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. Author manuscript; available in PMC: 2023 May 1.
Published in final edited form as: Neurotoxicology. 2022 Feb 18;90:1–9. doi: 10.1016/j.neuro.2022.02.006

Butyrylcholinesterase in SH-SY5Y human neuroblastoma cells

Seda Onder 1,2, Lawrence M Schopfer 1, Wei Jiang 3, Ozden Tacal 2, Oksana Lockridge 1,*
PMCID: PMC9124689  NIHMSID: NIHMS1782947  PMID: 35189179

Abstract

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.

Keywords: butyrylcholinesterase, mass spectrometry, human neuroblastoma cells, BiP chaperone, acetylcholinesterase, Western blot

1. Introduction

SH-SY5Y human neuroblastoma cells can be used in place of animals to study response to toxicants (Ehrich et al., 1997). Acute toxicity from organophosphate (OP) exposure is due to inhibition of acetylcholinesterase (Silman, 2021; Taylor, 2018), an enzyme present in the human brain, muscles, erythrocytes and in SH-SY5Y cells (Ehrich et al., 1997). Butyrylcholinesterase (BChE), an enzyme present in human plasma, brain, muscles, liver, lung, and other tissues plays a protective role against OP intoxication. Human blood contains 4–5 mg/L soluble BChE in plasma and 0.5 mg/L membrane bound AChE in erythrocytes (Bartels et al., 2000). BChE protects from OP toxicity by destroying OP (Lockridge et al., 2016; Saxena et al., 2015). The potency of newly synthesized inhibitors is assayed with commercially available AChE and BChE enzymes (Bayrak et al., 2017; Oztaskin et al., 2017).

Human SH-SY5Y neuroblastoma cells express catalytically active AChE (Adem et al., 1987; Ehrich et al., 1997; Kovalevich and Langford, 2013). Catalytically active BChE has been reported in SH-SY5Y cells by imaging the emission of a fluorescent substrate (Liu et al., 2018). In the present report we confirm the presence of catalytically active AChE and BChE enzymes in SH-SY5Y cells. In addition, we find unfolded AChE and BChE proteins.

2. Materials and Methods

2.1. Materials

Mouse anti-human erythrocyte AChE, AE-1 GenBank KY684073 and KY684074 (Dafferner et al., 2017; Fambrough et al., 1982)

Mouse anti-human plasma BChE B2 18–5, GenBank KT189143 and KT189144 (Brimijoin et al., 1983; Peng et al., 2016)

Mouse anti-denatured human BChE C191, GenBank KX278822 and KX278823 (Peng et al., 2016)

Mouse anti-human brain AChE HR2, Gene Tex TX22803 (Rakonczay and Brimijoin, 1988)

Mouse anti human AChE residues 481–614, A-11, Santa Cruz Biotechnology sc-373901.

Trypsin sequencing grade, Promega #V5113

Anti-mouse IgG-HRP, Cell Signaling #7076S

Halt protease inhibitor cocktail (AEBSF, Aprotinin, Bestatin, E64, Leupeptin, and Pepstatin A), Thermo Scientific #87786

Purified human butyrylcholinesterase prepared in-house (Schopfer et al., 2019)

Recombinant human AChE prepared in-house (Dafferner et al., 2017)

Trans retinoic acid, Sigma-Aldrich 554720

DMEM/F12 Glutamax, Gibco 10565–018

IP lysis buffer (25 mM TrisCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol), Pierce 87787 Thermo Fisher Scientific

Clarity Max Western Substrate, Bio-Rad #1705062

SH-SY5Y human neuroblastoma cells, ATCC CRL-2266

BCA (bicinchoninic acid) Protein Assay Kit, Thermo Scientific #23225

PVDF membrane 0.2 μm, Bio-Rad #162–0177

Pre-cast 4–20% gradient polyacrylamide gels, Bio-Rad #4568094

Durapore PVDF-0.45 μm centrifugal filter, Millipore UFC30HV00

2.2. Methods

2.2.1. SH-SY5Y cell culture and differentiation

SH-SY5Y cells (ATCC CRL-2266) were cultured and differentiated according to Encinas et al (Encinas et al., 2000). Cells in passage number 10 were grown in DMEM/F12 Glutamax, pen-strep, 15% FBS to 70–80% confluence in T75 flasks. Differentiation was initiated by addition of all trans-retinoic acid to 10 μM. After 5 days in retinoic acid and 15% FBS, adherent cells were washed with PBS. The washed cells were incubated with serum-free DMEM/F12 Glutamax, pen-strep containing 50 ng/mL BDNF. Neurite outgrowths were visible on day 3. Differentiated cells were harvested on day 5.

2.2.2. Activity assays for AChE and BChE

Enzyme activity in live whole cells was measured in a 96-well plate according to (Ehrich et al., 1997). Undifferentiated and differentiated cells in 50 μl PBS were added to a 96-well plate. For assay of AChE activity, 360,000 cells per well were incubated with 1 mM acetylthiocholine, 0.5 mM 5,5′-dithiobis (2-nitrobenzoic acid) in 0.1 M potassium phosphate pH 7.0, containing 20 μM ethopropazine to inhibit butyrylcholinesterase in a total volume of 200 μL. For assay of BChE activity 360,000 cells per well were incubated with 1 mM butyrylthiocholine, and 0.5 mM 5,5′-dithiobis (2-nitrobenzoic acid) in 0.1 M potassium phosphate pH 7.0. Butyrylthiocholine is a selective substrate for BChE. The microplate was covered with a lid to minimize evaporation while absorbance at 412 nm was recorded over a period of 120 min on a Tecan Spark microplate reader (Tecan Austria GmbH, Salzburg, Austria) set to 24°C. Autohydrolysis rates of acetylthiocholine and butyrylthiocholine were measured in wells that contained all reagents except cells. Observed increase in absorbance at 412 nm was corrected for substrate autohydrolysis.

Activity assays were also performed in a Gilford spectrophotometer interfaced to a MacLab data acquisition system (ADInstruments, Colorado Springs, CO), as described (Schopfer et al., 2019). A unit (u) of activity is one micromole of substrate hydrolyzed per minute.

2.2.3. Nondenaturing gel stained for cholinesterase activity

Nondenaturing polyacrylamide 4% to 22% gradient slab gels (thickness, 0.75 mm), with a 4% stacking gel were poured in a vertical slab gel apparatus (model SE600, Hoefer, Thermo Fisher Scientific). Electrophoresis was performed at 320 V constant voltage for 18 h at 4°C. The Karnovsky and Roots histochemical method (Karnovsky and Roots, 1964) for determination of cholinesterase activity was adapted for use with polyacrylamide gels (Li et al., 2005), using acetylthiocholine as substrate. Bands of cholinesterase activity had a red/brown color. Both AChE and BChE hydrolyze acetylthiocholine.

To confirm that the acetylthiocholine hydrolase activity in cell lysate was predominantly due to AChE, a gel shift assay was performed. Cell lysate was incubated with antibodies specific for native AChE, namely mouse monoclonal AE-1 and mouse monoclonal HR2. Migration of the antibody bound AChE complex was visualized on a nondenaturing gel stained for cholinesterase activity.

2.2.4. Western blot

The Western blot protocol is described (Biberoglu et al., 2019). The C191 antibody against denatured, truncated, monomeric recombinant human BChE was created at the University of Nebraska Medical Center (Peng et al., 2016). The HR2 monoclonal was created against catalytically active human brain AChE (Rakonczay and Brimijoin, 1988). The AE-1 and AE-2 monoclonals were created against catalytically active human erythrocyte AChE (Fambrough et al., 1982). We purified the AE-1 monoclonal from the culture medium of hybridoma cells obtained from the American Type Culture Collection. The A-11 monoclonal (Santa Cruz Biotechnology, Inc. sc-373901) was raised against amino acids 481–614 of human AChE.

Blots were hybridized with a secondary antibody conjugated to HRP at a dilution of 1:10000 for 1 h. Bound HRP activity was detected with Clarity Max Western ECL Substrate on an Azure c600 Biosystems device (Azure Biosystems Inc, Dublin, CA).

2.2.5. Cell lysis and protein concentration

Differentiated SH-SY5Y cells were lysed in 0.5 mL IP buffer (25 mM TrisCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) containing HALT protease inhibitor cocktail. The cell lysate was clarified by centrifugation. The protein concentration of the clarified cell lysate was determined in the BCA protein assay relative to bovine albumin standard. Cell lysate protein concentrations in repeat experiments were 3.2, 2.6, 2.6, and 1.5 mg/ml.

2.2.6. Immunopurification of AChE and BChE

Monoclonal antibodies B2 18–5, C191, and AE-1 were conjugated to CNBr-activated Sepharose 4B as described (Peng et al., 2016). Each 500 μl suspension contained 165 μg monoclonal antibody covalently bound to 100 μg Sepharose beads. A 500 μl suspension of each immobilized antibody was incubated with 600 to 1300 μg cell lysate on a rotating mixer for 16 h at room temperature. Beads were separated from unbound protein on a 0.45 μm PVDF spin filter. Beads were washed on the 0.45 μm filters with 0.4 mL PBS 3 times and water 3 times. Bound proteins were released from antibody beads with 2 × 200 μL of 50% acetonitrile, 0.1% formic acid. The 400 μL samples were dried and dissolved in 50 μL of 20 mM ammonium bicarbonate pH 8 in preparation for digestion with trypsin.

Monoclonal antibody A-11, specific for human AChE, was not immobilized, but was incubated directly with cell lysate in solution. The incubation mixture contained 50 μl antibody A-11 and 550 μL cell lysate (611 μg). The antibody-AChE complex was captured by addition of 100 μg Protein G agarose. The beads were washed on 0.45 μm spin filters with PBS and water. Bound proteins were released, dried, and prepared for digestion with trypsin as described above.

2.2.7. Trypsin digestion of immunopurified proteins

Immunopurified proteins in 50 μL of 20 mM ammonium bicarbonate were digested with 2.5 μL of 0.5 μg/μL Promega trypsin overnight at 37°C. The digests were acidified with 2 μL of 2.5% formic acid, a step that inactivates trypsin. Samples were centrifuged at 14,000xg for 30 min to pellet particulates before 10 μL aliquots were transferred to autosampler vials for submission to the mass spectrometer.

2.2.8. LC-MS/MS on the Orbitrap Fusion Lumos mass spectrometer.

Mass spectral data were acquired on an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) connected to a Thermo RSL Ultimate 3000 ultra high-pressure liquid chromatograph (Thermo Scientific). Five microliters of sample were loaded onto an Acclaim Pep Map 100 C18 trap column (75 μm × 2 cm; Thermo Scientific) and washed with 98% solvent A and 2% solvent B for 10 min at 4 μL/min. Solvent A was 0.1% formic acid in water. Solvent B was 70% acetonitrile and 0.1% formic. Flow was shunted onto a Thermo Easy-Spray Pep Map RSLC C18 separation column (75 μm × 50 cm; Thermo Scientific). The column was eluted at 0.3 μL/min with a two-step linear gradient: 3% B to 70% B over 50 min followed by 70% B to 99% B over 4 min. Then the column was washed with 99% B for 4 min before returning to 2% B for 19 min. Effluent from the column was sprayed directly into the mass spectrometer.

Data were acquired using information directed acquisition (IDA). A full mass spectral scan was performed from 350 to 1800 m/z. Parent ions were acquired in the Orbitrap at a resolution of 120,000 for 100 msec with the automatic gain control set to 4× 105. Up to 25 mass spectral fragmentation spectra were collected in the Orbitrap per 3 second cycle. Fragmentation was induced by high energy collision induced dissociation set to 35% normalized collision energy with a mass resolution of 30,000, a maximum ion filling time of 60 msec, and automatic gain control set to 5×104. IDA parameters included charge states from 2–6, dynamic exclusion for 30 sec with a 10-ppm mass window, positive mode, and a mass tolerance of 10 ppm.

Mass spectral data were interrogated using Batch Tag (Web)/Search Compare from Protein Prospector. Batch Tag parameters consisted of Database = User Protein; User Protein Sequence = Human BChE or Human AChE or Endoplasmic reticulum chaperone BiP; Digest = No enzyme; Max missed cleavages = 1; Constant modifications = none; Expectation calculation method = none; Max MSMS Pks = 80; Variable modifications = Oxidation (M); Precursor charge state = 2, 3, 4, and 5; Masses are = Monoisotopic; Parent Tolerance = 20 ppm; Fragment Tolerance = 30 ppm; Mass modifications = none; Crosslinking = No Link; Instrument = ESI-Q-high-res. Mass spectral fragmentation spectra shown below were taken from the Search Compare output.

3. Results

3.1. AChE and BChE activity in SH-SY5Y cells

Figure 1 compares the rates of hydrolysis of acetylthiocholine and butyrylthiocholine by live undifferentiated SH-SY5Y cells. The slope for AChE activity is 10-fold higher than the slope for BChE activity. AChE activity is 5.8 × 10−6 nmoles/min per cell; BChE activity is 0.5 × 10−6 nmoles/min per cell. Since the specific activity of human AChE is 5000 units/mg (Rosenberry and Scoggin, 1984) and that of human BChE is 500 units/mg (Schopfer et al., 2019), the protein concentration per cell is similar for catalytically active AChE and BChE proteins.

Figure 1.

Figure 1.

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Comparison of AChE and BChE activity in live undifferentiated SH-SY5Y cells. The slope for AChE activity is 10-fold higher than the slope for BChE activity. Autohydrolysis of acetylthiocholine (ATC) and butyrylthiocholine (BTC) in the absence of cells is represented by lines marked ATC and BTC.

The same assay was repeated for differentiated SH-SY5Y cells. AChE activity per cell was 1.8-fold higher in differentiated cells, whereas BChE activity was the same in differentiated and undifferentiated cells.

Das and Barone 1999 (Das and Barone, 1999) reported that BChE activity is 1.3 to 7% of AChE activity in rat PC12 cells. Ehrich and Veronesi (Ehrich and Veronesi, 1995) reported an AChE activity of 9 nmol/min per mg protein in SH-SY5Y cells and no BChE activity.

The serum free culture medium from differentiated SH-SY5Y cells had no AChE or BChE activity. It was concluded that catalytically active AChE and BChE are present in the cytoplasm of SH-SY5Y cells, but the enzymes are not secreted.

3.2. Nondenaturing gel stained for AChE activity

The gel shift assay in Figure 2 identifies the AChE in undifferentiated SH-SY5Y cell lysate as a monomer, based on a weak band of AChE activity in the cell lysate (Figure 2 lane 1) that migrates near the position of the BChE monomer. The acetylthiocholine substrate is hydrolyzed by both AChE and BChE. To definitively distinguish between AChE and BChE, cell lysate was incubated with antibodies that bind native AChE, but do not bind BChE (Dafferner et al., 2017). Monoclonals AE-1 and HR2 shifted the AChE activity band to slower migrating positions, consistent with a complex between AChE and antibody. It was concluded that the AChE in SH-SY5Y cells is a monomer. Higher molecular weight forms of AChE could be present, but at lower abundance than monomers.

Figure 2.

Figure 2.

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AChE in SH-SY5Y cell lysate is a monomer. The denaturing gel was stained for cholinesterase activity. Lane 1) 50 μL of 0.053 u/mL cell lysate. Lane 2) 50 μL cell lysate incubated with 4 μg anti-native human AChE monoclonal AE-1. Lane 3) 50 μL cell lysate incubated with 4 μg anti-native human AChE monoclonal HR2. Lane 4) 10 μl of fetal bovine serum, a marker for AChE tetramers. Lane 5) 5 μL of human plasma, a marker for BChE monomers (85 kDa), dimers(170 kDa), and tetramers (340 kDa). Bands for BChE activity are present in lane 5 because BChE hydrolyzes acetylthiocholine.

The 0.002 units of AChE activity loaded on the gel in Figure 2 lane 1 is a minimum detectable amount. The ten-fold lower BChE activity in cell lysate was not detectable on a nondenaturing gel stained for BChE activity.

3.4. Mass spectrometry results for immunopurified AChE and BChE in SH-SY5Y cell lysates

Proteins in SH-SY5Y cell lysate were immunopurified, digested with trypsin and subjected to LC-MS/MS followed by data searches using Protein Prospector software. Table 1 shows 3 peptides from native BChE and 4 peptides from unfolded BChE. Table 1 also shows 10 peptides from native AChE and 6 peptides from unfolded AChE. The results in Table 1 confirm the presence of both AChE and BChE proteins in SH-SY5Y cells. Furthermore, the results support the conclusion that catalytically active as well as unfolded, catalytically inactive forms of AChE and BChE are present in SH-SY5Y cells.

Table 1.

Human AChE and human BChE immunopurified from SH-SY5Y cell lysates

Peptide Accession Protein Antibody
EDDIIIATK P06276 BChE Anti-native BChE B2 18–5
QILVGVNK P06276 BChE Anti-native BChE B2 18–5
AILQSGSFNAPWAVTSLYEAR P06276 BChE Anti-native BChE B2 18–5
EDDIIIATK P06276 BChE Anti-denatured BChE C191
IFFPGYSEFGK P06276 BChE Anti-denatured BChE C191
AVTSLYEAR P06276 BChE Anti-denatured BChE C191
NTESTRIMTKL P06276 BChE Anti-denatured BChE C191
EDAELLVTVR P22303 AChE Anti-native AChE AE-1
VYAYVFEHR P22303 AChE Anti-native AChE AE-1
VGAFGFLALPGSR P22303 AChE Anti-native AChE AE-1
LGIPFAEPPMGPR P22303 AChE Anti-native AChE AE-1
LLSATDTLDEAER P22303 AChE Anti-native AChE AE-1
LISRAEFLAGVRVG P22303 AChE Anti-native AChE AE-1
PHGYEIEFIFGIPLDPSR P22303 AChE Anti-native AChE AE-1
LSATDTLDEAERQWKA P22303 AChE Anti-native AChE AE-1
APQWPPYTAGAQQYVSLDLRPLEVR P22303 AChE Anti-native AChE AE-1
VGVPQVSDLAAEAVVLHYTDWLHPEDPAR P22303 AChE Anti-native AChE AE-1
LFGESAGAASV P22303 AChE Anti-denatured AChE A-11
EAPGNVGLLDQR P22303 AChE Anti-denatured AChE A-11
VPQVSDLAAEAVVL P22303 AChE Anti-denatured AChE A-11
VPQVSDLAAEAVVLHY P22303 AChE Anti-denatured AChE A-11
RAQACAFWNRFLPKLLSATDTLDEAERQWKA P22303 AChE Anti-denatured AChE A-11
ALQWVQENVAAFGGDPTSVTLFGESAGAASVGMHLLSPPSR P22303 AChE Anti-denatured AChE A-11
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The unfolded protein chaperone BiP (accession number P11021) co-precipitated with immunopurified BChE and AChE proteins. This suggests that the catalytically active BChE and AChE enzymes as well as their unfolded forms are immature when they are present in the cytoplasm of SH-SY5Y cells. This observation is consistent with their sizes on Western blots, which are smaller than the mature secreted forms (see below). The smaller sizes suggest that BChE and AChE proteins located in the cytoplasm are incompletely glycosylated.

The mature human AChE tetramer has a molecular weight of 260 kDa for 583 amino acids and 3 N-glycans per subunit (P22303, residues 32–614). The mature human BChE tetramer (Leung et al., 2018) has a molecular weight of 340 kDa for 574 amino acids and 9 N-glycans per subunit (P06276, residues 29–602).

3.5. Mass spectrometry evidence for folded BChE in SH-SY5Y cells

Monoclonal antibody B2 18–5 recognizes human BChE in its catalytically active conformation and in partly unfolded conformations produced by exposure to 45°C (Peng et al., 2016). The MS/MS spectrum in Figure 3 is for BChE peptide EDDIIIATK, derived by immunopurification of BChE protein from SH-SY5Y cells with antibody B2 18–5. This spectrum and the fact that the peptide was immunoprecipitated with monoclonal B2 18–5 from SH-SY5Y cells strengthens the conclusion that SH-SY5Y cells express a low level of BChE in a native or nearly native conformation.

Figure 3.

Figure 3.

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MS/MS spectrum of human BChE peptide EDDIIIATK. The BChE protein was immunopurified from SH-SY5Y cell lysate by binding to immobilized anti-native BChE monoclonal B2 18–5. Bound protein was digested with trypsin and analyzed by LC-MS/MS. The doubly charged parent ion is at 509.30 m/z.

3.6. Mass spectrometry evidence for unfolded BChE in SH-SY5Y cells

Monoclonal antibody C191 recognizes completely denatured human BChE including 1) pure human BChE heated in a boiling water bath, 2) BChE in plasma that has been stored at desert temperatures (45°C) for 3 weeks or more, and 3) BChE in Western blots where proteins have been denatured in sodium dodecyl sulfate and dithiothreitol in a boiling water bath (Peng et al., 2016). Antibody C191 does not recognize native or partly unfolded BChE. The MS/MS spectrum in Figure 4A is for BChE peptide IFFPGVSEFGK, derived by immunopurification of BChE protein from SH-SY5Y cell lysate with antibody C191. This spectrum and the fact that the peptide was immunoprecipitated with antibody C191 strengthens the conclusion that SH-SY5Y cells express a low level of BChE in an unfolded conformation.

Figure 4.

Figure 4.

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Human BChE and BiP bound to immobilized anti-denatured BChE monoclonal C191. A) MS/MS spectrum of peptide IFFPGVSEFGK from BChE protein. The BChE protein had been captured from SH-SY5Y cell lysate on immobilized monoclonal C191. The doubly charged parent ion is at 614.32 m/z. B) MS/MS spectrum of peptide VEIIANDQGNR from BiP. The BiP had been captured from the same cell lysate on the same C191 beads as the BChE protein. The doubly charged parent ion is at 614.80 m/z.

The MS/MS spectrum in Figure 4B is for peptide VEIIANDQGNR, derived from BiP, that co-immunopurified with denatured BChE on immobilized monoclonal C191. This spectrum and the fact that the peptide was immunoprecipitated with C191 suggests that the unfolded BChE protein was in a complex with BiP, the unfolded protein chaperone.

3.7. Mass spectrometry evidence for folded AChE protein in SH-SY5Y cells

Monoclonal AE-1 recognizes human AChE in its catalytically active conformation (Dafferner et al., 2017; Fambrough et al., 1982). The MS/MS spectrum in Figure 5 is for AChE peptide APQWPPYTAGAQQYVSLDLRPLEVR, derived by immunopurification of AChE protein from SH-SY5Y cells with antibody AE-1. The presence of AChE peptides in the AE-1 immunoprecipitate is consistent with the conclusion that SH-SY5Y cells express AChE in a native conformation.

Figure 5.

Figure 5.

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MS/MS spectrum of human AChE peptide APQWPPYTAGAQQYVSLDLRPLEVR. The AChE protein was immunopurified from SH-SY5Y cell lysate by binding to immobilized anti-native AChE monoclonal AE-1. The triply charged parent ion has a mass of 952.50 m/z. Major, unlabeled red lines are internal fragments of the AChE peptide.

3.8. Mass spectrometry evidence for unfolded AChE protein in SH-SY5Y cells

Antibody A-11 was raised against amino acids 481–614 of human AChE. The A-11 antibody recognizes denatured AChE as shown in the Western blot below. The MS/MS spectrum in Figure 6A is for AChE peptide VPQVSDLAAEAVVL, immunopurified from SH-SY5Y cell lysate with antibody A-11. The MS/MS spectrum in Figure 6B is for BiP peptide IINEPTAAAIAYGLDKR. The unfolded protein chaperone BiP and AChE were co-immunopurified with antibody A-11 from SH-SY5Y cell lysate. It was concluded that unfolded AChE protein is present in the endoplasmic reticulum of SH-SY5Y cells.

Figure 6.

Figure 6.

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Human AChE and BiP bound to anti-denatured AChE antibody A-11. A) MS/MS spectrum of human AChE peptide VPQVSDLAAEAVVL. The doubly charged parent ion has a mass of 705.89 m/z. B) MS/MS spectrum of BiP. The triply charged parent ion has a mass of 606.00 m/z. Major, unlabeled red lines are internal fragments.

3.9. Western blot detects BChE in SH-SH5Y cell lysate

Monoclonal C191 was specifically made for denatured human BChE to allow detection on Western blots where proteins are reduced and denatured with sodium dodecyl sulfate in a boiling water bath. The Western blot in Figure 7 (lanes 1, 2 and 3) shows BChE bands in SH-SY5Y cell lysate at 65, 130 and 300 kDa. In contrast pure human plasma BChE (lanes 4 and 5) has bands at 85, 170, and 340 kDa. The smaller size of BChE in SH-SY5Y cell lysate is explained by incomplete glycosylation. The chemical bonds in nonreducible BChE dimers (170 kDa) and tetramers (340 kDa) have not yet been identified. BChE band intensities for 100 μg cell lysate (lanes 1 and 2) are similar to band intensities for 2 ng pure BChE (lane 5). This calculates to 24 × 10−15 moles of 85 kDa BChE in 100 μg SH-SY5Y cell lysate.

Figure 7.

Figure 7.

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Western blot hybridized with monoclonal C191 to detect human BChE in SH-SY5Y cell lysate. The bands for BChE in SH-SY5Y cell lysate are at 65, 130 and 300 kDa. The bands for pure human plasma BChE are at 85 kDa for the monomer, 170 kDa for the dimer, and 340 kDa for the tetramer. The smaller size of BChE in SH-SY5Y cell lysate suggests BChE is incompletely glycosylated.

3.9. Antibody to denatured human AChE

In Figure 8, we tested the ability of anti-AChE monoclonal antibody A-11 to detect denatured AChE in SH-SY5Y cell lysate, on Western blots. We used BChE and albumin as negative controls and recombinant human AChE as positive control.

Figure 8.

Figure 8.

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Western blots hybridized with anti-AChE antibody A-11. The A-11 monoclonal recognizes AChE in SH-SY5Y cells (lanes 1, 2, 3) and recombinant human AChE (lanes 7, 8 and 11), but does not recognize BChE (lanes 5 and 6), or human and mouse albumin (lanes 9 and 10). Recombinant human AChE has bands at 65 and 130 kDa, whereas AChE in SH-SY5Y cell lysate is slightly smaller at 60 kDa, suggesting incomplete glycosylation.

The A-11 monoclonal detects denatured AChE in SH-SY5Y cell lysate (Figure 8 lanes 1, 2, and 3) and denatured, recombinant human AChE (Figure 8 lanes 7, 8, and 11), but does not detect albumin (either human or mouse, Figure 8 lanes 9 and 10) or BChE (Figure 8 lanes 5 and 6). These factors make A-11 an attractive candidate for selectively immunopurifying denatured AChE. Recombinant human AChE has bands at 65 and 130 kDa, whereas AChE in the cell lysate is slightly smaller at 60 kDa. This suggests that AChE in the cell lysate of SH-SY5Y cells is incompletely glycosylated.

4. Discussion

4.1. Active BChE and AChE inside SH-SY5Y cells

SH-SY5Y cells do not secrete detectable quantities of catalytically active BChE and AChE. However, catalytically active BChE and AChE enzymes are present in the cytoplasm. Activity of BChE and AChE in intact cells was measured in a microtiter plate with butyrylthiocholine and acetylthiocholine, respectively. The rate of substrate hydrolysis was 10-fold higher for AChE than for BChE. Taking into consideration that the specific activity of AChE is 10-fold higher than the specific activity of BChE, we estimate that the protein concentrations of AChE and BChE are similar.

Active BChE in SH-SY5Y cell lysates was too low for detection on a nondenaturing gel stained for BChE activity. However, the higher specific activity of AChE allowed AChE activity in SH-SY5Y cell lysate to be visualized on a nondenaturing gel stained for AChE activity. It was found that intracellular AChE in SH-SY5Y cells was a monomer. Upon incubation with anti-native AChE antibodies, the AChE monomer shifted to slower migrating forms, evidence for complex formation with the antibodies. This supported the identity of the enzyme as AChE. The intracellular AChE monomer is an immature, unstable form. Stable forms of soluble AChE are tetramers (Dvir et al., 2004; Dvir et al., 2010), but AChE tetramers were not identified in the cytoplasm.

4.2. Unfolded and incompletely glycosylated BChE in cultured cells

Unfolded and incompletely glycosylated BChE is present in the endoplasmic reticulum of human SH-SY5Y cells. The following observations support this conclusion. 1) Monoclonal C191, which is specific for denatured BChE (Peng et al., 2016), co-immunopurified BChE and BiP/GRP78 from SH-SY5Y cell lysate. BiP/GRP78 is a chaperone protein in the endoplasmic reticulum that assists in folding newly synthesized proteins. Misfolded proteins in complex with BiP are translocated for degradation by the proteasome (Hetz et al., 2020; Ichhaporia and Hendershot, 2021). BiP/GRP78 is a master regulator of homeostasis in the endoplasmic reticulum, controlling the initiation of the unfolded protein response (Park et al., 2017). 2) BiP/GRP78 and BChE also co-immunopurified with the anti-native BChE antibody B2 18–5. The molecular weight of BChE (monomers and dimers) from these complexes is smaller than mature BChE suggesting they are immature. The lower molecular weight of these nascent forms of BChE is likely due to incomplete post-translational modifications that add nine N-glycans per 85 kDa subunit in the endoplasmic reticulum. The 9 N-glycans in mature BChE monomers account for 24% of its molecular weight (Lockridge et al., 1987). Such large differences in mass make it easy to distinguish between immature and mature BChE forms on a Western blot. 3) Pulse chase experiments with radiolabeled methionine and cysteine showed that 95% of the recombinant human BChE produced by CHO K1 cells was degraded inside the cell and only 5% was secreted (Altamirano et al., 2000). 4) Inactive BChE protein has been identified in the OLN-93 cell line derived from rat oligodendroglia (Robitzki et al., 2000).

4.3. Unfolded, inactive AChE in cultured cells and chicken brain

Unfolded, inactive AChE monomers and dimers have been reported in cultured muscle and brain cells. Up to 80% of newly synthesized AChE molecules are degraded inside the cell, with only 20% being processed through the Golgi apparatus and secreted (Rotundo, 1984, 2017). In chicken brain inactive AChE monomers and dimers occur under normal physiological conditions, where the inactive AChE forms represent 30% of the total (Chatel et al., 1993).

Proteasomal degradation of misfolded proteins is a universal phenomenon (Schubert et al., 2000). At least 30% of newly synthesized proteins never attain native structure despite the assistance of chaperones, glycosylating enzymes, and disulfide-forming enzymes.

Highlights.

  • Human neuroblastoma SH-SY5Y cells express AChE and BChE activity

  • Immature forms of AChE and BChE co-immunopurify with chaperone GRP78/BiP

  • Bands for AChE and BChE in SH-SY5Y cell lysates are present in Western blots

Acknowledgement

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.

Funding

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).

Abbreviations

AChE

acetylcholinesterase P22303

BChE

butyrylcholinesterase P06276

BiP

binding immunoglobulin protein

BDNF

brain derived neurotrophic factor that induces neurite outgrowth

FBS

fetal bovine serum

LC-MS/MS

liquid chromatography-tandem mass spectrometry

MS/MS

mass spectral fragmentation

OP

organophosphorus toxicant

PBS

phosphate buffered saline

Footnotes

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Conflict of interest

The authors declare no conflict of interest.

CRediT authorship contribution statement

SO - investigation, analysis, review and editing, OT – review and editing, WJ - review and editing, LMS – analysis, review and editing, OL- conceptualization, investigation, writing original draft

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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