RESUMO
The neuromuscular junction (NMJ) is a tripartite synapse in which not only presynaptic and post-synaptic cells participate in synaptic transmission, but also terminal Schwann cells (TSC). Acetylcholine (ACh) is the neurotransmitter that mediates the signal between the motor neuron and the muscle but also between the motor neuron and TSC. ACh action is terminated by acetylcholinesterase (AChE), anchored by collagen Q (ColQ) in the basal lamina of NMJs. AChE is also anchored by a proline-rich membrane anchor (PRiMA) to the surface of the nerve terminal. Butyrylcholinesterase (BChE), a second cholinesterase, is abundant on TSC and anchored by PRiMA to its plasma membrane. Genetic studies in mice have revealed different regulations of synaptic transmission that depend on ACh spillover. One of the strongest is a depression of ACh release that depends on the activation of α7 nicotinic acetylcholine receptors (nAChR). Partial AChE deficiency has been described in many pathologies or during treatment with cholinesterase inhibitors. In addition to changing the activation of muscle nAChR, AChE deficiency results in an ACh spillover that changes TSC signaling. In this mini-review, we will first briefly outline the organization of the NMJ. This will be followed by a look at the role of TSC in synaptic transmission. Finally, we will review the pathological conditions where there is evidence of decreased AChE activity.
RESUMO
Humans with the C5 genetic variant of butyrylcholinesterase (BChE) have 30-200% higher plasma BChE activity, low body weight, and shorter duration of action of the muscle relaxant succinylcholine. The C5 variant has an extra, slow-moving band of BChE activity on native polyacrylamide gel electrophoresis. This band is about 60 kDa larger than wild-type BChE. Umbilical cord BChE in 100% of newborn babies has a C5-like band. Our goal was to identify the unknown, 60 kDa protein in C5. Both wild-type and C5 BChE are under the genetic control of two independent loci, the BCHE gene on Chr 3q26.1 and the RAPH1 (lamellipodin) gene on Chr 2q33. Wild-type BChE tetramers are assembled around a 3 kDa polyproline peptide from lamellipodin. Western blot of boiled C5 and cord BChE showed a positive response with an antibody to the C-terminus of lamellipodin. The C-terminal exon of lamellipodin is about 60 kDa including an N-terminal polyproline. We propose that the unknown protein in C5 and cord BChE is encoded by the last exon of the RAPH1 gene. In 90% of the population, the 60 kDa fragment is shortened to 3 kDa during maturation to adulthood, leaving only 10% of adults with C5 BChE.
Assuntos
Butirilcolinesterase/química , Butirilcolinesterase/genética , Proteínas de Transporte/química , Variação Genética , Proteínas de Membrana/química , Multimerização Proteica , Sequência de Aminoácidos , Butirilcolinesterase/sangue , Sangue Fetal , Técnicas de Genotipagem , Humanos , Modelos Moleculares , Peso Molecular , Eletroforese em Gel de Poliacrilamida Nativa , Conformação ProteicaRESUMO
Inhibition of human AChE (acetylcholinesterase) and BChE (butyrylcholinesterase) by an alkylammonium derivative of 6-methyluracil, C-547, a potential drug for the treatment of MG (myasthenia gravis) was studied. Kinetic analysis of AChE inhibition showed that C-547 is a slow-binding inhibitor of type B, i.e. after formation of the initial enzyme·inhibitor complex (Ki=140 pM), an induced-fit step allows establishment of the final complex (Ki*=22 pM). The estimated koff is low, 0.05 min(-1) On the other hand, reversible inhibition of human BChE is a fast-binding process of mixed-type (Ki=1.77 µM; Ki'=3.17 µM). The crystal structure of mouse AChE complexed with C-547 was solved at 3.13 Å resolution. The complex is stabilized by cation-π, stacking and hydrogen-bonding interactions. Molecular dynamics simulations of the binding/dissociation processes of C-547 and C-35 (a non-charged analogue) to mouse and human AChEs were performed. Molecular modelling on mouse and human AChE showed that the slow step results from an enzyme conformational change that allows C-547 to cross the bottleneck in the active-site gorge, followed by formation of tight complex, as observed in the crystal structure. In contrast, the related non-charged compound C-35 is not a slow-binding inhibitor. It does not cross the bottleneck because it is not sensitive to the electrostatic driving force to reach the bottom of the gorge. Thus C-547 is one of the most potent and selective reversible inhibitors of AChE with a long residence time, τ=20 min, longer than for other reversible inhibitors used in the treatment of MG. This makes C-547 a promising drug for the treatment of this disease.
Assuntos
Acetilcolinesterase/química , Inibidores da Colinesterase/química , Simulação de Acoplamento Molecular , Miastenia Gravis , Compostos de Amônio Quaternário/química , Uracila/análogos & derivados , Animais , Células CHO , Inibidores da Colinesterase/uso terapêutico , Cricetinae , Cricetulus , Humanos , Camundongos , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/enzimologia , Compostos de Amônio Quaternário/uso terapêutico , Uracila/química , Uracila/uso terapêuticoRESUMO
Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at -80 °C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10(-9) M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal.
Assuntos
Anticorpos Monoclonais/metabolismo , Butirilcolinesterase/isolamento & purificação , Butirilcolinesterase/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Imobilizados/química , Anticorpos Imobilizados/metabolismo , Anticorpos Monoclonais/química , Sítios de Ligação de Anticorpos , Western Blotting , Butirilcolinesterase/sangue , Butirilcolinesterase/química , Proteínas de Transporte , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas Imunológicas , CamundongosRESUMO
Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ) in the form of asymmetric AChE (AChE/ColQ). The C-terminal domain of ColQ binds to MuSK, the muscle-specific receptor tyrosine kinase, that mediates a signal for acetylcholine receptor (AChR) clustering at the NMJ. ColQ also binds to heparan sulfate proteoglycans including perlecan. Congenital defects of ColQ cause endplate AChE deficiency. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq-/- mice rescued motor functions, synaptic transmission, and the ultrastructure of NMJ. We also injected AAV1-COLQ-IRES-EGFP to the left tibialis anterior and observed colocalization of AChE/ColQ at all the examined NMJs of the non-injected limbs. Additionally, injection of purified recombinant AChE/ColQ protein complex into gluteus maximus accumulated AChE in non-injected forelimbs. These observations suggest that the tissue-targeting signal of ColQ can be exploited to specifically deliver the transgene product to the target tissue. MuSK antibody-positive myasthenia gravis (MG) accounts for 5-15% of autoimmune MG. As AChR deficiency is typically mild and as cholinesterase inhibitors are generally ineffective or worsen myasthenic symptoms, we asked if the patient's MuSK-IgG interferes with binding of ColQ to MuSK. In vitro overlay of AChE/ColQ to muscle sections of Colq-/- mice revealed that MuSK-IgG blocks binding of ColQ to the NMJ. In vitro plate-binding of MuSK to ColQ disclosed that MuSK-IgG exerts a dose-dependent block of MuSK-ColQ interaction. In addition, passive transfer of MuSK-IgG to mice reduced the size and density of ColQ to â¼10% of controls and had a lesser effect on the sizes and densities of AChR and MuSK. Elucidation of molecular mechanisms of specific binding of ColQ to the NMJ enabled us to ameliorate devastating myasthenic symptoms of Colq-/- mice and to reveal bases of anti-MuSK MG.
Assuntos
Acetilcolinesterase/metabolismo , Colágeno/metabolismo , Proteínas Musculares/metabolismo , Síndromes Miastênicas Congênitas/terapia , Junção Neuromuscular/metabolismo , Acetilcolinesterase/administração & dosagem , Acetilcolinesterase/deficiência , Acetilcolinesterase/genética , Animais , Colágeno/administração & dosagem , Colágeno/deficiência , Colágeno/genética , Dependovirus/genética , Feminino , Proteínas Ligadas por GPI/metabolismo , Terapia Genética , Humanos , Imunoglobulina G/administração & dosagem , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/administração & dosagem , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Miastenia Gravis Autoimune Experimental/genética , Miastenia Gravis Autoimune Experimental/metabolismo , Miastenia Gravis Autoimune Experimental/terapia , Síndromes Miastênicas Congênitas/genética , Síndromes Miastênicas Congênitas/metabolismo , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transmissão SinápticaRESUMO
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.
Assuntos
Acetilcolinesterase/metabolismo , Colágeno/genética , Colágeno/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Doenças da Junção Neuromuscular/terapia , Junção Neuromuscular/metabolismo , Acetilcolinesterase/genética , Animais , Dependovirus/genética , Terapia Genética , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Junção Neuromuscular/genética , Doenças da Junção Neuromuscular/genética , Doenças da Junção Neuromuscular/fisiopatologia , Transmissão SinápticaRESUMO
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.
Assuntos
Acetilcolinesterase/metabolismo , Colágeno/metabolismo , Dictyocaulus/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/metabolismo , Multimerização Proteica , Acetilcolinesterase/química , Acetilcolinesterase/genética , Acetilcolinesterase/isolamento & purificação , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Schwartz-Jampel syndrome (SJS) is a recessive neuromyotonia with chondrodysplasia. It results from hypomorphic mutations of the gene encoding perlecan, leading to a decrease in the levels of this heparan sulphate proteoglycan in basement membranes (BMs). It has been suggested that SJS neuromyotonia may result from endplate acetylcholinesterase (AChE) deficiency, but this hypothesis has never been investigated in vivo due to the lack of an animal model for neuromyotonia. We used homologous recombination to generate a knock-in mouse strain with one missense substitution, corresponding to a human familial SJS mutation (p.C1532Y), in the perlecan gene. We derived two lines, one with the p.C1532Y substitution alone and one with p.C1532Y and the selectable marker Neo, to down-regulate perlecan gene activity and to test for a dosage effect of perlecan in mammals. These two lines mimicked SJS neuromyotonia with spontaneous activity on electromyogramm (EMG). An inverse correlation between disease severity and perlecan secretion in the BMs was observed at the macroscopic and microscopic levels, consistent with a dosage effect. Endplate AChE levels were low in both lines, due to synaptic perlecan deficiency rather than major myofibre or neuromuscular junction disorganization. Studies of muscle contractile properties showed muscle fatigability at low frequencies of nerve stimulation and suggested that partial endplate AChE deficiency might contribute to SJS muscle stiffness by potentiating muscle force. However, physiological endplate AChE deficiency was not associated with spontaneous activity at rest on EMG in the diaphragm, suggesting that additional changes are required to generate such activity characteristic of SJS.
Assuntos
Acetilcolinesterase/deficiência , Acetilcolinesterase/genética , Síndrome de Isaacs/enzimologia , Síndrome de Isaacs/genética , Placa Motora/enzimologia , Osteocondrodisplasias/enzimologia , Osteocondrodisplasias/genética , Alelos , Animais , Modelos Animais de Doenças , Eletromiografia , Feminino , Dosagem de Genes , Proteoglicanas de Heparan Sulfato/deficiência , Proteoglicanas de Heparan Sulfato/genética , Humanos , Síndrome de Isaacs/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Placa Motora/fisiopatologia , Contração Muscular/genética , Contração Muscular/fisiologia , Mutação de Sentido Incorreto , Osteocondrodisplasias/fisiopatologia , FenótipoRESUMO
Congenital myasthenic syndromes are caused by mutations in molecules expressed at the neuromuscular junction. Collagen Q (ColQ) makes a triple helical structure and anchors the catalytic subunit of acetylcholinesterase (AChE) to the synaptic basal lamina in the form of asymmetric AChE. Mutations in the collagen Q gene (COLQ) cause endplate AChE deficiency. As an initial step to develop a novel therapeutic strategy for endplate acetylcholinesterase deficiency, we expressed AChE species in cultured cells using retrovirus and adeno-associated virus (AAV). The retroviral vectors carried human ACHE and COLQ either in a single construct (EF1alpha-ACHE-IRES-COLQ) or in two separate constructs (EF1alpha-ACHE and EF1alpha-COLQ). We produced high-titer retroviruses using the PLAT-E retrovirus packaging cells. We also confirmed expression of asymmetric AChE in the PLAT-E cells. We infected NIH3T3 and confirmed expression of the transgenes by RT-PCR. The AAV vector carried human COLQ-IRES-EGFP downstream of the CMV promoter (pAAV-CMV-COLQ-IRES-EGFP). We produced recombinant AAV using HEK293 cells carrying pDF6 encoding the AAV6 capsid gene. We infected AAVHT1080 cells and confirmed expression of COLQ by RT-PCR and EGFP by flow cytometry. We are currently trying to achieve further higher expression levels of transgenes in cultured cells to make the current strategy applicable to an animal model.
Assuntos
Acetilcolinesterase/genética , Colágeno/genética , Dependovirus/genética , Vetores Genéticos , Proteínas Musculares/genética , Retroviridae/genética , Animais , Humanos , Camundongos , Células NIH 3T3RESUMO
To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE. Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChE(T) subunit, which should be able to produce all three globular forms of AChE: monomers (G(1)), dimers (G(2)), and tetramers (G(4)), and assemble into asymmetric forms in association with the collagenic subunit collagen Q. Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium. In cell extracts, amphiphilic monomers (G(1)(a)) and non-amphiphilic tetramers (G(4)(na)) are found. Amphiphilic dimers (G(2)(a)) and non-amphiphilic tetramers (G(4)(na)) are secreted into the medium. Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A(12) form of the enzyme. Collagenase digestion of the A(12) AChE produces a lytic G(4) form. Notably, only globular forms are present in vivo. This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms. We also performed a phylogenetic analysis of the sequence. We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates.
Assuntos
Acetilcolinesterase/metabolismo , Ciona intestinalis/enzimologia , Colágeno/metabolismo , Acetilcolinesterase/química , Acetilcolinesterase/genética , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Colágeno/química , Colágeno/genética , Colagenases/química , DNA Complementar/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Filogenia , Conformação Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
As a tetramer, acetylcholinesterase (AChE) is anchored to the basal lamina of the neuromuscular junction and to the membrane of neuronal synapses. We have previously shown that collagen Q (ColQ) anchors AChE at the neuromuscular junction. We have now cloned the gene PRiMA (proline-rich membrane anchor) encoding the AChE anchor in mammalian brain. We show that PRiMA is able to organize AChE into tetramers and to anchor them at the surface of transfected cells. Furthermore, we demonstrate that AChE is actually anchored in neural cell membranes through its interaction with PRiMA. Finally, we propose that only PRiMA anchors AChE in mammalian brain and muscle cell membranes.