranes and this shedding is stimulated by cholinergic agonists. Immunocytochemical studies on rat brain have shown that membrane-associated PRiMA immunofluorescence is located selectively at cholinergic neurons of the basal forebrain and striatum. A strong association of AChE with the membrane via PRiMA seems therefore to be a specific requirement of forebrain cholinergic neurons. α7 nicotinic acetylcholine receptors are also associated with lipid rafts where they undergo rapid internalisation on stimulation. We are currently probing the mechanism(s) of AChE shedding, and whether this process and its apparent association with α7 nicotinic acetylcholine receptors and metabolism of the Alzheimer's amyloid precursor protein is determined by its association with lipid raft domains either in normal or pathological situations.     

Assembly of acetylcholinesterase tetramers by peptidic motifs from the proline-rich membrane anchor, PRiMA: competition between degradation and secretion pathways of heteromeric complexes

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, P4LP10), 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 (T4-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 (RP4LP10RL), 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.     

Decay-accelerating factor (DAF) shares a common carbohydrate determinant with the variant surface glycoprotein (VSG) of the African Trypanosoma brucei

Decay-accelerating factor (DAF) is an integral membrane protein that inhibits amplification of the complement cascade on the cell surface. We and other investigators have shown that DAF is part of a newly characterized family of proteins that are anchored to the cell membrane by phosphatidylinositol (PI). The group includes the variant surface glycoprotein (VSG) of African trypanosomes, the p63 protein of Leishmania, acetylcholinesterase (AChE), alkaline phosphatase, Thy-1, 5'-nucleotidase, and RT6.2--an alloantigen from rat T cells. The structure of the membrane anchor has been best characterized for VSG, but chemical studies of the membrane anchors of AChE and Thy-1 suggest that similar glycolipid moieties anchor these proteins to the cell surface. In the VSG, the membrane anchor consists of an ethanolamine linked covalently to an oligosaccharide and glucosamine; the entire complex is anchored to the cell membrane by PI. Immunologically, this glycolipid defines an epitope, the cross-reacting determinant (CRD), that is only revealed after removal of the diacyl glycerol anchor by a phospholipase C. By Western blotting, we show here that DAF-S (DAF released from the membrane by PI-specific phospholipase C [PIPLC]) also contains CRD. Using a newly developed immunoradiometric assay (IRMA) in which the solid-phase capturing antibody is a monoclonal antibody to DAF and the second antibody is anti-CRD, we have been able to quantitate DAF-S. By IRMA, we show that the reaction between anti-CRD and DAF-S is specific, since the binding is competitively inhibited only by the soluble form of the VSG. These observations further support the concept that the glycolipid anchors of this new family of proteins have similar structures. DAF is also found as a soluble protein in various tissue fluids as well as in Hela cell supernatants. No evidence for the presence of the CRD epitope was found on these proteins, suggesting that these forms of DAF are not released from the surface of cells by endogenous phospholipases.     

Developmental Adaptation of Central Nervous System to Extremely High Acetylcholine Levels

Acetylcholinesterase (AChE) is a key enzyme in termination of fast cholinergic transmission. In brain, acetylcholine (ACh) is produced by cholinergic neurons and released in extracellular space where it is cleaved by AChE anchored by protein PRiMA. Recently, we showed that the lack of AChE in brain of PRiMA knock-out (KO) mouse increased ACh levels 200–300 times. The PRiMA KO mice adapt nearly completely by the reduction of muscarinic receptor (MR) density. Here we investigated changes in MR density, AChE, butyrylcholinesterase (BChE) activity in brain in order to determine developmental period responsible for such adaptation. Brains were studied at embryonal day 18.5 and postnatal days (pd) 0, 9, 30, 120, and 425. We found that the AChE activity in PRiMA KO mice remained very low at all studied ages while in wild type (WT) mice it gradually increased till pd120. BChE activity in WT mice gradually decreased until pd9 and then increased by pd120, it continually decreased in KO mice till pd30 and remained unchanged thereafter. MR number increased in WT mice till pd120 and then became stable. Similarly, MR increased in PRiMA KO mice till pd30 and then remained stable, but the maximal level reached is approximately 50% of WT mice. Therefore, we provide the evidence that adaptive changes in MR happen up to pd30. This is new phenomenon that could contribute to the explanation of survival and nearly unchanged phenotype of PRiMA KO mice.     

Cholinesterases: anchored enzymes in membranes and basal laminae

Two proteins, ColQ and PRiMA, organize tetramers of acetylcholinesterase (AChE) and of butyrylcholinesterase (BChE) through peptide interactions. A short proline rich sequence in the N-terminal domain of ColQ or PRiMA associates four C-terminal extension of AChE or BChE. ColQ targets the enzymes in the basal lamina, PRiMA targets the enzymes at the plasma membrane. These complexes represent the mature proteins. The unassembled C-terminal extention of AChE is the key determinant recognized during the "quality control" of protein synthesis. Unassembled catalytic subunits are then degraded by the proteasome pathway. At the neuromuscular junction, ColQ/AChE represents the concentrated enzyme. The clusterisation of AChE depends upon ColQ through three sites of interactions: two different heparin binding domains in the collagen domain interact with heparan sulfate proteoglycan particularly the perlecan and the C-terminal non collagenic domain interacts with MuSK, the tyrosine kinase receptor organiser of the neuromuscular junction. The absence of ColQ and AChE has revealed that the excess of Ach stimulates more nicotinic receptors but probably not until their desensitization. Several morphological modifications may help the clearance of Ach. Conversely the synapse transmission fails during high frequency nerve stimulation.     

Two distinct proteins are associated with tetrameric acetylcholinesterase on the cell surface

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.     

The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers: HYBRID MOLECULES COMPOSED OF ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE DIMERS ARE UP-REGULATED DURING DEVELOPMENT OF CHICKEN BRAIN*

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE_T and BChE_T with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q_N-GPI). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q_N-GPI always consist of AChE_T and BChE_T homodimers. The dimer formation of AChE_T and BChE_T depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal “t-peptides” in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE_T or BChE_T homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.     

The origin of the molecular diversity and functional anchoring of cholinesterases

Vertebrates possess two cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) which both hydrolyze acetylcholine, but differ in their specificity towards other substrates, and in their sensitivity to inhibitors. In mammals, the AChE gene produces three types of coding regions through the choice of 3' splice acceptor sites, generating proteins which possess the same catalytic domain, associated with distinct C-terminal peptides. AChE subunits of type R ('readthrough') produce soluble monomers; they are expressed during development and induced by stress in the mouse brain. AChE subunits of type H ('hydrophobic') produce GPI-anchored dimers, but also secreted molecules; they are mostly expressed in blood cells. Subunits of type T ('tailed') exist for both AChE and BChE. They represent the enzyme forms expressed in brain and muscle. These subunits generate a variety of quaternary structures, including homomeric oligomers (monomers, dimers, tetramers), as well as hetero-oligomeric assemblies with anchoring proteins, ColQ and PRiMA. Mutations in the four-helix bundle (FHB) zone of the catalytic domain indicate that subunits of type H and T use the same interaction for dimerization. On the other hand, the C-terminal T peptide is necessary for tetramerization. Four T peptides, organized as amphiphilic alpha helices, can assemble around proline-rich motifs of ColQ or PRiMA. The association of AChE(T) or BChE subunits with ColQ produces collagen-tailed molecules, which are inserted in the extracellular matrix, e.g. in the basal lamina of neuromuscular junctions. Their association with PRiMA produces membrane-bound tetramers which constitute the predominant form of cholinesterases in the mammalian brain; in muscles, the level of PRiMA-anchored tetramers is regulated by exercise, but their functional significance remains unknown. In brain and muscles, the hydrolysis of acetylcholine by cholinesterases, in different contexts, and their possible noncatalytic functions clearly depend on their localization by ColQ or PRiMA.     

Rapid protein anchoring into the membranes of Mammalian cells using oleyl chain and poly(ethylene glycol) derivatives

The cell membrane is an important interface for communication with extracellular events, and incorporation of bioactive substances, such as antibodies and receptors, into the cell membrane may enhance the potential abilities of cells. Gene manipulation, chemical modification of membrane proteins, and cell surface painting using a GPI anchor have been performed to introduce substances into cell membranes. Furthermore, many lipid anchors have also been used to modify lipid membranes such as liposomes. In this study, we have focused on developing an easy and rapid method for anchoring of substances including macromolecular proteins into the membranes of living mammalian cells. We employed a single oleyl chain derivative coupled with hydrophilic poly(ethylene glycol) (PEG90, the ethyleneoxide (EO) unit is 90) to facilitate solubilization in water. This water-soluble derivative was designated Biocompatible Anchor for Membrane (BAM). Some proteins (streptavidin, EGFP and an antibody) were coupled with BAM90 at the distal terminal of PEG and rapidly (within a few minutes) anchored into the membranes of various cells (NIH3T3, 32D, Ba/F3, hybridoma 9E10). However, the anchored BAM90 disappeared from the cell membranes within 4-5 h in serum-free culture media, and moreover, the retention time of anchoring was shortened (1-2 h) in culture medium containing 10% FBS. We further prepared a dioleylphosphatidylethanolamine (DOPE)-PEG derivative (DOPE-BAM80, the EO unit is 80) as a double oleyl chain derivative for comparison with the single oleyl chain derivative, BAM90. The retention time of anchored DOPE-BAM80 was longer than that of BAM90 and more than 8 h in culture media with and without 10% serum. Furthermore, the treatment time of DOPE-BAM80 for anchoring was nearly as short (within a few minutes) as that of BAM90. In addition, both types of BAMs, BAM90 and DOPE-BAM80, showed no cytotoxicity. Therefore, DOPE-BAM80 is useful for protein anchoring to cells. Although the utilization of BAM90 is considered to be limited, it is expected to useful in restricted environments, for example, in tissues such as the cornea, peritoneum, bladder, and various mucosae, which are less exposed to serum. Thus, we suggest the possibility that both types of BAM can be applied to cell surface engineering.     

Conversion of human erythrocyte acetylcholinesterase from an amphiphilic to a hydrophilic form by phosphatidylinositol-specific phospholipase C and serum phospholipase D

Each catalytic subunit in the amphiphilic dimer of human erythrocyte acetylcholinesterase (AChE) is anchored in the plasma membrane exclusively by a glycoinositol phospholipid. In contrast to erythrocyte AChEs in other mammalian species, the human enzyme is resistant to direct cleavage by phosphatidylinositol-specific phospholipase C (PtdIns-specific PLC). The resistance is due to the existence of an additional fatty acyl chain on the inositol ring which blocks the action of PtdIns-specific PLC [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. In this report, nondenaturing polyacrylamide gel electrophoresis was applied to permit rapid and unambiguous distinction between amphiphilic AChE, in which each catalytic subunit binds one nonionic detergent micelle, and hydrophilic AChE, which does not interact with detergent. Deacylation of human erythrocyte AChE by an alkaline treatment with hydroxylamine rendered the amphiphilic AChE susceptible to PtdIns-specific PLC with the consequent release of hydrophilic AChE. Although serum anchor-specific phospholipase D (PLD) cleaves the intact human erythrocyte AChE anchor, this treatment, as judged by nondenaturing electrophoresis, did not release hydrophilic AChE. Hydroxylamine treatment before or after PLD digestion was necessary to achieve the conversion. These observations indicate that binding of a single detergent micelle was maintained when any of the three fatty acyl or alkyl groups in the human erythrocyte AChE anchor phospholipid were retained. For proteins that can be identified following nondenaturing gel electrophoresis, these procedures provide methods both for detecting glycoinositol phospholipid anchors resistant to PtdIns-specific PLC and for indicating fatty acyl and/or alkyl chains in these anchors.     

A novel membrane anchor for FtsZ is linked to cell wall hydrolysis in Caulobacter crescentus

In most bacteria, the tubulin‐like GTPase FtsZ forms an annulus at midcell (the Z‐ring) which recruits the division machinery and regulates cell wall remodeling. Although both activities require membrane attachment of FtsZ, few membrane anchors have been characterized. FtsA is considered to be the primary membrane tether for FtsZ in bacteria, however in Caulobacter crescentus, FtsA arrives at midcell after stable Z‐ring assembly and early FtsZ‐directed cell wall synthesis. We hypothesized that additional proteins tether FtsZ to the membrane and demonstrate that in C. crescentus, FzlC is one such membrane anchor. FzlC associates with membranes directly in vivo and in vitro and recruits FtsZ to membranes in vitro. As for most known membrane anchors, the C‐terminal peptide of FtsZ is required for its recruitment to membranes by FzlC in vitro and midcell recruitment of FzlC in cells. In vivo, overproduction of FzlC causes cytokinesis defects whereas deletion of fzlC causes synthetic defects with dipM, ftsE and amiC mutants, implicating FzlC in cell wall hydrolysis. Our characterization of FzlC as a novel membrane anchor for FtsZ expands our understanding of FtsZ regulators and establishes a role for membrane‐anchored FtsZ in the regulation of cell wall hydrolysis.     

Phosphatidylinositol is involved in the attachment of tailed asymmetric acetylcholinesterase to neuronal membranes

1. We analyzed the mode of attachment of 16 S tailed acetylcholinesterase (AChE; EC 3.1.1.7) to rat superior cervical ganglion (SCG) neuronal membranes. Using extractions by high-salt (HS) and nonionic detergent (Triton X-100), we found two pools of 16 S AChE. 2. The detergent-extracted (DE) 16 S AChE was tightly bound to membranes through detergent-sensitive, high-salt insensitive interactions and was distinct from high-salt-soluble 16 S AChE. The detergent-extracted (DE) 16 S AChE constituted a significant proportion of about one-third of the total 16 S AChE. 3. Treatment of the neuronal membranes by a phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the release of some, but not all DE 16 S AChE, indicating that a significant amount of the neuronal DE 16 S AChE, about one-third, is anchored to membranes through a phosphatidylinositol containing residue. Thus, a covalent association of a glycolipid and catalytic or structural AChE polypeptidic chains occurs not only for dimeric AChE but also for the asymmetric species of AChE. 4. The complex polymorphism of AChE is due not only to different globular or asymmetric associations of catalytic and structural subunits but also to the alternative existence of a transmembrane domain or a glycolipid membrane anchor.