Xavier Cousin

ESTHER, the database of the α/β-hydrolase fold superfamily of proteins

The α/β-hydrolase fold is characterized by a β-sheet core of five to eight strands connected by α-helices to form a α/β/α sandwich. In most of the family members the β-strands are parallels, but some show an inversion in the order of the first strands, resulting in antiparallel orientation. The members of the superfamily diverged from a common ancestor into a number of hydrolytic enzymes with a wide range of substrate specificities, together with other proteins with no recognized catalytic activity. In the enzymes the catalytic triad residues are presented on loops, of which one, the nucleophile elbow, is the most conserved feature of the fold. Of the other proteins, which all lack from one to all of the catalytic residues, some may simply be ‘inactive’ enzymes while others are known to be involved in surface recognition functions. The ESTHER database (http://bioweb.ensam.inra.fr/esther) gathers and annotates all the published information related to gene and protein sequences of this superfamily, as well as biochemical, pharmacological and structural data, and connects them so as to provide the bases for studying structure–function relationships within the family. The most recent developments of the database, which include a section on human diseases related to members of the family, are described.

Acetylcholinesterases from Elapidae snake venoms: biochemical, immunological and enzymatic characterization

We analyzed 45 batches of venom from 20 different species belonging to 11 genera from the 3 main families of venomous snakes (Elapidae, Viperidae and Crotalidae). We found high acetylcholinesterase (AChE) activity in all venoms from Elapidae, except in those from the Dendroaspis genus. AChE was particularly abundant in Bungarus venoms which contain up to 8 mg of enzyme per gram of dried venom. We could not detect acetylcholinesterase activity in any batch of venom from Viperidae or Crotalidae. Titration of active sites with an organophosphorous agent (MPT) revealed that the AChE of all venoms have similar turnovers (6000 to 8000 s(-1)) which are clearly higher than those of Torpedo and mammalian enzymes but lower than that of Electrophorus. AChEs from the venom of elapid snakes of the Bungarus, Naja, Ophiophagus and Haemacatus genera were purified by affinity chromatography. SDS-PAGE analysis and sucrose gradient centrifugation demonstrated that AChE is exclusively present as a nonamphiphilic monomer. These enzymes are true AChEs, hydrolyzing acetylthiocholine faster than propionylthiocholine and butyrylthiocholine and exhibiting excess substrate inhibition. Twenty-seven different monoclonal antibodies directed against AChE from Bungarus fasciatus venom were raised in mice. Half of them recognized exclusively the Bungarus enzyme while the others cross-reacted with AChEs from other venoms. Polyspecific mAbs were used to demonstrate that venoms from Dendroaspis, which contain the AChE inhibitor fasciculin but lack AChE activity, were also devoid of immunoreactive AChE protein. AChE inhibitors acting at the active site (edrophonium, tacrine) and at the peripheral site (propidium, fasciculin), as well as bis-quaternary ligands (BW284C51, decamethonium), were tested against the venom AChEs from 11 different species. All enzymes had a very similar pattern of reactivity with regard to the different inhibitors, with the exception of fasciculin. AChEs from Naja and Haemacatus venoms were relatively insensitive to fasciculin inhibition (IC50 >> 10(-6) M), while Bungarus (IC50 approximately 10(-8) M) and especially Ophiophagus (IC50 < 10(-10) M) AChEs were inhibited very efficiently. Ophiophagus and Bungarus AChEs were also efficiently inhibited by a monoclonal antibody (Elec-410) previously described as a specific ligand for the Electrophorus electricus peripheral site. Taken together, these results show that the venoms of most Elapidae snakes contain large amounts of a highly active non-amphiphilic monomeric AChE. All snake venom AChEs show strong immunological similarities and possess very similar enzymatic properties. However, they present quite different sensitivity to peripheral site inhibitors, fasciculin and the monoclonal antibody Elec-410.

Acetylcholinesterase from Bungarus venom: a monomeric species.

The venom of Bungarus fasciatus, an Elapidae snake, contains a high level of AChE activity. Partial peptide sequences show that it is closely homologous to other AChEs. Bungarus venom AChE is a non‐amphiphilic monomeric species, a molecular form of AChE which has not been previously found in significant levels in other tissues. The composition of carbohydrates suggests the presence of N‐glycans of the ‘complex’ and ‘hybrid’ types. Ion exchange chromatography, isoelectric focusing and electrophoresis in non‐denaturing and denaturing conditions reveal a complex microheterogeneity of this enzyme, which is partly related to its glycosylation.

The α/β fold family of proteins database and the cholinesterase gene server ESTHER

ESTHER (for esterases, alpha/betahydrolase enzyme and relatives) is a database of sequences phylogenetically related to cholinesterases. These sequences define a homogeneous group of enzymes (carboxylesterases, lipases and hormone-sensitive lipases) sharing a similar structure of a central beta-sheet surrounded by alpha-helices. Among these proteins a wide range of functions can be found (hydrolases, adhesion molecules, hormone precursors). The purpose of ESTHER is to help comparison of structures and functions of members of the family. Since the last release, new features have been added to the server. A BLAST comparison tool allows sequence homology searches within the database sequences. New sections are available: kinetics and inhibitors of cholinesterases, fasciculin-acetylcholinesterase interaction and a gene structure review. The mutation analysis compilation has been improved with three-dimensional images. A mailing list has been created.

A Cholinesterase Genes Server (ESTHER): A Database of Cholinesterase-Related Sequences for Multiple Alignments, Phylogenetic Relationships, Mutations and Structural Data Retrieval

We have built a database of sequences phylogenetically related to cholinesterases (ESTHER) for esterases, alpha/beta hydrolase enzymes and relatives). These sequences define a homogeneous group of enzymes (carboxylesterases, lipases and hormone-sensitive lipases) with some related proteins devoid of enzymatic activity. The purpose of ESTHER is to help comparison and alignment of any new sequence appearing in the field, to favour mutation analysis of structure-function relationships and to allow structural data recovery. ESTHER is a World Wide Web server with the URL http://www.montpellier.inra.fr:70/cholinesterase.

Acetylcholinesterase in Elapid Snakes

Acetylcholinesterase (AChE) plays a key role in cholinergic transmission, but is also present in non-cholinergic contexts where its function is not clearly known (1). This is the case in pulmonary and intestinal epitheliums, salivary glands, or blood cells surface. AChE is a very abundant protein in the venom of some Elapids snakes (2–3).

Identification of Residues Involved in Naja Naja Oxiana AChE Insensitivity to Fasciculin

AChEs from Elapid venoms have been recently characterized in detail, they present several specific properties when compared to other vertebrates enzymes. Venom AChEs are monomers and their sensitivity to fasciculin, a peripheral site inhibitor purified from Dendroaspis venom, varies greatly following the species (1).