Oligomerization of chicken acetylcholinesterase does not require intersubunit disulfide bonds.
Acetylcholinesterase (AChE) is secreted from muscle and nerve cells and associates as multimers through intermolecular covalent and noncovalent bonds. The amino acid sequence of the C-terminus is thought to play an important role in these interactions. We generated mutants in the C-terminus of the catalytic T-subunit of chicken AChE to determine the importance of this region to oligomerization and to the amphipathic character of the protein. Wild-type recombinant chicken AChE secreted from human embryonic kidney 293 cells was assembled into dimers and tetramers exclusively. Mutants lacking the C-terminal Cys764, the only cysteine involved in interchain disulfide bonds, showed lower but significant levels of the secreted dimeric and tetrameric forms. A truncated mutant, lacking the C-terminal 39 amino acids, exhibited a severe decrease in content of the multimeric forms, yet small amounts of the dimer were detectable. The amphipathic character was dependent on the state of oligomerization. When analyzed by sucrose gradients, the sedimentation of tetramers was not affected by detergent, but monomers and dimers sedimented more slowly in the presence of detergent. Most of the recombinant wild-type enzyme, shown to be dimeric and tetrameric by sedimentation analysis, was monomeric when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that much of the secreted oligomeric AChE was not disulfide bonded. These data suggest that disulfide bonding of Cys764 is not required for the catalytic subunit of chicken AChE to form oligomers and that regions outside of the C-terminus contribute to the hydrophobic interactions that are important for stabilizing the oligomeric forms.
Gough
N R
NR
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine and Medical Biotechnology Center, Baltimore 21201, USA.
Randall
W R
WR
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002645:Chickens; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics; D014162:Transfection
D004220:Disulfides; D010446:Peptide Fragments; D000110:Acetylcholinesterase