No abstract

Torpedo acetyicholinesterase (AcChoEase, EC 3.1.1.7) and human butyrylcholnesterase (BtChoEase, EC 3.1.1.8), while clearly differing in substrate specificity and sensitivity to inhibitors, possess 53% sequence homology; this permitted modeling human BtChoEase on the basis of the three-dimensional structure of Torpedo AcChoEase. The modeled BtChoEase structure closely resembled that of AcChoEase in overall features. However, six conserved aromatic residues that line the active-site gorge, which is a prominent feature ofthe AcChoEase structure, are absent in BtChoEase. Modeling showed that two such residues, Phe-288 and Phe-290, replaced by leucine and valine, respectively, in BtChoEase, may prevent entrance of butyrylcholine into the acyl-binding pocket. Their mutation to leucine and valine in AcChoEase, by site-directed mutagenesis, produced a double mutant that hydrolyzed butyrylthiocholine almost as well as acetylthiocholine. The mutated enzyme was also inhibited well by the bulky, BtChoEaseselective organophosphate inhibitor (tetraisopropylpyrophosphoramide, iso-OMPA). Trp-279, at the entrance of the activesite gorge in AcChoEase, is absent in BtChoEase. Modeling designated it as part of the "peripheral" anionic site, which is lacking in BtChoEase. The mutant W279A displayed strongly reduced inhibition by the peripheral site-specific ligand propidium relative to wild-type Torpedo AcChoEase, whereas inhibition by the catalytic-site inhibitor edrophonium was unaffected.

Acetylcholinesterase (AChE) occurs in both asymmetric forms, covalently associated with a collagenous subunit called Q (ColQ), and globular forms that may be either soluble or membrane associated. At the skeletal neuromuscular junction, asymmetric AChE is anchored to the basal lamina of the synaptic cleft, where it hydrolyzes acetylcholine to terminate synaptic transmission. AChE has also been hypothesized to play developmental roles in the nervous system, and ColQ is also expressed in some AChE-poor tissues. To seek roles of ColQ and AChE at synapses and elsewhere, we generated ColQ-deficient mutant mice. ColQ −/− mice completely lacked asymmetric AChE in skeletal and cardiac muscles and brain; they also lacked asymmetric forms of the AChE homologue, butyrylcholinesterase. Thus, products of the ColQ gene are required for assembly of all detectable asymmetric AChE and butyrylcholinesterase. Surprisingly, globular AChE tetramers were also absent from neonatal ColQ −/− muscles, suggesting a role for the ColQ gene in assembly or stabilization of AChE forms that do not themselves contain a collagenous subunit. Histochemical, immunohistochemical, toxicological, and electrophysiological assays all indicated absence of AChE at ColQ −/− neuromuscular junctions. Nonetheless, neuromuscular function was initially robust, demonstrating that AChE and ColQ do not play obligatory roles in early phases of synaptogenesis. Moreover, because acute inhibition of synaptic AChE is fatal to normal animals, there must be compensatory mechanisms in the mutant that allow the synapse to function in the chronic absence of AChE. One structural mechanism appears to be a partial ensheathment of nerve terminals by Schwann cells. Compensation was incomplete, however, as animals lacking ColQ and synaptic AChE failed to thrive and most died before they reached maturity.

The collagen-tailed or asymmetric forms (A) represent a major component of acetylcholinesterase (AChE) in the neuromuscular junction of higher vertebrates. They are hetero-oligomeric molecules, in which tetramers of catalytic subunits of type T (AChE T ) are attached to the subunits of a triple-stranded collagen "tail." We report the cloning of a rat AChE-associated collagen subunit, Q. We show that collagen tails are encoded by a single gene, COLQ. The ColQ subunits form homotrimers and readily form collagen-tailed AChE, when coexpressed with rat AChE T . We found that the same ColQ subunits are incorporated, in vivo, in asymmetric forms of both AChE and butyrylcholinesterase. A splice variant from the COLQ gene encodes a proline-rich AChE attachment domain without the collagen domain but does not represent the membrane anchor of the brain tetramer. The COLQ gene is expressed in cholinergic tissues, brain, muscle, and heart, and also in noncholinergic tissues such as lung and testis.Acetylcholinesterase (AChE, EC 3.1.1.7)1 is highly concentrated at vertebrate neuromuscular junctions. This enzyme is encoded by a single gene, and adult mammalian muscles express a single splice variant, corresponding to the catalytic subunit of type T (AChE T ) (1, 2). At the post-translational level, however, quaternary interactions introduce a considerable diversity of molecular forms that are characterized by distinct localizations in cellular structures. These molecules include amphiphilic monomers (G 1 a ) and dimers (G 2 a ), nonamphiphilic tetramers (G 4 na ), as well as hetero-oligomeric structures in which tetramers of catalytic subunits are disulfide-linked with a hydrophobic "tail" (20 kDa) in the membrane-bound G 4 a forms (3, 4) or with a collagenous "tail" in the collagen-tailed or asymmetric (A) forms. The latter molecules consist of one, two, or three tetramers (A 4 , A 8 , A 12 ), which are disulfide-linked to the strands of the triple helical collagen tail (see Fig. 1A). G 1 a and G 2 a forms appear to remain mostly intracellular and represent precursors of more complex molecules. The G 4 na form is secreted and hydrophobic-tailed tetramers (G 4 a ) are attached to the plasma membrane. The collagen-tailed molecules are tethered in the basal lamina, and are largely responsible for the high concentration of AChE at the neuromuscular junction.To understand the biosynthesis of the various AChE forms and its regulation, it is necessary to analyze the association of AChE T catalytic subunits with anchoring subunits, particularly the collagen subunits, which have been named Q, according to the nomenclature of AChE-associated proteins (5). Cloning and expression of the collagen tail subunit of the asymmetric AChE forms from Torpedo electric organ (tQ 1 ) allowed us to show that the structural and catalytic subunits assemble into collagen-tailed molecules when coexpressed in COS cells (6). The primary sequence of the Q subunit comprises an N-terminal region, Q N , a collagen domain, and a C-terminal domain, Q C . We showe...

At the neuromuscular junction, acetylcholinesterase (AChE) is mainly present as asymmetric forms in which tetramers of catalytic subunits are associated to a specific collagen, collagen Q (ColQ). The accumulation of the enzyme in the synaptic basal lamina strictly relies on ColQ. This has been shown to be mediated by interaction between ColQ and perlecan, which itself binds dystroglycan. Here, using transfected mutants of ColQ in a ColQ-deficient muscle cell line or COS-7 cells, we report that ColQ clusterizes through a more complex mechanism. This process requires two heparin-binding sites contained in the collagen domain as well as the COOH terminus of ColQ. Cross-linking and immunoprecipitation experiments in Torpedo postsynaptic membranes together with transfection experiments with muscle-specific kinase (MuSK) constructs in MuSK-deficient myotubes or COS-7 cells provide the first evidence that ColQ binds MuSK. Together, our data suggest that a ternary complex containing ColQ, perlecan, and MuSK is required for AChE clustering and support the notion that MuSK dictates AChE synaptic localization at the neuromuscular junction.

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.

The asymmetric forms of cholinesterases are synthesized only in differentiated muscular and neural cells of vertebrates. These complex oligomers are characterized by the presence of a collagen-like tail, associated with one, two or three tetramers of catalytic subunits. The collagenic tail is responsible for ionic interactions, explaining the insertion of these molecules in extraceliular basal lamina, e.g. at neuromuscular endplates. We report the cloning of a collagenic subunit from Torpedo marmorata acetylcholinesterase (AChE). The predicted primary structure contains a putative signal peptide, a proline-rich domain, a collagenic domain, and a C-terminal domain composed of proline-rich and cysteine-rich regions. Several variants are generated by alternative splicing. Apart from the collagenic domain, the AChE tail subunit does not present any homology with previously known proteins. We show that coexpression of catalytic AChE subunits and collagenic subunits results in the production of asymmetric, collagen-tailed AChE forms in transfected COS cells. Thus, the assembly of these complex forms does not depend on a specific cellular processing, but rather on the expression of the collagenic subunits.

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.