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

Cibby Varkey Altamirano, Oksana Lockridge

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

Human butyrylcholinesterase (BChE) in serum is composed predominantly of tetramers. The tetramerization domain of each subunit is contained within 40 C-terminal residues. To identify key residues within this domain participating in tetramer stabilization, the interaction between C-terminal 46 residue peptides was quantitated in the yeast two-hybrid system. The wild- type peptide interacted strongly with another wild-type peptide in the yeast two-hybrid system. The C571A mutant peptides interacted to a similar degree as the wild-type. However, the mutant in which seven conserved aromatic residues (Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564) and C571 were altered to alanines showed only 12% of the interaction seen with the wild-type peptide. The seven mutations (aromatics-off) were incorporated into the complete BChE molecule, with or without the C571A mutation, and expressed in 293T and CHO-K1 cells. Expression of wild-type BChE in these cell lines yielded 10% tetramers. The aromatics-off mutant formed dimers and monomers but no tetramers. The aromatics-off/C571A mutant yielded only monomers. Addition of poly-L-proline to culture medium, or coexpression with the N-terminus of COLQ including the proline-rich attachment domain (Q(N)PRAD), increased the amount of tetrameric wild-type BChE from 10 to 70%, but had no effect on the G534stop (lacking 41 C-terminal residues) and the aromatics-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 stabilization of BChE tetramers is mediated through the interaction of the seven conserved aromatic residues and that poly-L-proline and PRAD act through these aromatic residues to induce tetramerization.

Original languageEnglish (US)
Pages (from-to)13414-13422
Number of pages9
JournalBiochemistry
Volume38
Issue number40
DOIs
StatePublished - Oct 5 1999

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Butyrylcholinesterase
Peptides
Two-Hybrid System Techniques
Hybrid systems
Yeast
Stabilization
Monomers
Column chromatography
Mutation
CHO Cells
Proline
Alanine
Dimers
Culture Media
Chromatography
Cells
Cell Line
Molecules
Serum

ASJC Scopus subject areas

  • Biochemistry

Cite this

Conserved aromatic residues of the C-terminus of human butyrylcholinesterase mediate the association of tetramers. / Altamirano, Cibby Varkey; Lockridge, Oksana.

In: Biochemistry, Vol. 38, No. 40, 05.10.1999, p. 13414-13422.

Research output: Contribution to journalArticle

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title = "Conserved aromatic residues of the C-terminus of human butyrylcholinesterase mediate the association of tetramers",
abstract = "Human butyrylcholinesterase (BChE) in serum is composed predominantly of tetramers. The tetramerization domain of each subunit is contained within 40 C-terminal residues. To identify key residues within this domain participating in tetramer stabilization, the interaction between C-terminal 46 residue peptides was quantitated in the yeast two-hybrid system. The wild- type peptide interacted strongly with another wild-type peptide in the yeast two-hybrid system. The C571A mutant peptides interacted to a similar degree as the wild-type. However, the mutant in which seven conserved aromatic residues (Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564) and C571 were altered to alanines showed only 12{\%} of the interaction seen with the wild-type peptide. The seven mutations (aromatics-off) were incorporated into the complete BChE molecule, with or without the C571A mutation, and expressed in 293T and CHO-K1 cells. Expression of wild-type BChE in these cell lines yielded 10{\%} tetramers. The aromatics-off mutant formed dimers and monomers but no tetramers. The aromatics-off/C571A mutant yielded only monomers. Addition of poly-L-proline to culture medium, or coexpression with the N-terminus of COLQ including the proline-rich attachment domain (Q(N)PRAD), increased the amount of tetrameric wild-type BChE from 10 to 70{\%}, but had no effect on the G534stop (lacking 41 C-terminal residues) and the aromatics-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 stabilization of BChE tetramers is mediated through the interaction of the seven conserved aromatic residues and that poly-L-proline and PRAD act through these aromatic residues to induce tetramerization.",
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