Conserved aromatic residues of the C-terminus of human butyrylcholinesterase mediate the association of tetramers

Human butyrylcholinesterase (BChE) in serum is composed predominantly of te tramers. The tetramerization domain of each subunit is contained within 40 C-terminal residues. To identify key residues within this domain participat ing in tetramer stabilization, the interaction between C-terminal 46 residu e peptides was quantitated in the yeast two-hybrid system. The wild-type pe ptide interacted strongly with another wild-type peptide in the yeast two-h ybrid system. The C571A mutant peptides interacted to a similar degree as t he wild-type. However, the mutant in which seven conserved aromatic residue s (Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564) and C 571 were altered to alanines showed only 12% of the interaction seen with t he wild-type peptide. The seven mutations (aromatics-off) were incorporated into the complete BChE molecule, with or without the C571A mutation, and e xpressed in 293T and CHO-K1 cells. Expression of wild-type BChE in these ce ll lines yielded 10% tetramers. The aromatics-off mutant formed dimers and monomers but no tetramers. The arornatics-off/C571A mutant yielded only mon omers. Addition of poly-L-proline to culture medium, or coexpression with t he N-terminus of COLQ including the proline-rich attachment domain (Q(N)PRA D), increased the amount of tetrameric wild-type BChE from 10 to 70%, but: bad no effect on the G534stop (lacking 41 C-terminal residues) and the arom atics-off mutants. Recombinant BChE produced by coexpression with Q(N)PRAD was purified by column chromatography. The purified tetramers contained the FLAG-tagged Q(N)PRAD peptide. These observations suggest that the stabiliz ation of BChE tetramers is mediated through the interaction of the seven co nserved aromatic residues and that poly-L-proline and PRAD act through thes e aromatic residues to induce tetramerization.