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Cholinesterase-Like Domains in Enzymes and Structural Proteins

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Cholinesterase-Like Domains in Enzymes and Structural Proteins Proc. Natl. Acad. Sci. USA Vol. 88, pp. 6647-6651, August 1991 Neurobiology Cholinesterase-like domains in enzymes and structural proteins: Functional and evolutionary relationships and identification of a catalytically essential aspartic acid (choLinesterase/esterase/lipase/neurotactin/glutactin) ERIC KREJCI*, NATHALIE DUVAL*, ARNAUD CHATONNETt, PIERRE VINCENSf, AND JEAN MASSOULIV* *Laboratoire de Neurobiologie, Centre National de la Recherche Scientifique Unite Associde 295, Ecole Normale Supdrieure, 75005 Paris, France; tPhysiologie Animale, Institut National de la Recherche Agronomique, 34060 Montpellier, France; and tGroupe de Bioinformatique, Laboratoire de Biochimie et Physiologie du DIeloppement, Centre National de la Recherche Scientifique Unite Associde 686, Ecole Normale Suporieure, 75005 Paris, France Communicated by David D. Sabatini, April 1, 1991 (receivedfor review December 27, 1990) ABSTRACT Primary sequences of cholinesterases and re- ChoEases now appear as prototypes of a family of pro- lated proteins have been systematically compared. The cholin- teins. In this report, we compare the primary structures ofthe esterase-like domain of these proteins, about 500 amino acids, various elements of this family. We define an alignment of may fulfill a catalytic and a structural function. We identified amino acid sequences, use it to identify common features, an aspartic acid residue that is conserved among esterases and and discuss functional and evolutionary implications. lipases (Asp-397 in Torpedo acetylcholinesterase) but that had not been considered to be involved in the catalytic mechanism. RESULTS AND DISCUSSION Site-directed mutagenesis demonstrated that this residue is necessary for activity. Analysis of evolutionary relationships Alignment of Peptide Sequences. Fig. 1 shows an alignment shows that the noncatalytic members of the family do not of ChoEases and homologous protein domains. This align- constitute a separate subgroup, suggesting that loss ofcatalytic ment was obtained by visual inspection on the basis of activity occurred independently on several occasions, probably strongly conserved peptides or isolated amino acid motifs. from bifunctional molecules. Cholinesterases may thus be Essentially the same peptide motifs were found by using the involved in cell-cell interactions in addition to the hydrolysis of multialign program developed by Corpet (38). Some se- acetylcholine. This would explain their specific expression in quences present insertions, e.g., large hydrophilic peptides in well-defined territories during embryogenesis before the for- Drosophila AcChoEase (RGANGGEHPNGKQADTHLIH- mation ofcholinergic synapses and their presence in noncholin- NGNPQNTTNG) (9) and Anopheles AcChoEase (GLNFG- ergic tissues. SNDYFQDDDDFQRQHQSKGG) (12). Such additional peptides are likely to be located at the periphery of the Acetylcholinesterases (AcChoEases, EC 3.1.1.7) and bu- tertiary structure of the protein between distinct elements of tyrylcholinesterases (BtChoEases, EC 3.1.1.8) (1-3) consti- secondary structure, so as not to disturb its organization. In tute a closely homologous family of proteins (4-13). These Drosophila AcChoEase, this peptide is normally removed enzymes consist of a major common catalytic domain and a from the mature subunit, producing two fragments of 18 and small C-terminal variable region. In vertebrate AcChoEase, 55 kDa from the 75-kDa precursor (39, 40). two types of C-terminal regions are encoded by alternative Fig. 2 illustrates the position of the ChoEase-like domain exons, differentiating the subunits that participate in the in various proteins. This domain is represented as a grey bar, collagen-tailed, hydrophobic-tailed, or soluble tetramers in which we have indicated the approximate position of (exon T) and in the glycolipid-anchored dimers (exon H) residues that seem to play a role in the catalytic mechanism (refs. 14 and 15; for review, see ref. 3). A number ofproteins of esterases (see below). In addition, we have marked the have been shown to present homology with the common position of intracatenary disulfide bridges, deduced from domain of cholinesterases (ChoEases). In addition to serine homology with Torpedo AcChoEase (36). Note that the hydrolases such as carboxylesterases (16-23), pancreatic ChoEase-like domain constitutes the predominant part of the lysophospholipase or cholesterol esterase (24, 25), and lipase protein in esterases but is associated with other large peptide from Geotrichum candidum (26), this family includes non- domains in the nonenzymatic proteins (thyroglobulin, glu- catalytic proteins; the C-terminal domain of bovine (27), rat tactin, and neurotactin). (28), and human (29) thyroglobulins (4, 30, 31); and two Identification of an Essential Aspartic Acid Residue. The Drosophila proteins that seem to be involved in cellular present analysis shows that Asp-397 (numbering of Torpedo interactions, glutactin (32) and neurotactin (33, 34). AcChoEase) is present in all ChoEase-like esterases, except The hydrolysis of acetylcholine by AcChoEase operates in the first primary sequence determined for Torpedo mar- near the theoretical limit set by the diffusion of the substrate morata, which contained asparagine at this position (codon (35). Its catalytic mechanism remains obscure, however; in AAC) (5). This was probably due to an error in the construc- particular, the presence of a charge-relay system, Asp/His/ tion of the cDNA since independent clones contained aspar- Ser, is not established. We presently show that Asp-397 of tic acid (codon GAC) at this position. Torpedo AcChoEase, which, to our knowledge, has not Transfections of COS cells with CDM8 constructions (41, hitherto been considered to be involved in the catalytic 42) that contained either Asp-397 or Asn-397 but were mechanism of ChoEases, is strictly conserved among Cho- otherwise identical showed that AcChoEase activity was Ease-like esterases and is essential for activity. produced exclusively in the first case, although the synthesis and the stability of the protein were equivalent (Fig. 3). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: ChoEase, cholinesterase; AcChoEase, acetylcho- in accordance with 18 U.S.C. §1734 solely to indicate this fact. linesterase; BtChoEase, butyrylcholinesterase. 6647 Downloaded by guest on September 30, 2021 6648 Neurobiology: Krejci et aL Proc NatL Acad. Sci USA 88 (1991) -, f- %,: ... X - .. 1z, -- _~ C.C.C.C.C.C. 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