TY - JOUR
T1 - Butyrylcholinesterase in SH-SY5Y human neuroblastoma cells
AU - Onder, Seda
AU - Schopfer, Lawrence M.
AU - Jiang, Wei
AU - Tacal, Ozden
AU - Lockridge, Oksana
N1 - Funding Information:
Supported by the Fred & Pamela Buffet Cancer Center Support Grant P30CA036727 , NIH grant 1R21ES030132-01A1 (to OL). National Natural Science Foundation of China grant 81903290 (to WJ).
Funding Information:
Mass spectrometry data were obtained by the Mass Spectrometry and Proteomics Core Facility at the University of Nebraska Medical Center, which is supported by state funds from the Nebraska Research Initiative. Mass spectral data were interrogated using Batch Tag (Web) and Search Compare programs from Protein Prospector. These programs are available at no cost https://prospector.ucsf.edu [prospector.ucsf.edu]. Programs were developed in the University of California San Francisco Mass Spectrometry Facility, directed by Dr. Alma Burlingame.
Publisher Copyright:
© 2022 The Authors
PY - 2022/5
Y1 - 2022/5
N2 - Cultured SH-SY5Y human neuroblastoma cells are used in neurotoxicity assays. These cells express markers of the cholinergic and dopaminergic systems. Acetylcholinesterase (AChE) activity has been reported in these cells. Neurotoxic organophosphate compounds that inhibit AChE, also inhibit butyrylcholinesterase (BChE). We confirmed the presence of AChE in the cell lysate by activity assays, Western blot, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of immunopurified AChE. A nondenaturing gel stained for AChE activity identified the catalytically active AChE in SH-SY5Y cells as the unstable monomer. We also identified immature BChE in the cell lysate. The concentration of active BChE protein was similar to that of active AChE protein. The rate of substrate hydrolysis by AChE was 10-fold higher than substrate hydrolysis by BChE. The higher rate was due to the 10-fold higher specific activity of AChE over BChE (5000 units/mg for AChE; 500 units/mg for BChE). Neither cholinesterase was secreted. Tryptic peptides of immunopurified AChE and BChE were identified by LC-MS/MS on an Orbitrap Lumos Fusion mass spectrometer. The unfolded protein chaperone, binding immunoglobulin protein BiP/GRP78, was identified in the mass spectral data from all cholinesterase samples, suggesting that BiP was co-extracted with cholinesterase. This suggests that the cytoplasmic cholinesterases are immature forms of AChE and BChE that bind to BiP. It was concluded that SH-SY5Y cells express active AChE and active BChE, but the proteins do not mature to glycosylated tetramers.
AB - Cultured SH-SY5Y human neuroblastoma cells are used in neurotoxicity assays. These cells express markers of the cholinergic and dopaminergic systems. Acetylcholinesterase (AChE) activity has been reported in these cells. Neurotoxic organophosphate compounds that inhibit AChE, also inhibit butyrylcholinesterase (BChE). We confirmed the presence of AChE in the cell lysate by activity assays, Western blot, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of immunopurified AChE. A nondenaturing gel stained for AChE activity identified the catalytically active AChE in SH-SY5Y cells as the unstable monomer. We also identified immature BChE in the cell lysate. The concentration of active BChE protein was similar to that of active AChE protein. The rate of substrate hydrolysis by AChE was 10-fold higher than substrate hydrolysis by BChE. The higher rate was due to the 10-fold higher specific activity of AChE over BChE (5000 units/mg for AChE; 500 units/mg for BChE). Neither cholinesterase was secreted. Tryptic peptides of immunopurified AChE and BChE were identified by LC-MS/MS on an Orbitrap Lumos Fusion mass spectrometer. The unfolded protein chaperone, binding immunoglobulin protein BiP/GRP78, was identified in the mass spectral data from all cholinesterase samples, suggesting that BiP was co-extracted with cholinesterase. This suggests that the cytoplasmic cholinesterases are immature forms of AChE and BChE that bind to BiP. It was concluded that SH-SY5Y cells express active AChE and active BChE, but the proteins do not mature to glycosylated tetramers.
KW - Acetylcholinesterase
KW - BiP chaperone
KW - Butyrylcholinesterase
KW - Human neuroblastoma cells
KW - Mass spectrometry
KW - Western blot
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U2 - 10.1016/j.neuro.2022.02.006
DO - 10.1016/j.neuro.2022.02.006
M3 - Article
C2 - 35189179
AN - SCOPUS:85124876327
SN - 0161-813X
VL - 90
SP - 1
EP - 9
JO - NeuroToxicology
JF - NeuroToxicology
ER -