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Acetylcholinesterase associates differently with its anchoring proteins ColQ and PRiMA.

ABSTRACT: 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.

SUBMITTER: Noureddine H

PROVIDER: S-EPMC3258952 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A tetrameric acetylcholinesterase from the parasitic nematode Dictyocaulus viviparus associates with the vertebrate tail proteins PRiMA and ColQ.

Project description:Dictyocaulus viviparus causes a serious lung disease of cattle. Similar to other parasitic nematodes, D. viviparus possesses several acetylcholinesterase (AChE) genes, one of which encodes a putative neuromuscular AChE, which contains a tryptophan (W) amphiphilic tetramerization (WAT) domain at its C-terminus. In the current study, we describe the biochemical characterization of a recombinant version of this WAT domain-containing AChE. To assess if the WAT domain is biologically functional, we investigated the association of the recombinant enzyme with the vertebrate tail proteins, proline-rich membrane anchor (PRiMA) and collagen Q (ColQ), as well as the synthetic polypeptide poly-l-proline. The results indicate that the recombinant enzyme hydrolyzes acetylthiocholine preferentially and exhibits inhibition by excess substrate, a characteristic of AChEs but not butyrylcholinesterases (BChEs). The enzyme is inhibited by the AChE inhibitor, BW284c51, but not by the BChE inhibitors, ethopropazine or iso-OMPA. The enzyme is able to assemble into monomeric (G(1)), dimeric (G(2)), and tetrameric (G(4)) globular forms and can also associate with PRiMA and ColQ, which contain proline-rich attachment domains (PRADs). This interaction is likely to be mediated via WAT-PRAD interactions, as the enzyme also assembles into tetramers with the synthetic polypeptide poly-l-proline. These interactions are typical of AChE(T) subunits. This is the first demonstration of an AChE(T) from a parasitic nematode that can assemble into heterologous forms with vertebrate proteins that anchor the enzyme in cholinergic synapses. We discuss the implications of our results for this particular host/parasite system and for the evolution of AChE.

| S-EPMC3222274 | biostudies-literature

COOH-terminal collagen Q (COLQ) mutants causing human deficiency of endplate acetylcholinesterase impair the interaction of ColQ with proteins of the basal lamina.

Project description:Collagen Q (ColQ) is a key multidomain functional protein of the neuromuscular junction (NMJ), crucial for anchoring acetylcholinesterase (AChE) to the basal lamina (BL) and accumulating AChE at the NMJ. The attachment of AChE to the BL is primarily accomplished by the binding of the ColQ collagen domain to the heparan sulfate proteoglycan perlecan and the COOH-terminus to the muscle-specific receptor tyrosine kinase (MuSK), which in turn plays a fundamental role in the development and maintenance of the NMJ. Yet, the precise mechanism by which ColQ anchors AChE at the NMJ remains unknown. We identified five novel mutations at the COOH-terminus of ColQ in seven patients from five families affected with endplate (EP) AChE deficiency. We found that the mutations do not affect the assembly of ColQ with AChE to form asymmetric forms of AChE or impair the interaction of ColQ with perlecan. By contrast, all mutations impair in varied degree the interaction of ColQ with MuSK as well as basement membrane extract (BME) that have no detectable MuSK. Our data confirm that the interaction of ColQ to perlecan and MuSK is crucial for anchoring AChE to the NMJ. In addition, the identified COOH-terminal mutants not only reduce the interaction of ColQ with MuSK, but also diminish the interaction of ColQ with BME. These findings suggest that the impaired attachment of COOH-terminal mutants causing EP AChE deficiency is in part independent of MuSK, and that the COOH-terminus of ColQ may interact with other proteins at the BL.

| S-EPMC4024244 | biostudies-literature

The association of tetrameric acetylcholinesterase with ColQ tail: a block normal mode analysis.

Project description:Acetylcholinesterase (AChE) rapidly hydrolyzes acetylcholine in the neuromuscular junctions and other cholinergic synapses to terminate the neuronal signal. In physiological conditions, AChE exists as tetramers associated with the proline-rich attachment domain (PRAD) of either collagen-like Q subunit (ColQ) or proline-rich membrane-anchoring protein. Crystallographic studies have revealed that different tetramer forms may be present, and it is not clear whether one or both are relevant under physiological conditions. Recently, the crystal structure of the tryptophan amphiphilic tetramerization (WAT) domain of AChE associated with PRAD ([WAT]4PRAD), which mimics the interface between ColQ and AChE tetramer, became available. In this study we built a complete tetrameric mouse [AChE(T)]4-ColQ atomic structure model, based on the crystal structure of the [WAT]4PRAD complex. The structure was optimized using energy minimization. Block normal mode analysis was done to investigate the low-frequency motions of the complex and to correlate the structure model with the two known crystal structures of AChE tetramer. Significant low-frequency motions among the catalytic domains of the four AChE subunits were observed, while the [WAT]4PRAD part held the complex together. Normal mode involvement analysis revealed that the two lowest frequency modes were primarily involved in the conformational changes leading to the two crystal structures. The first 30 normal modes can account for more than 75% of the conformational changes in both cases. The evidence further supports the idea of a flexible tetramer model for AChE. This model can be used to study the implications of the association of AChE with ColQ.

| S-EPMC1285061 | biostudies-literature

Protein-anchoring strategy for delivering acetylcholinesterase to the neuromuscular junction.

Project description:Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ). Congenital defects of ColQ cause endplate AChE deficiency and myasthenic syndrome. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq(-/-) mice recovered motor functions, synaptic transmission, as well as the morphology of the NMJ. ColQ-tailed AChE was specifically anchored to NMJ and its amount was restored to 89% of the wild type. We next characterized the molecular basis of this efficient recovery. We first confirmed that ColQ-tailed AChE can be specifically targeted to NMJ by an in vitro overlay assay in Colq(-/-) mice muscle sections. We then injected AAV1-COLQ-IRES-EGFP into the left tibialis anterior and detected AChE in noninjected limbs. Furthermore, the in vivo injection of recombinant ColQ-tailed AChE protein complex into the gluteus maximus muscle of Colq(-/-) mice led to accumulation of AChE in noninjected forelimbs. We demonstrated for the first time in vivo that the ColQ protein contains a tissue-targeting signal that is sufficient for anchoring itself to the NMJ. We propose that the protein-anchoring strategy is potentially applicable to a broad spectrum of diseases affecting extracellular matrix molecules.

| S-EPMC3392993 | biostudies-literature

Human endplate acetylcholinesterase deficiency caused by mutations in the collagen-like tail subunit (ColQ) of the asymmetric enzyme.

Project description:In skeletal muscle, acetylcholinesterase (AChE) exists in homomeric globular forms of type T catalytic subunits (ACHET) and heteromeric asymmetric forms composed of 1, 2, or 3 tetrameric ACHET attached to a collagenic tail (ColQ). Asymmetric AChE is concentrated at the endplate (EP), where its collagenic tail anchors it into the basal lamina. The ACHET gene has been cloned in humans; COLQ cDNA has been cloned in Torpedo and rodents but not in humans. In a disabling congenital myasthenic syndrome, EP AChE deficiency (EAD), the normal asymmetric species of AChE are absent from muscle. EAD could stem from a defect that prevents binding of ColQ to ACHET or the insertion of ColQ into the basal lamina. In six EAD patients, we found no mutations in ACHET. We therefore cloned human COLQ cDNA, determined the genomic structure and chromosomal localization of COLQ, and then searched for mutations in this gene. We identified six recessive truncation mutations of COLQ in six patients. Coexpression of each COLQ mutant with wild-type ACHET in SV40-transformed monkey kidney fibroblast (COS) cells reveals that a mutation proximal to the ColQ attachment domain for ACHET prevents association of ColQ with ACHET; mutations distal to the attachment domain generate a mutant approximately 10.5S species of AChE composed of one ACHET tetramer and a truncated ColQ strand. The approximately 10.5S species lack part of the collagen domain and the entire C-terminal domain of ColQ, or they lack only the C-terminal domain, which is required for formation of the triple collagen helix, and this likely prevents their insertion into the basal lamina.

| S-EPMC21394 | biostudies-literature

Mutation in the human acetylcholinesterase-associated collagen gene, COLQ, is responsible for congenital myasthenic syndrome with end-plate acetylcholinesterase deficiency (Type Ic).

Project description:Congenital myasthenic syndrome (CMS) with end-plate acetylcholinesterase (AChE) deficiency is a rare autosomal recessive disease, recently classified as CMS type Ic (CMS-Ic). It is characterized by onset in childhood, generalized weakness increased by exertion, refractoriness to anticholinesterase drugs, and morphological abnormalities of the neuromuscular junctions (NMJs). The collagen-tailed form of AChE, which is normally concentrated at NMJs, is composed of catalytic tetramers associated with a specific collagen, COLQ. In CMS-Ic patients, these collagen-tailed forms are often absent. We studied a large family comprising 11 siblings, 6 of whom are affected by a mild form of CMS-Ic. The muscles of the patients contained collagen-tailed AChE. We first excluded the ACHE gene (7q22) as potential culprit, by linkage analysis; then we mapped COLQ to chromosome 3p24.2. By analyzing 3p24.2 markers located close to the gene, we found that the six affected patients were homozygous for an interval of 14 cM between D3S1597 and D3S2338. We determined the COLQ coding sequence and found that the patients present a homozygous missense mutation, Y431S, in the conserved C-terminal domain of COLQ. This mutation is thought to disturb the attachment of collagen-tailed AChE to the NMJ, thus constituting the first genetic defect causing CMS-Ic.

| S-EPMC1377491 | biostudies-other

Targeting acetylcholinesterase to membrane rafts: a function mediated by the proline-rich membrane anchor (PRiMA) in neurons.

Project description:In the mammalian brain, acetylcholinesterase (AChE) is anchored in cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor). We present evidence that at least part of the PRiMA-linked AChE is integrated in membrane microdomains called rafts. A significant proportion of PRiMA-linked AChE tetramers from rat brain was recovered in raft fractions; this proportion was markedly higher at low rather than at high concentrations of cold Triton X-100. The detergent-resistant fraction increased during brain development. In NG108-15 neuroblastoma cells transfected with cDNAs encoding AChE(T) and PRiMA, PRiMA-linked G(4) AChE was found in membrane rafts and showed the same sensitivity to cold Triton X-100 extraction as in the brain. The association of PRiMA-linked AChE with rafts was weaker than that of glycosylphosphatidylinositol-anchored G(2) AChE or G(4) Q(N)-H(C)-linked AChE. It was found to depend on the presence of a cholesterol-binding motif, called CRAC (cholesterol recognition/interaction amino acid consensus), located at the junction of transmembrane and cytoplasmic domains of both PRiMA I and II isoforms. The cytoplasmic domain of PRiMA, which differs between PRiMA I and PRiMA II, appeared to play some role in stabilizing the raft localization of G(4) AChE, because the Triton X-100-resistant fraction was smaller with the shorter PRiMA II isoform than that with the longer PRiMA I isoform.

| S-EPMC2857032 | biostudies-literature

Leisure-Time and Occupational Physical Activity Associates Differently with Epigenetic Aging.

Project description:PurposeGreater leisure-time physical activity (LTPA) associates with healthier lives, but knowledge regarding occupational physical activity (OPA) is more inconsistent. DNA methylation (DNAm) patterns capture age-related changes in different tissues. We aimed to assess how LTPA and OPA are associated with three DNAm-based epigenetic age estimates, namely, DNAm age, PhenoAge, and GrimAge.MethodsThe participants were young adult (21-25 yr, n = 285) and older (55-74 yr, n = 235) twin pairs, including 16 pairs with documented long-term LTPA discordance. Genome-wide DNAm from blood samples was used to compute DNAm age, PhenoAge, and GrimAge Age acceleration (Acc), which describes the difference between chronological and epigenetic ages. Physical activity was assessed with sport, leisure-time, and work indices based on the Baecke Questionnaire. Genetic and environmental variance components of epigenetic age Acc were estimated by quantitative genetic modeling.ResultsEpigenetic age Acc was highly heritable in young adult and older twin pairs (~60%). Sport index was associated with slower and OPA with faster DNAm GrimAge Acc after adjusting the model for sex. Genetic factors and nonshared environmental factors in common with sport index explained 1.5%-2.7% and 1.9%-3.5%, respectively, of the variation in GrimAge Acc. The corresponding proportions considering OPA were 0.4%-1.8% and 0.7%-1.8%, respectively. However, these proportions were minor (<0.5%) after adjusting the model for smoking status.ConclusionsLTPA associates with slower and OPA with faster epigenetic aging. However, adjusting the models for smoking status, which may reflect the accumulation of unhealthy lifestyle habits, attenuated the associations.

| S-EPMC7886335 | biostudies-literature

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

Project description: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.

| S-EPMC3190869 | biostudies-literature

Single nucleotide polymorphisms alter kinase anchoring and the subcellular targeting of A-kinase anchoring proteins.

Project description:A-kinase anchoring proteins (AKAPs) shape second-messenger signaling responses by constraining protein kinase A (PKA) at precise intracellular locations. A defining feature of AKAPs is a helical region that binds to regulatory subunits (RII) of PKA. Mining patient-derived databases has identified 42 nonsynonymous SNPs in the PKA-anchoring helices of five AKAPs. Solid-phase RII binding assays confirmed that 21 of these amino acid substitutions disrupt PKA anchoring. The most deleterious side-chain modifications are situated toward C-termini of AKAP helices. More extensive analysis was conducted on a valine-to-methionine variant in the PKA-anchoring helix of AKAP18. Molecular modeling indicates that additional density provided by methionine at position 282 in the AKAP18? isoform deflects the pitch of the helical anchoring surface outward by 6.6°. Fluorescence polarization measurements show that this subtle topological change reduces RII-binding affinity 8.8-fold and impairs cAMP responsive potentiation of L-type Ca2+ currents in situ. Live-cell imaging of AKAP18? V282M-GFP adducts led to the unexpected discovery that loss of PKA anchoring promotes nuclear accumulation of this polymorphic variant. Targeting proceeds via a mechanism whereby association with the PKA holoenzyme masks a polybasic nuclear localization signal on the anchoring protein. This led to the discovery of AKAP18?: an exclusively nuclear isoform that lacks a PKA-anchoring helix. Enzyme-mediated proximity-proteomics reveal that compartment-selective variants of AKAP18 associate with distinct binding partners. Thus, naturally occurring PKA-anchoring-defective AKAP variants not only perturb dissemination of local second-messenger responses, but also may influence the intracellular distribution of certain AKAP18 isoforms.

| S-EPMC6298107 | biostudies-literature

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