Abstract
As deduced from cDNA clones, the catalytic domain of Bungarus fasciatus venom acetylcholinesterase (AChE) is highly homologous to those of other AChEs. It is, however, associated with a short hydrophilic carboxyl-terminal region, containing no cysteine, that bears no resemblance to the alternative COOH-terminal peptides of the GPI-anchored molecules (H) or of other homomeric or heteromeric tailed molecules (T). Expression of complete and truncated AChE in COS cells showed that active hydrophilic monomers are produced and secreted in all cases, and that cleavage of a very basic 8-residue carboxyl-terminal fragment occurs upon secretion. The COS cells produced Bungarus AChE about 30 times more efficiently than an equivalent secreted monomeric rat AChE. The recombinant Bungarus AChE, like the natural venom enzyme, showed a distinctive ladder pattern in nondenaturing electrophoresis, probably reflecting a variation in the number of sialic acids. By mutagenesis, we showed that two differences (methionine instead of tyrosine at position 70; lysine instead of aspartate or glutamate at position 285) explain the low sensitivity of Bungarus AChE to peripheral site inhibitors, compared to the Torpedo or mammalian AChEs. These results illustrate the importance of both the aromatic and the charged residues, and the fact that peripheral site ligands (propidium, gallamine, D-tubocurarine, and fasciculin 2) interact with diverse subsets of residues.
Highlights
As deduced from cDNA clones, the catalytic domain of Bungarus fasciatus venom acetylcholinesterase (AChE) is highly homologous to those of other AChEs
Expression of complete and truncated AChE in COS cells showed that active hydrophilic monomers are produced and secreted in all cases, and that cleavage of a very basic 8-residue carboxyl-terminal fragment occurs upon secretion
Modeling—We modeled the B. fasciatus venom enzyme, using the first approach protocol proposed by Swiss-Model (Peitsch, 1995), with the structure of Torpedo californica AChE as template (1ace.pdb; Sussman et al, 1991)
Summary
A NEW TYPE OF COOH-TERMINAL DOMAIN; INVOLVEMENT OF A POSITIVELY CHARGED RESIDUE IN THE PERIPHERAL SITE*. In the AChE genes of vertebrates, Torpedo, and mammals, the signal peptide and the catalytic domain are encoded by common exons, followed by alternatively spliced sequences which encode distinct types of short COOH-terminal regions, characterizing R (“read-through”), H (“hydrophobic”), or T (“tailed”) subunits. These subunits possess identical catalytic activity, but generate different molecular forms. 3 Cousin, X., Creminon, C., Grassi, J., Meflah, K., Cornu, G., Saliou, B., Bon, S., Massoulie, J., and Bon, C. By site-directed mutagenesis, the role of two residues that differ between Bungarus AChE and Torpedo or mammalian AChEs, in the inhibition by peripheral site ligands
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