Engineering of a monomeric and low-glycosylated form
of human butyrylcholinesterase
Expression, puri®cation, characterization and crystallization
Florian Nachon
1
, Yvain Nicolet
2
, Nathalie Viguie
Â
1
, Patrick Masson
1
, Juan C. Fontecilla-Camps
2
and Oksana Lockridge
3
1
Centre de Recherches du Service de Sante
Â
des Arme
Â
es, Unite
Â
d'Enzymologie, La Tronche, France;
2
Laboratoire de Cristallographie
et Cristalloge
Â
ne
Á
se des Prote

3
per
Da (estimated 60% solvent) for a single molecule of
recombinant BChE i n the asymmetric unit. The crystal
structure of butyrylcholinesterase will h elp elucidate
unsolved issues concerning cholinesterase mechanisms in
general.
Keywords: butyrylcholinesterase; crystallization; N-glycosy-
lation; site-directed mutagenesis; X-ray diraction.
Acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcho-
linesterase (BChE; EC 3.1.1.8) are closely related serine
hydrolases with different s ubstrate speci®city and inhibitor
sensitivity. AChE terminates the action of the neurotrans-
mitter acetylcholine at postsynaptic membranes and neuro-
muscular junctions. Altho ugh BChE i s found in various
vertebrate tissues (liver, intestine, lung, heart, muscle, brain,
serum), its physiological role remains undetermined. How-
ever, plasma BChE is o f pharmacological and t oxicological
importance because it hydrolyzes ester-containing dru gs
such as succinylcholine and cocaine. Consequently, puri®ed
BChE has been used for treatment of succinylcholine-
induced apnea in humans [1] and it is known to protect
rodents from the toxic effects of cocaine [2,3]. To improve
the rate of hydrolysis of cocaine, a mutated enzyme has been
designed [4]. However, a h igher catalytic rate may be
necessary if BC hE is to be used therapeutically in severe
cocaine overdoses.
Human BChE is also kno wn to be a good scavenger of
organophosphorus (OP) pesticides and chemical warfare
nerve agents [5]. For example, injections of puri®ed BChE as

Â
es, Unite
Â
d'enzymologie, 24 Avenue des Maquis du
Gre
Â
sivaudan, BP 87±38702 La Tronche Ce
Â
dex, France.
Fax:+33476636961,Tel.:+33476636988,
E-mail: ¯
Abbreviations: A C hE, acetylcholinesterase; BChE, butyrylcholinest-
erase, CCD, charge coupled device; ChE, cholinesterase; C HO, Chi-
nese hamster ovary; DMEM, Dulbecco's modi®ed Eagle's medium;
Nbs
2
,5,5¢-dithiobis-2-nitrobenzoic acid; HEK, human embryonic
kidney cells; OP, organophosphorus ester.
(Received 6 A ugust 2 001, revised 19 November 2 001, accepted 20
November 2001)
Eur. J. Biochem. 269, 630±637 (2002) Ó FEBS 2002
During the past decade, the crystallization of puri®ed
plasma BChE has not been successful, despite an exhaus-
tive screening program in one of our laboratories. Human
BChE is a heavily glycosylated homotetramer of 340 kDa
with nine N-glycosylation sites per catalytic subunit
representing almost 25% of its m ass [17,18]. It i s known
that the glycan m oieties o ften perturb crystallization
[19,20]. Human BChE o ligosaccharides, which are o f the
complex biantennary type [21,22], could shield the protein

Plasmid p GS has the CMV p romoter and rat glutamine
synthetase for selection.
Other mutants from which carbohydrate a ttachment sites
were deleted were also constucted by PCR. In each case, a
codon for Asn was replaced by a c odon for Gln. The
expression plasmid pGS was suitable for both transient and
stable expression.
Transient expression
BChE mutants w ere t ransiently express ed i n human
embryonic kidney cell line 293T/17, used with permission
from D. Baltimore (Rockefeller University of New York;
ATCC No CRL 11268). Cells were grown to 80±90%
con¯uence i n 100 mm dishes and then transfected by
calcium phosphate co-precipitation of 20 lg plasmid DNA
per dish. Four days after transfection, the culture medium
[5% f etal bovine serum in Dulbecco's modi®ed Eagle's
medium (DMEM)] was harvested for a BChE activity
assay. Each mutant BChE was transfected into ®ve dishes.
Large scale production of recombinant human BChE
4sugOff
17/455/481/486
BChE
D
inpGSwasexpressedinCHO
cells and stably transfected as previously described [11].
Selective p ressure to retain the plasmid was provided by
25 l
M
methionine sulfoximine. Secreted BChE was collected
into serum-free and glutamine-free culture medium, Ultra-

were conducted at 4 °C.
Serum-free culture medium was collected from roller
bottles over a period o f 6 months. Twenty-six liters of
culture medium containing 100 mg of 4sugOff
17/455/481/486
BChE
D
were loaded onto 400 mL o f procainamide±
Sepharose p acked in a XK50/30 Pharmacia column
(diameter, 5 cm; ¯ow rate of 1 Láh
)1
). The column was
washed with 20 m
M
potassium phosphate, pH 7.0, 1 m
M
EDTA (until D
280
 0)andthenwith0.1,0.2and0.3
M
NaCl in buffer. The BChE activity was eluted with buffer
containing 0.3
M
NaCl and 0.1
M
N(Me)
4
Br. The eluted
enzyme was 21% pure as judged from speci®c activity.
Then, the 4sugOff

NaCl in 20 m
M
Tris/HCl pH 7.4, then
dialyzed against 5 m
M
Mes pH 6.5 and concentrated to
10 mg ámL
)1
(7200 UámL
)1
) in an Amicon Dia¯o appa-
ratus with a PM10 membrane. The dialyzed, concentrated
sample was ®ltered through a 0.2- lm®lterandstoredat4 °C.
Determination of kinetic parameters
Hydrolysis of butyrylthiocholine iodide at 25 °Cwas
measured at concentrations ranging from 0.010 to 50 m
M
according to the method of Ellman [27]. The buffer w as
0.1
M
sodium phosphate at pH 7.0 and contained
0.1 mgámL
)1
Nbs
2
and 0.1% BSA. The active sites
were titrated by the method of residual activity u sing
diisopropyl phosphoro ¯uoridate (DFP) as titrant [28].
Kinetic parameters (k
cat

Catalytic activity in the crystals
A recombinant BChE crystal grown at pH 6.5 was washed
twice for 5 min in a 100 lLdropof0.1
M
Mes pH 6.5 buffer
containing 2.4
M
(NH
4
)
2
SO
4
. Then the crystal was soaked in
a 20-lL drop of the same buffer containing 0.1 mgámL
)1
Nbs
2
and 5 m
M
butyrylthiocholine iodide (Sigma). The
change in crystal coloration (turning yellow) was followed
under a binocular magnifying glass. No spontaneous
hydrolysis of the substrate i n t he soaking liquor was
observed when monitored by spectrophotometry at
412 nm.
Data collection
Diffraction data were collected at k  0.932 A
Ê
wavelength

crystallization of BChE by designing an enzyme with the
fewest possible glycosylation sites, while preserving its
solubility, stability a nd functional properties. Amino-acid
sequences of AChE and BChE from different species were
aligned to pinpoint the conserved N-glycosylation sites
(Table 1). BChEs are generally more glycosylated than
AChEs. AChEs from different species contain three to six
N-glycosylation s ites, t hree of which are conserved in
BChE. Therefore, our ®rst attempt w as to construct a
recombinant BChE containing only these three glycosyla-
tion sites (positions 256, 341 and 455). This was achieved by
mutating six Asn residues in Asn-X-Ser/Thr recognition
sites into Gln residues.
These studies overlooked the possibility that a muta-
tion of Asn486 might unmask a glycosylation site at
Asn485. Three gly cosylation r ecognition s ites are present
in the sequence N
481
ETQNNSTS
489
, but the peptide
sequencing of human BChE showed that positions 481
and 486 were glyc osylated, and position 485 was not [18].
Because Asn485 and Asn486 are adjacent, the nongly-
cosylation of Asn485 may be due to steric hindrance.
Therefore, w e assume that all of th e constructs with the
double m utation N 481Q/N486Q should be glycosylated
at position 485.
Table 1. C omparison of the N-glycosylation positions for various cholinesterases.
Enzyme

nine sites off) due to retention of the protein inside the cell,
as shown by Western blotting. As the expression level of
these c lones w as not high enough to p roduce large amounts
of BChE, new constructs were tested in which the
N-glycosylation sites were suppressed empirically.
Suppression of sites N481 and N486 (Table 2, two s ites
off) led to 45% higher expression levels than native BChE.
Suppression of sites N455, N481 and 486 led to a 15%
greater expression level than the native enzyme (Table 2,
three s ites off). When an additional site was suppressed at
position N256, the expression level was similar to t hat of the
native enzyme (Table 2, four sites off; oligomeric domain:
Ôyes Õ). The additional N 341Q mutation resulted in a ®vefold
lower active enzyme (Tables 2, ®ve sites off). Consequently,
the ® ve glycosylation sites mutant was not used any further.
Interestingly, the N341 s ite is also conserved i n Candida
rugosa lipase, where it plays an important role in the
stabilization of the open con formation of the enzyme [37].
Such a role has not yet been observed in cholinesterases.
The tetramerization domain is located at the C-termini of
AChE and B ChE. In human BChE, this domain comprises
40 amino acids, e ncoded by exon 4. I ts deletion leads to
higher levels of secretion into the culture medium and
expression of monomers [ 25]. Crystallization o f monomeric
cholinesterases is more favorable t han for oligomeric forms,
even if they form a noncovalent dimer by association of a
four-helix bundle ( helices 383±372 and 526±543; human
Table 2. I n¯uence of the number and position of N-glycosylation sites on the expression level of secreted human BChE. The presence or absence of the
oligomerization domain at the C-terminus is indicated by yes or no. Transient transfection in 293T cells was repeated in ®ve dishes. The r elative
expression unit corresponds to 0 .2 lmol butyrylthiocholine hydrolyzed p er minute.

BChE
D
, human BChE, and crystallized forms of human AChE, mouse AChE and
Torpedo c alifornica AChE. 4sugO
17/455/481/486
BChE
D
(Rec. B ChE), human B ChE [18], human AChE [44], mouse AChE [38] an d T. californica
AChE (Torc a AChE) [45] w ere aligned using
CLUSTALW
. Asterisks denote identity, and full s tops show high similarity.
Ó FEBS 2002 Butyrylcholinesterase designed for crystallization (Eur. J. Biochem. 269) 633
AChE numbering) under the protein concentrations used
for crystallization [31,38]. Therefore, a truncated BChE
lacking both the tetramerization domain and the N256,
N455, N481 and N 486 N-glycosylation sites was c on-
structed. Deletion of the tetramerization domain was
achieved by introducing a stop codon at position 530
according to Blong et al.[25].Asexpected,activity
measured in culture media was about sevenfold higher for
the monomeric form (BChE
D
) than for the o ligomeric form
(Tables 2, four sites off; oligomeric domain: ÔnoÕ). In
another effort, a second truncated clone also lacking four
N-glycosylation sites (N17, N455, N481 and N486) was
constructed. The activity level of this enzyme was slightly
lower than t hat of t he previous clone but suf®ciently h igh to
produce signi®cant amounts of enzyme. Consequently, this
clone (4sugOff

D
were selected for large-scale
production. Puri®cation was carried out by anion-exchange
and af®nity chromatography. Axelsen et al. reported that
decamethonium, used during the last af®nity chromatogra-
phy step of T. californica AChE, was present in t he crystals
despite extensive dialysis of the puri®ed enzyme [39]. Thus,
to avoid contamination by a ligand, NaCl was used for
elution of BChE f rom af®nity chromatography gels. T he
purity of the ®nal enzyme preparation was estimated to be
greater than 98% based on its speci®c activity and the
presence of a single band on SDS/PAGE.
Characterization of 4sugOff
17/455/481/486
BChE
D
The kinetics of butyrylthiocholine hydrolysis by recombi-
nant BChE under standard conditions (0.1
M
phosphate
buffer, pH 7.0) can b e d escribed by the model of Radic
[29]. The kinetic parameters are very close to the values
reported previously for the native BChE [40], with
k
cat
 28 000 min
)1
and K
m
 25.6  0.4 l

BChE
D
still
displays a signi®cant glycosylation-related heterogeneity.
This issue was addressed using IEF analysis. The carbohy-
drate chains of BChE are partly capped by sialic acids [22],
which directly in¯uence the pI of the enzyme. Whereas
plasma BChE displayed a continuous smear e xtending from
pH 4.0 t o 5.7, thus re¯ecting h igh sialylation h eterogeneity,
4sugOff
17/455/481/486
BChE
D
displayed 10 well-resolved
bands between p H 5.0 and 6.5 (Fig. 2B). T his was a de®nite
improvement of the enzyme homogeneity, and encouraged
us to start BChE crystallization trials.
Crystallization of 4sugOff
17/455/481/486
BChE
D
and data collection
Initial crystallization conditions were screened according to
Jancarick & Kim [30] using the hanging drop method.
Tetragonal crystals appeared within 1 week in a pH 6.5
0.1
M
Mes buffer solution containing 2.1
M
(NH

2
and the product of the Ellman's reaction
may easily diffuse in a short period of time inside and
outside the crystals. However w e cannot rule out the
possibility that t he substrate might have been hydrolyzed by
the protein located in the crystal surface, which is likely to
solubilize during the soaking experiment.
The crystals that m easured up to 0 .3 mm in their longest
dimension diffracted to 2.2±2.3 A
Ê
resolution at 100 K, using
15% glycerol (v/v) as a cryoprotectant, and synchrotron
radiation at the ESRF ID14-eh1 beamline. As recrystalli-
zation improved the quality of human AChE crystals [31],
we reproduced the procedure by transferring crystal-
containing drops over reservoirs of water until the crystals
dissolved. The drops were then placed over the original
reservoir solution, or a solution with slightly lower precip-
itant concentration, for r ecrystallization. As reported for
human AChE, these new crystals were fewer but larger with
longest dimensions of up to 0.6 mm. They diffracted to
2.0 A
Ê
at 100 K, using 15% glycerol (v/v) as a cryoprotec-
tant, and synchrotron radiation at the E SRF ID14-eh2
beamline. Analysis of the c ollected data (Table 3) indicated
that BChE crystals belong to the tetragonal space group
I422 with unit cell dimensions a  b  154.7 A
Ê
,

)
2
SO
4
as the p recipitant. A fter their quality was
improved by recrystallization, they diffracted to 2.0 A
Ê
resolution. The ®rst three-dimensional structure of a
butyrylcholinesterase is expected to improve our knowledge
regarding ChE mechanism, such as allosteric modulation,
product clearance outside th e active site go rge and motion
of water molecules. Moreover, the three-dimensional struc-
ture of human BChE sho uld provide a template for the
design of new mutants capable of hydrolyzing nerve agents
and drugs such as cocaine with increased ef®ciency.
ACKNOWLEDGEMENTS
This work was supported by the US Army Medical Research and
Materiel Command under contract DAMD 17-97-1-7349 to O. L. and
Fig. 3. Te tragonal crystals of 4sugO
17/455/481/486
BChE
D
. (A) The
larger crystal has dimension of 0.5 ´ 0.5 ´ 0.3 mm
3
. (B) Crystal after a
10-min soaking in Ellman's buer with precipitant and 5 mM butyryl-
thiocholine.
Table 3. D ata collection and processing. Values fo r the high est reso-
lution shell a re giv en in parentheses.

Â
gation Ge
Â
ne
Â
rale de l'Armement under contract DGA/DSP/
STTC-PEA 990802/99 CO 029 (ODCA, Washington, DC, 00-2-032-0-
00) to P. M. W e thank, respectively, Hassan Belrhali and Jo anne
McCarthy for the opportunity to collect data at the ID14-eh1 and
ID14-eh2 be amline at the ESRF in G renoble.
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