Research Article Proline-rich chaperones are compared computationally and experimentally for their abilities to facilitate recombinant butyrylcholinesterase tetramerization in CHO cells† Qiong Wang1, Charles H. Chen2,3, Cheng-yu Chung1, Joseph Priola1, Jeffrey H. Chu1, Juechun Tang1, Martin B. Ulmschneider2,3, Michael J. Betenbaugh1 1
-Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 221 Maryland Hall, 3400 N. Charles St. Baltimore, Maryland, 21218, USA
2
-Department of Materials Science and Engineering, Johns Hopkins University, 204C Shaffer Hall, 3400 N. Charles St. Baltimore, Maryland, 21218, USA 3
-Department of Chemistry, King’s College London, Britannia House, 7 Trinity Street, London, SE1 1DB, UK. Correspondence to Michael J. Betenbaugh: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 221 Maryland Hall, 3400 N. Charles St. Baltimore, Maryland, 21218, USA,
[email protected]
†
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/biot.201700479].
This article is protected by copyright. All rights reserved Received: July 15, 2017 / Revised: September 26, 2017 / Accepted: October 10, 2017
Abstract Human butyrylcholinesterase (BChE), predominantly tetramers with a residence time of days, offers the potential to scavenge organophosphorus pesticides and chemical warfare agents.
Efficient assembly of human BChE into tetramers requires an association with proline-rich peptide
chaperones. In this study, the incorporation of different proline-rich peptide chaperones into BChE was investigated computationally and experimentally. First, we applied molecular dynamic (MD) simulations to interpret the interactions between proline-rich chaperones with human BChE
tetramer domains. The P24 chaperone which contains 24 prolines, promoted the association of BChE tetramer with a 74 % simulated helicity of BChE subunits, whereas the control without
chaperone and BChE with an 8-proline chaperone (P8) complex exhibited 55.8 % and 60.6 %
predicted helicity, respectively. The interaction of proline-rich chaperones with BChE subunits (B-P) provides a conduit to facilitate the interactions between BChE subunits (B-B) of the complex, which is mainly attributed to hydrophobic interactions and hydrogen-bond binding. Experimental
assessment of these two proline-rich chaperones plus a 14-proline chaperone (P14) was performed and confirmed that P24 has superior capability to facilitate recombinant BChE (rBChE)
tetramerization with >60 % rBChE tetramer in P24-transfected rBChE cells, whereas P14- and P8transfected rBChE cells have 44 % and 33 % rBChE tetramer respectively. The rBChE control had 14% tetramer. Finally, we developed a stable rBChE tetramer expression system in CHO cells by enriching P24 expression in rBChE expressing cells. Overall, our simulations provided a design
concept for identifying proline-rich peptides that promote the rBChE tetramerization in CHO cells. Keywords:
butyrylcholinesterase tetramerization; BChE; proline-rich chaperones; molecular dynamic simulations; Chinese hamster ovary;
1. Introduction Butyrylcholinesterase (BChE) is a serine hydrolase, which can hydrolyze cocaine, heroin,
aspirin, bambuterol and succinylcholine and can also scavenge various organophosphorus
pesticides and chemical warfare agents, making it an organophosphorus bioscavenger [1] [2] [3].
Organophosphorus agents are acutely neurotoxic because they can inhibit acetylcholinesterase
(AChE), which is essential for the control of transmitter acetylcholine (ACh) of nervous impulses. Inhibition of AChE results in excessive ACh accumulation at synapses [4], leading to
overstimulation that can cause headaches, insomnia, muscle twitches and even convulsions, coma
or respiratory failure depending on the organophosphorus agent type and the dosage [5]. Although
BChE’s exact physiological role is unclear, its substrate and inhibitor selectivity are similar to the AChE. As a result, BChE can scavenge and further act to counter administered or inhaled poisons that target AChE and other physiological targets in mammals [6].
The BChE enzyme isolated from human plasma is a tetrameric glycoprotein with each subunit comprising 574 residues (MW: 85 kDa per subunit) and nine N-linked carbohydrates with no
indication of O-linked carbohydrates [7, 8] [9]. Pharmacokinetic properties of expressed
recombinant BChE have been observed to be affected by a number of factors including the size of
the recombinant protein and its glycosylation characteristics, including sialic acid content [10, 11].
Especially important to the pharmacokinetic properties is the formation of oligomers including the full tetrameric unit [12]. Indeed, the half-life of BChE in circulation is strongly dependent on the
quaternary form of the protein. Human BChE with predominantly tetramers has a half-life of ∼46 h
in mice [10, 13, 14] and 12-14 days in human plasma [15], whereas the BChE monomer’s half-life is on the order of minutes in the circulation of mice [14, 16, 17].
The predominant form of human BChE in blood plasma, vascular system and brain is
composed of the tetrameric forms complexed with proline-rich chaperones. The tetrameric forms
of human BChE in brain have been identified as either inserted in the basal lamina of
neuromuscular junctions through the interaction of the four C-terminal peptides of BChE subunits
with the proline-rich attachment domain (PRAD) of cholinesterase-associated collagen Q (ColQ) or anchored in neural cell membranes by the PRAD of the transmembrane protein proline-rich
membrane anchor (PRiMA) [18, 19]. The difference between PRADs of ColQ and PRiMA is the
length of their proline-rich motifs (10 and 15 residues, respectively) shown in Table 1. Moreover, Li
et al extracted a series of additional proline-rich peptides from denatured human plasma BChE that
share the same proline-rich motif but varied in different peptide length (Table 1) [18]. These
chaperones, including PRAD of ColQ (P8), PRAD of PRiMA (P14) and proline-rich core sequence extracted from human BChE (P24), are observed to bind and be complexed to the C-terminal tpeptide of BChE. Unlike AChE, which exists as multiple variants in humans, human BChE is
observed as a single form [16, 20]. The major function of the t-peptides is to allow association of
BChE subunits with their proline-rich chaperones and facilitate tetramerization. The t-peptide, also called the tryptophan amphiphilic tetramerization (WAT) domain, is an α-helix located within the
last 40 C-terminal residues of human BChE, containing seven strictly conserved aromatic residues
(Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564) in which the tryptophans are
evenly spaced [21] [22].These seven aromatic residues are responsible, in part, for stabilizing
human BChE and mediating the interactions of the monomer with the proline-rich chaperones. The crystal structure of human BChE tetramer with P8 chaperone reveals a single antiparallel proline-
rich peptide surrounded by four parallel alpha-helical peptides[23].
Recombinant human BChE (rBChE) expressed from CHO cell culture is composed
principally of dimers and monomers with few tetramers, resulting in their rapid clearance in blood [24]. Subsequently, several researchers reported that coexpression of rBChE with the PRAD of
ColQ (P8) in CHO cells led to a decrease in the formation of monomers concomitant with an
increase in the percentage of tetramers [8][14]. However, the tetramer assembly catalyzed by exogenous P8 was not completely effective either in vivo or in vitro studies [8, 25].
Remarkably, previous researchers have not yet examined the effectiveness either computationally
or experimentally of the alternative proline-rich chaperones including the PRAD of PRiMA (P14)
and the proline-rich core sequence extracted from human BChE (P24) in facilitating recombinant BChE tetramer assembly in mammalian cells. In vitro studies have demonstrated that purified
recombinant BChE (65 uM) could be assembled into tetramers when incubated with synthetic 17mer or 50-mer polyproline peptides (100uM) [8]. In vivo assembly of recombinant BChE by
coexpression with proline-rich chaperones could avoid the high concentration of polyproline
peptide required for in vitro assembly. Therefore, the goal of this project is to compare
computationally and in vivo alternative proline-rich domains in order to develop a CHO cell
expression system to produce more stable recombinant BChE tetramer complexes. Here, we first
applied molecular dynamics (MD) simulations to decipher how these proline-rich chaperones
stabilize and interact with BChE tetramer complex. We found that specific proline-rich sequences exhibited superior capability to assemble BChE tetramerization due to the differences in their
amino acid sequences. Next, in order to assess the simulation predictions, we optimized the
independent expression of rBChE in CHO cells, and experimentally evaluated and compared the relative performances of proline-rich sequences -PRAD of ColQ(P8), PRAD of PRiMA (P14) and proline-rich core sequence extracted from human BChE (P24) on their capabilities to facilitate
rBChE tetramer assembly. Finally, by promoting the proline-rich chaperone expression in rBChE expressing cells, we developed a stable rBChE tetramer expression system in CHO cells.
2. Materials and Methods 2.1 Construction of expression plasmids Gene constructs, pcDNA3.1/v5-his-TOPO-huBChE, was kindly obtained from Dr. Ashima
Saxema at the Walter Reed Army Institute of Research in Silver Spring, MD. The DNA fragment encoding the open reading frame of huBChE (human BChE) was amplified by PCR using a 5’ primer
containing an EcoRV site, 5’-GCCGATATCGCCACCATGGATAGCAAAGTCACAATCA-3’ and a 3’ primer
containing a PacI site, 5’-GCCTTAATTAACTATTATCAAGACCCACACAACTTTCTTTC-3’, and the
pcDNA3.1/v5-his-TOPO-huBCHE construct as a template. The sequences of human BChE and P8,
P14 and P24 were codon optimized to fit expression in CHO cells from Biomatik. BChE-6His was constructed by fusing the C-terminus of BChE with a 6x his tag. A set of BChE combinations
including BChE, BChE-6His, codon-optimized BChE(O-BChE) and codon-optimized BChE-6His (OBChE-6His), were inserted into pcDNA3.1 vector though HindIII and BamHI separately. The same
set of BChE combinations (BChE, BChE-6His, O-BChE, O-BChE-6His) were inserted into pEF6/V5his TOPO TA vector (Addgene) through BamHI and NotI and pCHO1.0 vector (Life Technologies) through EcoRV and PacI respectively. These plasmids constructs are shown in Figure 3A.
Besides, pcDNA-(proline-rich chaperone) plasmids were constructed by inserting the
corresponding sequences (P8, P14 and P24) into pcDNA3/Hgy(+) through HindIII and BamHI sites individually. Because no P24 DNA sequence available, we designed the CHO codon optimized P24
cDNA sequence based on its amino acid sequence using a mixture of Pro codons in order to avoid using same Pro codon consistent repeatedly for p24 [18]. For p8 and p14, DNA sequences were
available but we also used a variety of codons for the Pro peptide expression in CHO cells [18, 19].
For pcDNA3.1 P24-IRES-GFP construct, codon optimized P24 sequence followed with IRES and GFP
sequences was introduced into pcDNA3.1 vector through NhelI and Xhol sites.
2.2 Cell culture and transfection Adherent CHO-K1 cell lines (Sigma-Aldrich, St. Louis, MO) were maintained in adherent
growth flasks (Sarstedt, Numbrect, Germany) in Ham’s F-12K (Life Technologies, Carlsbad, CA)
Medium supplemented with 10% fetal bovine serum, FBS (Thermo Scientific, Logan, UT), 20 mM L-
glutamine (Corning Cellgro Manassas, VA), and 1% non-essential amino acids (Invitrogen, Carlsbad,
CA). Cells were grown at 37ºC with 5% CO2 in a Series 8000 – Direct Heat and Water Jacket CO2
incubator (Thermo Scientific, Waltham, MA). For transfection, cells were seeded onto a 6-well plate
at appropriate densities in 2 ml of media each well 24 hours prior to transfection. When the cells
were at around 90% confluency, DNA constructs were transfected into cells using lipofectamine 3000 kit (Invitrogen, Carlsbad, CA) and Optimum media (Invitrogen, Carlsbad, CA), according to the
manufacturer’s instructions. At each stage, transfected stable pools were obtained based on antibiotic selection using blasticidin (Invitrogen, Carlsbad, CA) for BCHE, Zeocin (Invitrogen,
Carlsbad, CA) for P24. Stable single clones were established by seeding 0.8 cell/well in 96-well plates (Corning, Tewksbury, MA) for limited dilution. 2.3 Immunobloting
Conditioned cell media was harvested from the adherent rBChE cells, and the supernatant
was removed for analysis of secreted protein. For SDS-PAGE, the media samples were loaded onto
10% polyacrylamide gels. Proteins were separated using gel electrophoresis run at 70 V constant voltage through the stacking gel, and then 110V constant voltage through the resolving gel for 90
minutes at room temperature. Proteins were transferred from the gel to PVDF membranes (BIO-
RAD, Hercules, CA) by blotting for 75 minutes at constant 100 V. Transferred membranes were
blocked in 5% milk solution in Phosphate Buffered Saline (Corning Cellgro, Manassas, VA) with 0.1% Tween-20 (PBST) for 1 h. Membranes were washed with PBST for 10 minutes and then
incubated in a 1% milk in PBST solution containing the appropriate dilution of anti-huBChE rabbit
serum (kindly provided by Dr. Ashima Saxena) as primary antibody. Membranes were then washed
with PBST. A horseradish peroxidase-linked anti-rabbit IgG antibody (Amersham, Louisville, CO) was used as s a secondary antibody. Chemiluminescent detection of the bound antibody was
achieved using Immun-Star WesternC Chemiluminescent Kit (BIO-RAD, Hercules, CA) on the
Molecular Imager® ChemiDoc™ XRS (BIO-RAD, Hercules, CA) with Quantity One Software (BIO-
RAD, Hercules, CA).
2.4 Native gel electrophoresis For native gel electrophoresis, media samples were mixed with a native sample buffer (BIO-
RAD, Hercules, CA). Electrophoresis was at 75 V constant voltage for 1 hour and 110 V constant voltage for 3 hours at 4 ºC. Buffer conditions for running native gel electrophoresis are without SDS.
BChE activity was detected on the gel by a method developed by Karnowsky and Roots [26]. Briefly, gels were incubated in 100 mM phosphate buffer, pH 7.0, containing 5 mg of thiocholine ester, 0.5 ml of 100 mM sodium citrate, 1.0 ml of 30 mM copper sulphate, and 1.0 ml of 5 mM potassium ferricyanide in a total volume of 10 ml for 20 mins. The gel is then treated with ammonium sulphide solution for 5 mins to form brown bands on the gel. 2.5 Ellman’s activity assay
Conditioned cell media was harvested from adherent CHO-K1 cells. Reagents for Ellman’s
Assay were kindly obtained from Dr. Ashima Saxena. 10 µl of media was added in duplicate to wells
of a 96-well microtiter plate (Corning, Manassas, VA), with each well contained 240 µl of 50 mM Phosphate Buffer (PB), pH 8.0 and 1.3mM DTNB (PB-DTNB) [27]. Mixture was incubated at room
temperature for 20 minutes, and then 50 µl of 6 mM BTC in PB was added. The rate of BTC hydrolysis was read at 405 nm over 10 minutes using a SPECTRAmax PLUS plate reader (Molecular
Devices Corp., Sunnyvale, CA). Wells that contained only PB-DTNB without cell media were measured as blanks.
2.6 Fluorescence activated cell sorting (FACS) After transfecting pcDNA3.1 P24-IRES-GFP into rBChE stably expressing cell line, stable
pools were obtained based on antibiotic selections including blasticidin (Invitrogen, Carlsbad, CA) for BCHE, Zeocin (Invitrogen) for P24. And stable pools were passaged several times to ensure
appropriate expression of GFP. Then cells in the stable pool were seeded at 1x106 cells per dish for
three dishes. When the confluency was above 90%, cells were pelleted at 500xg for 15 mins, PBS
washed twice and resuspended in 1.5ml PBS in 12 x 75 mm Tube with Cell Strainer Cap (BD Falcon)
and were subjected to fluorescence activated cell sorting (BD FACSAria II). After cells with strong GFP expression were sorted, sorted cells were moved to 50 ml tube with culture media up to 40 ml,
centrifuge 2100xg for 5 mins to remove sorting buffer and culture media and then resuspended cells in a dish with antibiotic selection. Three days later, the sorted cells have more than 50%
recovery, which means a successful sorting. Passage cells for one more time, then cells were ready for further analysis.
2.7 Molecular dynamics (MD) simulations The structure of human butyrylcholinesterase tetramer with a proline-rich attachment
domain ([BChE-6His]4-(Proline peptide)) complex was based on a homology model, with the
acetylcholinesterase-associated collagen ([AChE]4-ColQ) complex serving as a template (PDB code
1VZJ: a crystal structure of soman-aged human butyrylcholinesterase in complex with the substrate analog butyrylthiocholine). In our simulations, we studied two proline-rich peptides with sequences of CCLLMPPPPPLFPPPFF (P8) and APSPPLPPPPPPPPPPPPPPPPPPPPLP (P24). We
aligned the sequence of human BChE-6His (with a 6xHis tag at the C-terminal of BChE) with
EeAChE,2 and replaced amino acids in the [AChET]4ColQ complex to [BChE-6His]4-(Proline peptide) complexes with two different proline-rich peptides (P8 and P24) and one complex without proline-
rich chaperone (Control), which aligned with the template of ColQ, using Hippo BETA simulation package (see supplemental Table S1, Table S2, and Table S3). These initial structures were relaxed
in the NPT ensemble using atomic detail Monte Carlo (MC) simulations for 200 MC steps, and water was treated implicitly using the Generalized Born theory of solvation.
After relaxation, the [BChE-6His]4-(Proline peptide) complex was setup in atomic detail
water with 100 mM Na and Cl ions at 40 ˚C. The system was neutralized by additional Cl ions and
equilibrated for 10 ns and while applying position restraints to the peptide. Multi-microsecond timescale MD simulations were performed and analyzed with GROMACS 4.6.5 and Hippo BETA simulation packages , using the CHARMM22* force field, in
conjunction with the TIP3P water model. Electrostatic interactions were computed using PME, and a cutoff of 10 Å was used for van der Waals interactions. Bonds involving hydrogen atoms were
constrained using LINCS. The integration time-step was 2 fs and neighbor lists were updated every
5 steps. All simulations were performed in the NPT ensemble without any restraints or biasing potentials. Water, and the protein were each coupled separately to a heat bath with time constant τT = 0.5 ps using velocity rescale temperature coupling. Atmospheric pressure of 1 bar was
maintained using weak semi-isotropic pressure coupling with compressibility κz = κxy = 4.6 · 10−5 bar−1 and time constant τP = 1 ps. The final simulations were analyzed by the Gromacs package.
3. Results 3.1 MD Simulations characterizing the interactions between proline-rich chaperones with BChE tetramerization domain In order to elucidate and explore the potential interactions between proline-rich sequences
and human BChE and to better characterize the tetramerization efficiency of human BChE together
with its proline-rich chaperones, we applied computational MD simulation modeling. Among the
main proline-rich sequences identified in humans (Table 1), PRAD of ColQ (P8) is the most extensively utilized and widely-studied proline-rich sequence but it contains a relatively short
proline length. Alternatively, the human plasma proline-rich core sequence (P24) contains a much longer proline sequence and has not been utilized in any in vivo coexpression studies. Studies showed that the BChE tetramerization domain is located in the last 40 amino acids (residues: 534-
574) in the C-terminus of BChE, which protrudes away from the catalytic domain of BChE (1-533)
[28] [23, 29]. Here, we constructed models of human BChE-6His tetramerization domains (residues:
534-574) with either P8 or P24 proline-rich peptides or compared BChE-6His tetramerization
against a control case of BChE-6His self-assembly without any chaperone. We studied protein-
protein interactions using unbiased all-atom MD simulations in which all protein-folding simulations reach their equilibrium states in less than 1 µs. We found the helicity of the BChE-6His tetramer contains the predominant protein-protein interactions and protein binding between the
chaperone and BChE-6His, and the models suggest that the degree of helicity is facilitated by the
interactions between chaperone and BChE-6His subunits. Indeed, the simulated helicity of [BChE6His]4 (Control; no proline-rich sequence), [BChE-6His]4-P8, and [BChE-6His]4-P24 complexes are
55.8 %, 60.6 %, and 74.0 %, respectively (Figure 1A and Figure 1B), which is correlated to the
length of the different chaperones. The detailed binding sequences within BChE-6His are shown in
Table 2. In addition, we found that the helicity of BChE-6His subunits is correlated to their binding strength between protein domains.
We assumed a two-state process between folding and
unfolding states and applied ∆Gfolding = -RT ln(Kf) with Kf = xfolding/xunfolding in order to evaluate the binding strength of each subunit residue in the [BChE-6His]4 complex (Figure 1C). BChE-6His
subunits for the Control showed 27 amino acids (residues: 539-565) with ∆Gfolding P14 >P8. It is worth considering whether non-human-originated proline-rich sequence
containing more than 24 proline residues, such as 35 contiguous proline residues P35, could
provide an even stronger association in vivo assembling than P24. Larson et al. reported that culturing rBChE using CHO cell expression with synthetic 0.1 mM 50-mer polyproline peptides for 3
days can achieve highly efficient tetramerization assembly [8]. These results suggest a longer
proline length of the chaperone, e.g. P35, may further optimize the stability of rBChE
tetramerization and extend the lifetime of the complexes.
The application of fluorescence activated cell sorting (FACS) allowed us to select higher P24 expressing rBChE cells based on GFP fluorescent levels linked with the P24 gene through internal
ribosome entry sites (IRES) (supplementary figures S1 and S2), which further promoted rBChE
tetramer association as shown in Figure 5. These results suggest that higher P24 expression resulted in increases rBChE tetramer association, which may be attributed to the proline-rich
peptide interacting with the rBChE tetramer domain and increasing subunit interactions. In addition, the development of higher rBChE-expressing cells would also be beneficial for rBChE tetramer assembly as shown in Figure 3B. Indeed, an in vitro study reported that a high
concentration of purified rBChE (65 uM) in the presence of 50-mer polyproline peptides (100 uM)
is sufficient for efficient conversion into tetramers [8]. Therefore, both proline length of proline-
rich peptides as well as the overall monomer and chaperone concentration are likely critical for efficient rBChE tetramer assembly.
Collectively, the combined simulation and experimental work demonstrates that by coexpression of
proline-rich sequences in recombinant BChE cell line, the monomers and dimers can be more effectively assembled into tetramers in vivo. The introduction of a proline-rich core sequence
extracted from human BChE (P24) into rBChE cell line can significantly promote tetramerization (>60% tetramers). Alternatively, PRAD of PRiMA (P14) and PRAD of ColQ (P8) are less effective (48% and 33%, respectively) at organizing tetramer assembly but all three provide substantial
improvement over the absence of any proline-rich sequences. In addition, the assistance of the
computational simulation of P8 and P24 chaperones with BChE tetramerization domain allowed us
to study the atomistic details of this assembly and decipher how proline-rich sequences interact
with BChE and stabilize both inter-subunit interactions and peptide-subunit interactions.
Furthermore, these simulations support the experimental observations that tetramerization and
the longer proline-rich sequence, e.g. P24, has higher capacity to enhance tetramer assembly than
P8. Surprisingly, the major impact of the longer P24 sequence was due to its effect in enhancing
inter-subunit interactions while both sequences were similarly impactful on B-P interactions.
Nonetheless, the addition of any proline-rich sequence is superior to rBChE self-assembly without
any chaperone. These MD simulation may in the future be used to help design the ideal sequence of
proline-rich chaperones or other amino acids for optimal rBChE tetramer assembly. Finally, this
study indicates that CHO cells expressing high rBChE levels together with high concentration of P24 peptide as a mediator can be a suitable recombinant BChE bioscavenger production host in the
future.
Acknowledgements We would like to acknowledge National Science Foundation (NSF) for support of this research (BES grant no. 1264802) .We thank Tania Perestrelo for her assistance with the microscopy experiments. Conflicts of Interest: The authors declare no financial or commercial conflict of interest. 5 Reference
[1] Hou, S., Xue, L., Yang, W., Fang, L., Zheng, F., Zhan, C. G., Substrate selectivity of high-activity mutants of human butyrylcholinesterase. Org Biomol Chem 2013, 11, 7477-7485. [2] Nicolet, Y., Lockridge, O., Masson, P., Fontecilla-Camps, J. C., Nachon, F., Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products. J Biol Chem 2003, 278, 4114141147. [3] Wandhammer, M., Carletti, E., Van der Schans, M., Gillon, E., Nicolet, Y., Masson, P., Goeldner, M., Noort, D., Nachon, F., Structural Study of the Complex Stereoselectivity of Human Butyrylcholinesterase for the Neurotoxic V-agents. Journal of Biological Chemistry 2011, 286, 16783-16789. [4] Delfino, R. T., Ribeiro, T. S., Figueroa-Villar, J. D., Organophosphorus compounds as chemical warfare agents: a review. Journal of the Brazilian Chemical Society 2009, 20, 407-428. [5] Munro, N., Toxicity of the Organophosphate Chemical Warfare Agents GA, GB, and VX: Implications for Public Protection. Environmental Health Perspectives 1994, 102, 18-37. [6] Ralph, E. C., Xiang, L., Cashman, J. R., Zhang, J., His-tag truncated butyrylcholinesterase as a useful construct for in vitro characterization of wild-type and variant butyrylcholinesterases. Protein expression and purification 2011, 80, 22-27. [7] Kolarich, D., Weber, A., Pabst, M., Stadlmann, J., Teschner, W., Ehrlich, H., Schwarz, H. P., Altmann, F., Glycoproteomic characterization of butyrylcholinesterase from human plasma. Proteomics 2008, 8, 254263.
[8] Larson, M. A., Lockridge, O., Hinrichs, S. H., Polyproline promotes tetramerization of recombinant human butyrylcholinesterase. Biochem J 2014, 462, 329-335. [9] Lockridge, O., Bartels, C. F., Vaughan, T. A., Wong, C. K., Norton, S. E., Johnson, L. L., Complete amino acid sequence of human serum cholinesterase. J Biol Chem 1987, 262, 549-557. [10] Saxena, A., Ashani, Y., Raveh, L., Stevenson, D., Patel, T., Doctor, B. P., Role of oligosaccharides in the pharmacokinetics of tissue-derived and genetically engineered cholinesterases. Molecular pharmacology 1998, 53, 112-122. [11] Rosenberg, Y. J., Saxena, A., Sun, W., Jiang, X., Chilukuri, N., Luo, C., Doctor, B. P., Lee, K. D., Demonstration of in vivo stability and lack of immunogenicity of a polyethyleneglycol-conjugated recombinant CHO-derived butyrylcholinesterase bioscavenger using a homologous macaque model. Chemico-biological interactions 2010, 187, 279-286. [12] Ilyushin, D. G., Smirnov, I. V., Belogurov, A. A., Jr., Dyachenko, I. A., Zharmukhamedova, T., Novozhilova, T. I., Bychikhin, E. A., Serebryakova, M. V., Kharybin, O. N., Murashev, A. N., Anikienko, K. A., Nikolaev, E. N., Ponomarenko, N. A., Genkin, D. D., Blackburn, G. M., Masson, P., Gabibov, A. G., Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo. Proceedings of the National Academy of Sciences of the United States of America 2013, 110, 1243-1248. [13] Zhou, Q., Shankara, S., Roy, A., Qiu, H., Estes, S., McVie-Wylie, A., Culm-Merdek, K., Park, A., Pan, C., Edmunds, T., Development of a simple and rapid method for producing non-fucosylated oligomannose containing antibodies with increased effector function. Biotechnol Bioeng 2008, 99, 652-665. [14] Duysen, E. G., Bartels, C. F., Lockridge, O., Wild-type and A328W mutant human butyrylcholinesterase tetramers expressed in Chinese hamster ovary cells have a 16-hour half-life in the circulation and protect mice from cocaine toxicity. Journal of Pharmacology and Experimental Therapeutics 2002, 302, 751-758. [15] Huang, Y. J., Huang, Y., Baldassarre, H., Wang, B., Lazaris, A., Leduc, M., Bilodeau, A. S., Bellemare, A., Cote, M., Herskovits, P., Touati, M., Turcotte, C., Valeanu, L., Lemee, N., Wilgus, H., Begin, I., Bhatia, B., Rao, K., Neveu, N., Brochu, E., Pierson, J., Hockley, D. K., Cerasoli, D. M., Lenz, D. E., Karatzas, C. N., Langermann, S., Recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning. Proceedings of the National Academy of Sciences of the United States of America 2007, 104, 13603-13608. [16] Blong, R. M., Bedows, E., Lockridge, O., Tetramerization domain of human butyrylcholinesterase is at the C-terminus. The Biochemical journal 1997, 327 ( Pt 3), 747-757. [17] Pan, Y. M., Gao, D. Q., Yang, W. C., Cho, H., Yang, G. F., Tai, H. H., Zhan, C. G., Computational redesign of human butyrylcholinesterase for anticocaine medication. Proceedings of the National Academy of Sciences of the United States of America 2005, 102, 16656-16661. [18] Li, H., Schopfer, L. M., Masson, P., Lockridge, O., Lamellipodin proline rich peptides associated with native plasma butyrylcholinesterase tetramers. Biochem J 2008, 411, 425-432. [19] Noureddine, H., Carvalho, S., Schmitt, C., Massoulie, J., Bon, S., Acetylcholinesterase associates differently with its anchoring proteins ColQ and PRiMA. J Biol Chem 2008, 283, 20722-20732. [20] Liang, D., Blouet, J. P., Borrega, F., Bon, S., Massoulie, J., Respective roles of the catalytic domains and C-terminal tail peptides in the oligomerization and secretory trafficking of human acetylcholinesterase and butyrylcholinesterase. The FEBS journal 2009, 276, 94-108. [21] Bon, S., Dufourcq, J., Leroy, J., Cornut, I., Massoulie, J., The C-terminal t peptide of acetylcholinesterase forms an alpha helix that supports homomeric and heteromeric interactions. European journal of biochemistry / FEBS 2004, 271, 33-47. [22] Altamirano, C. V., Lockridge, O., Conserved aromatic residues of the C-terminus of human butyrylcholinesterase mediate the association of tetramers. Biochemistry 1999, 38, 13414-13422.
[23] Pan, Y., Muzyka, J. L., Zhan, C.-G., Model of Human Butyrylcholinesterase Tetramer by Homology Modeling and Dynamics Simulation. The Journal of Physical Chemistry B 2009, 113, 6543-6552. [24] Lockridge, O., Review of human butyrylcholinesterase structure, function, genetic variants, history of use in the clinic, and potential therapeutic uses. Pharmacology & therapeutics 2015, 148, 34-46. [25] Duysen, E. G., Bartels, C. F., Lockridge, O., Wild-type and A328W mutant human butyrylcholinesterase tetramers expressed in Chinese hamster ovary cells have a 16-hour half-life in the circulation and protect mice from cocaine toxicity. The Journal of pharmacology and experimental therapeutics 2002, 302, 751-758. [26] Karnovsky, M. J., Roots, L., A "DIRECT-COLORING" THIOCHOLINE METHOD FOR CHOLINESTERASES. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 1964, 12, 219-221. [27] Ellman, G. L., Courtney, K. D., Andres, V., Jr., Feather-Stone, R. M., A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical pharmacology 1961, 7, 88-95. [28] Dvir, H., Harel, M., Bon, S., Liu, W. Q., Vidal, M., Garbay, C., Sussman, J. L., Massoulie, J., Silman, I., The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix. Embo j 2004, 23, 4394-4405. [29] Altamirano, C. V., Lockridge, O., Conserved Aromatic Residues of the C-Terminus of Human Butyrylcholinesterase Mediate the Association of Tetramers. Biochemistry 1999, 38, 13414-13422. [30] Huang, Y. J., Lundy, P. M., Lazaris, A., Huang, Y., Baldassarre, H., Wang, B., Turcotte, C., Cote, M., Bellemare, A., Bilodeau, A. S., Brouillard, S., Touati, M., Herskovits, P., Begin, I., Neveu, N., Brochu, E., Pierson, J., Hockley, D. K., Cerasoli, D. M., Lenz, D. E., Wilgus, H., Karatzas, C. N., Langermann, S., Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin. BMC biotechnology 2008, 8, 50. [31] Bouabe, H., Fassler, R., Heesemann, J., Improvement of reporter activity by IRES-mediated polycistronic reporter system. Nucleic Acids Res 2008, 36, e28. [32] Larson, M. A., Lockridge, O., Hinrichs, S. H., Polyproline promotes tetramerization of recombinant human butyrylcholinesterase. Biochemical Journal 2014, 462, 329-335. [33] Schopfer, L. M., Lockridge, O., Tetramer-organizing polyproline-rich peptides differ in CHO cellexpressed and plasma-derived human butyrylcholinesterase tetramers. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2016, 1864, 706-714.
Table legends Table 1 Three main BChE tetramer-associating chaperones identified in humans Table 2 Protein-binding sequences of [BChE-6His]4-(Proline peptide) complexes and [BChE-6His]4 Control complex. The result showed that proline-rich peptide can stabilize the BChE-6His tetramer and expand the protein binding domain of the BChE-6His subunits. Longer P24 peptide includes higher overall helicity and the overall helicity corresponds to the protein-protein binding. The binding sequences were determined by the helicity per residue that has more than 50 % helical fraction content. The additional binding residues of P8 and P24 to the control are shown as bold red. The aromatic residues are highlighted using underline.
Table 3 Quantification of rBChE monomer and oligomer distribution by determining band intensity using ImageJ analysis (n>3). Table 4 BChE activity assay of sorted and unsorted rBChE-P24 stable pools (n>3). Cells from sorted and unsorted rBChE-P24 stable pools, rBChE expressing cells and control CHOK1 cells were seeded at same density. After a 3-day incubation, equal volumes of conditioned media were subjected to Ellman’s assay to determine BChE activity.
Figure legends Figure 1 Model of [BChE-6His]4-(Proline peptide) complexes with 100 mM NaCl in TIP3P water model. (A) Top view and side view of [BChE-6Hhis]4 associated proline-rich peptides (Control, P8, and P24) from the microsecond all-atom MD simulations, and their helicity of [BChE-6His]4 complexes at the equilibrium state (average of the last 100 ns). The four BChE-6His subunits are denoted as color blue, purple, green and gray. Proline-rich chaperones (P8 or P24) are shown as red at the center of the BChE-6His tetramer. Polar and non-polar residues are yellow and light blue, respectively. (B) Overall helicity of [BChE-6His]4 complex against the timeline in MD simulations. (C) Protein-protein binding free energy of amino acid of BChE-6His subunits between sequences 534-574 [9]. The negative values mean the protein binding is more favorable. Figure 2 Number of H-bond formation during microsecond MD simulations between (A) separated BChE-6His subunits (B-B), (B) BChE-6His subunits and proline-rich peptide (B-P). Aromatic residues of BChE-6His subunits (Trp543, Trp550, Trp557, and Tyr564) that form hydrogen bonds with (C) P8 and (D) P24 are presented. Figure 3 Assessment of recombinant BChE expression levels under various promoters, gene codon optimization and 6xhis-tag conditions. (A) Expression plasmids used in developing an efficient rBChE system. Inserts included human BChE, human BChE with a his tag at C-terminal (BChE-6His), Codon-optimized human BChE(O-BChE) and codon-optimized human BChE-6His (O-BChE-6His) respectively (B) Ellman’s assay analysis of rBChE activity from 12 transfectants, CMV: pcDNA3.1 vector with CMV promotor, labeled in blue, EF1α: pEF6 vector with EF1α promotor, labeled in orange, pCHO: pCHO vector with CMV/ EF-1α hybrid promoter, labeled in green, CHOK1 cells were used as negative control, labeled in grey; (C) Western Blot detection of rBChE expression from pEF6/EF/O-BChE-6His stable cell line Figure 4 Assembly state of rBChE using different proline-rich chaperones. Lane1, rBChE from rBChE producing CHO cell line as negative control; Lane2, purified human BChE from plasma; Lane3-5, transient expression P8, P14 and P24 sequences on rBChE-producing CHO cells respectively; Lane6, transient expression of empty pcDNA3.1 vector on rBChE producing CHO cells
Figure 5 Analysis of rBChE tetramer enrichment for sorted and unsorted rBChE-P24 cells. P24-IRES-GFP was overexpressed in rBChE cells. The rBChE-P24 cells were subjected to fluorescence-activated cell sorting (FACS) and analyzed by native gel electrophoresis. Lane 1, purified human BChE from plasma; Lane2, supernatant from sorted rBChE-P24 stable pool; Lane 3, supernatant from unsorted rBChE-P24 stable pool, Lane 4, supernatant from rBChE expressing cells; Lane 5, supernatant from CHOK1 cells.
Table 1. Three main BChE tetramer-associating chaperones identified in humans
Table 2. Protein-binding sequences of [BChE-6His]4-(Proline peptide) complexes and [BChE-6His]4 Control complex.
Table 3. Quantification of rBChE monomer and oligomer distribution
Table 4. BChE activity assay of sorted and unsorted rBChE-P24 stable pools (n>3).
