Suzanne Bon

Molecular and cellular biology of cholinesterases

Abbreviations

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

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.

Quaternary Associations of Acetylcholinesterase II. THE POLYPROLINE ATTACHMENT DOMAIN OF THE COLLAGEN TAIL

In transfected COS cells, we analyzed the formation of heteromeric associations between rat acetylcholinesterase of type T (AChET) and various constructions derived from the NH2-terminal region of the collagen tail of asymmetric forms, QN. Using a series of deletions and point mutations in QN, we showed that the binding of AChET to QN does not require the cysteines that normally establish intersubunit disulfide bonds with catalytic subunits and that it essentially relies on the presence of stretches of successive prolines, although adjacent residues also contribute to the interaction. We thus defined a roline-ich ttachment omain or PRAD, which recruits AChET subunits to form heteromeric associations. Such molecules, consisting of one PRAD associated with a tetramer of AChET, are exported efficiently by the cells. Using the proportion of AChET subunits engaged in heteromeric tetramers, we ranked the interaction efficiency of various constructions. From these experiments we evaluated the contribution of different elements of the PRAD to the quaternary assembly of AChET subunits in the secretory pathway. The PRAD remained functional when reduced to six residues followed by a string of 10 prolines (Glu-Ser-Thr-Gly3-Pro10). We then showed that synthetic polyproline itself can associate with AChET subunits, producing well defined tetramers, when added to live transfected cells or even to cell extracts. This is the first example of an in vitro assembly of AChE tetramers from monomers and dimers. These results open the way to a chemical-physical exploration of the formation of these quaternary associations, both in the secretory pathway and in vitro.

Chapter 15: Structure and functions of acetylcholinesterase and butyrylcholinesterase

Publisher Summary Vertebrates possess two cholinesterases, acetylcholinesterase and butyrylcholinesterase. Cholinesterases catalyze a very simple reaction, hydrolysis of the ester bond of acetylcholine. The role of AChE in cholinergic transmission, although admittedly secondary to that of the pre-synaptic machinery responsible for the synthesis and release of acetylcholine, and of the postsynaptic receptors, is crucial for synaptic function. The role of ChE is not clear; this enzyme is, in fact, dispensable, since its absence in humans does not correlate with any physiological abnormality. Despite the fact that they do not, at first sight, appear as glamorous as receptors, cholinesterases display a number of fascinating features, and pose important questions concerning their structure and functions. AChE is one of the fastest enzymes known and possesses an unusual molecular structure. Both AChE and BChE display a repertoire of molecular forms, which differ in their quaternary structure and may be anchored in different ways to synaptic structures. Cholinesterases are thought to exert non-cholinergic functions, e.g. in morphogenesis, during early embryonic development, in the modulation of neuronal activity and in the elimination of various toxic compounds, which may explain their presence outside the context of cholinergic transmission. They are also expressed abnormally in some tumors and in other pathological states. This chapter discusses the following aspects: the atomic structure of cholinesterases and their catalytic mechanism; the structure and biosynthesis of their molecular forms; the possibility that these enzymes participate in cellular interactions in addition to their catalytic activity.

Primary structure of a collagenic tail peptide of Torpedo acetylcholinesterase: co-expression with catalytic subunit induces the production of collagen-tailed forms in transfected cells.

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 extracellular 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 co‐expression 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.

Complex alternative splicing of acetylcholinesterase transcripts in Torpedo electric organ; primary structure of the precursor of the glycolipid-anchored dimeric form

In this paper, we show the existence of alternative splicing in the 3′ region of the coding sequence of Torpedo acetylcholinesterase (AChE). We describe two cDNA structures which both diverge from the previously described coding sequence of the catalytic subunit of asymmetric (A) forms (Schumacher et al., 1986; Sikorav et al., 1987). They both contain a coding sequence followed by a non‐coding sequence and a poly(A) stretch. Both of these structures were shown to exist in poly(A)+ RNAs, by S1 mapping experiments. The divergent region encoded by the first sequence corresponds to the precursor of the globular dimeric form (G2a), since it contains the expected C‐terminal amino acids, Ala‐Cys. These amino acids are followed by a 29 amino acid extension which contains a hydrophobic segment and must be replaced by a glycolipid in the mature protein. Analyses of intact G2a AChE showed that the common domain of the protein contains intersubunit disulphide bonds. The divergent region of the second type of cDNA consists of an adjacent genomic sequence, which is removed as an intron in A and Ga mRNAs, but may encode a distinct, less abundant catalytic subunit. The structures of the cDNA clones indicate that they are derived from minor mRNAs, shorter than the three major transcripts which have been described previously (14.5, 10.5 and 5.5 kb). Oligonucleotide probes specific for the asymmetric and globular terminal regions hybridize with the three major transcripts, indicating that their size is determined by 3′‐untranslated regions which are not related to the differential splicing leading to A and Ga forms.

Cloning and Expression of a Rat Acetylcholinesterase Subunit: Generation of Multiple Molecular Forms and Complementarity with a Torpedo Collagenic Subunit

Abstract: We obtained a cDNA clone encoding one type of catalytic subunit of acetylcholinesterase (AChE) from rat brain (T subunit). The coding sequence shows a high frequency of (G + C) at the third position of the codons (66%), as already noted for several AChEs, in contrast with mammalian butyrylcholinesterase. The predicted primary sequence of rat AChE presents only 11 amino acid differences, including one in the signal peptide, from that of the mouse T subunit. In particular, four alanines in the mouse sequence are replaced by serine or threonine. In northern blots, a rat AChE probe indicates the presence of major 3.2‐and 2.4‐kb mRNAs, expressed in the CNS as well as in some peripheral tissues, including muscle and spleen. In vivo, we found that the proportions of G1, G2, and G4 forms are highly variable in different brain areas. We did not observe any glycolipid‐anchored G2 form, which would be derived from an H subunit. We expressed the cloned rat AChE in COS cells: The transfected cells produce principally an amphiphilic G1a form, together with amphiphilic G2a and G4a forms, and a nonamphiphilic G4na form. The amphiphilic G1a and G2a forms correspond to type II forms, which are predominant in muscle and brain of higher vertebrates. The cells also release G4na, G2a, and G1a in the culture medium. These experiments show that all the forms observed in the CNS in vivo may be obtained from the T subunit. By cotransfecting COS cells with the rat T subunit and the Torpedo collagenic subunit, we obtained chimeric collagentailed forms. This cross‐species complementarity demonstrates that the interaction domains of the catalytic and structural subunits are highly conserved during evolution.

The polymorphism of acetylcholinesterase: post-translational processing, quaternary associations and localization.

The molecular forms of acetylcholinesterase (AChE) correspond to various quaternary structures and modes of anchoring of the enzyme. In vertebrates, these molecules are generated from a single gene: the catalytic domain may be associated with several types of C-terminal peptides, that define distinct types of catalytic subunits (AChE(S), AChE(H), AChE(T)) and determine their post-translational maturation. AChE(S) generates soluble monomers, in the venom of Elapid snakes. AChE(H) generates GPI-anchored dimers, in Torpedo muscles and on mammalian blood cells. AChE(T) is the only type of catalytic subunit that exists in all vertebrate cholinesterases; it produces the major forms in adult brain and muscle. AChE(T) generates multiple structures, ranging from monomers and dimers to collagen-tailed and hydrophobic-tailed forms, in which catalytic tetramers are associated with anchoring proteins that attach them to the basal lamina or to cell membranes. In the collagen-tailed forms, AChE(T) subunits are associated with a specific collagen, ColQ, which is encoded by a single gene in mammals. ColQ contains a short peptidic motif, the proline-rich attachment domain (PRAD), that triggers the formation of AChE(T) tetramers, from monomers and dimers. The critical feature of this motif is the presence of a string of prolines, and in fact synthetic polyproline shows a similar capacity to organize AChE(T) tetramers. Although the COLQ gene produces multiple transcripts, it does not generate the hydrophobic tail. P, which anchors AChE in mammalian brain membranes. The coordinated expression of AChE(T) subunits and anchoring proteins determines the pattern of molecular forms and therefore the localization and functionality of the enzyme.

Polymorphism of Pseudocholinesterase in Torpedo marmorata Tissues: Comparative Study of the Catalytic and Molecular Properties of this Enzyme with Acetylcholinesterase

Abstract: We report the existence, in Torpedo marmorata tissues, of a cholinesterase species (sensitive to 10−5M eserine) that differs from acetylcholinesterase (AChE, EC 3.1.1.7) in several respects: (a) The enzyme hydrolyzes butyrylthiocholine (BuSCh) at about 30% of the rate at which it hydrolyzes acetylthiocholine (AcSCh), whereas Torpedo AChE does not show any activity on BuSCh. (b) It is not inhibited by 10−5M BW 284C51, but rapidly inactivated by 10−8M diisopropyl‐fluorophosphonate. (c) It does not exhibit inhibition by excess substrate up to 5 × 10−3M AcSCh. (d) It does not cross‐react with anti‐AChE antibodies raised against purified Torpedo AChE. This enzyme is obviously homologous to the “nonspecific” or pseudocholinesterase (pseudo‐ChE, EC 3.1.1.8) that exists in other species, although it is closer to “true” AChE than classic pseudo‐ChE in several respects. Thus, it shows the highest Vmax with acetyl‐, and not propionyl‐ or butyrylthiocholine, and it is not specifically sensitive to ethopropazine. Pseudo‐ChE is apparently absent from the electric organs, but represents the only cholinesterase species in the heart ventricle. Pseudo‐ChE and AChE coexist in the spinal cord and in blood plasma, where they contribute to AcSCh hydrolysis in comparable proportions. Pseudo‐ ChE exists in several molecular forms, including collagen‐tailed forms, which can be considered as homologous to those of AChE. In the heart the major component of pseudo‐ChE appears to be a soluble monomeric form (G1). This form is inactivated by Triton X‐100 within days. In addition, it is converted, on storage of the extracts, into more rapidly sedimenting, Triton X‐100‐resistant forms (G2 and G4). A G4 form exists in spinal cord and plasma. Collagen‐tailed forms, A12 and A8, were characterized in high‐salt extracts from spinal cord and heart ventricle. Pseudo‐ChE molecular forms sediment slightly slower than the corresponding AChE forms. The two polymorphic enzyme systems, AChE and pseudo‐ChE, therefore arose as early as elasmobranchs during the evolution of vertebrates.

An immunoglobulin M monoclonal antibody, recognizing a subset of acetylcholinesterase molecules from electric organs of Electrophorus and Torpedo, belongs to the HNK-1 anti-carbohydrate family.

An immunoglobulin M (IgM) monoclonal antibody (mAb Elec‐39), obtained against asymmetric acetyl‐cholinesterase (AChE) from Electrophorus electric organs, also reacts with a fraction of globular AChE (amphiphilic G2 form) from Torpedo electric organs. This antibody does not react with asymmetric AChE from Torpedo electric organs or with the enzyme from other tissues of Electrophorus or Torpedo. The corresponding epitope is removed by endoglycosidase F, showing that it is a carbohydrate. The subsets of Torpedo G2 that react or do not react with Elec‐39 (Elec‐39+ and Elec‐39‐) differ in their electrophoretic mobility under nondenaturing conditions; the Elec‐39+ component also binds the lectins from Pisum sativum and Lens culinaris. Whereas the Elec‐39‐ component is present at the earliest developmental stages examined, an Elec‐39+ component becomes distinguishable only around the 70‐mm stage. Its proportion increases progressively, but later than the rapid accumulation of the total G2form. In immunoblots, mAt Elec‐39 recognizes a number of proteins other than AChE from various tissues of several species. The specificity of Elec‐39 resembles that of a family of anti‐carbohydrate antibodies that includes HNK‐1, L2, NC‐1, NSP‐4, as well as IgMs that occur in human neuropathies. Although some human neuropathy IgMs that recognize the myelin‐associated glycoprotein did not react with, Elec‐39+ AChE, mAbs HNK‐1, NC‐1, and NSP‐4 showed the same selectivity as Elec‐39 for Torpedo G2 AChE, but differed in the formation of immune complexes.