Association of tetramers of human butyrylcholinesterase is mediated by conserved aromatic residues of the carboxy terminus

Human butyrylcholinesterase (BChE) is composed predominantly of tetramers. Our laboratory has shown that up to 40 carboxy terminal residues of each su bunit contribute to the stabilization of tetramers (R.M. Blong, E. Bedows, O. Lockridge, The tetramerization domain of butyrylcholinesterase is at the carboxy-terminus, Biochem. J. 327 (1997) 747-757). To better define the re sidues which participate in tetramer stabilization, the in vivo interaction of the BChE C-terminus 46 residue peptide was quantitated for wild type an d mutant BChE using the yeast two-hybrid system. The wild type C-terminal p eptides interacted with one another in this system. The K-variant (A539T) a nd C571A peptides showed interaction similar to that of the wild type. Howe ver, only 11.7% of the interaction seen with the wild type peptide was obse rved with the mutant in which seven conserved aromatic residues (Trp 543, P he 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564) had been altered t o alanines (aromatics off mutant). When these seven mutations were incorpor ated into the complete BChE molecule and expressed in 293T cells, only mono mers and dimers were observed. The addition of poly-L-proline to the medium of 293T cells expressing wild type BChE resulted in the increase of the te trameric form, similar to that observed by Bon et al. (S. Bon, F. Coussen, J. Massoulie, Quaternary associations of acetylcholinesterase II. The polyp roline attachment domain of the collagen tail, J. Biol. Chem. 272 (1997) 30 16-3021) for acetylcholinesterase expressed in COS cells. However, no incre ase in tetramers was observed with poly-L-proline addition to the medium of 293T cells expressing the aromatics off BChE mutant. These observations su ggest that the stabilization of BChE tetramers is mediated through the inte raction of the seven conserved aromatic residues, Trp 543, Phe 547, Trp 550 , Tyr 553, Trp 557, Phe 561, and Tyr 564, and that the poly-L-proline induc ed increase in tetrameric BChE is mediated through these seven aromatic res idues. (C) 1999 Published by Elsevier Science Ireland Ltd. All rights reser ved.