The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant.
Site-directed mutagenesis was used to study the cysteine residue involved in the assembly of human acetylcholinesterase (HuAChE) catalytic subunits. Substitution of the cysteine at position 580 by alanine resulted in impairment of interchain disulfide bridge formation; the mutagenized enzyme (C580A) was secreted from recombinant cells in the monomeric form and failed to assemble into dimers. The mutant monomeric HuAChE did not differ from the native oligomeric enzyme neither in rate of catalysis nor in affinity to acetylthiocholine. Mutant monomers were also shown to retain the acetylcholinesterase characteristic sensitivity to high substrate concentrations. The mutation did not seem to affect the efficiencies of either synthesis or secretion of recombinant HuAChE polypeptides, as was demonstrated in cell lines derived from human embryonic kidney (293 cells) as well as from a human neuroblastoma (SK-N-SH). Furthermore, the mutation did not lead to an increase in accumulation of intracellular HuAChE polypeptides, suggesting that export of acetylcholinesterase from cells may not be coupled to subunit assembly.
Velan
B
B
Department of Biochemistry, Israel Institute for Biological Research, Ness-Ziona.
Grosfeld
H
H
Kronman
C
C
Leitner
M
M
Gozes
Y
Y
Lazar
A
A
Flashner
Y
Y
Marcus
D
D
Cohen
S
S
Shafferman
A
A
D000110:Acetylcholinesterase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003545:Cysteine; D004220:Disulfides; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D013816:Thermodynamics; D014162:Transfection
D004220:Disulfides; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000110:Acetylcholinesterase; D003545:Cysteine; D000409:Alanine