Our aim was to obtain satisfactory amounts of the ECDs of the non-a sub-units of human muscle AChR for use as starting material for the determin-ation of the 3D structure of the receptor
Trang 1extracellular domains of the b, c and e subunits of the
human muscle acetylcholine receptor
Kalliopi Kostelidou1, Nikolaos Trakas1, Marios Zouridakis1,2, Kalliopi Bitzopoulou1,2,
Alexandros Sotiriadis1, Ira Gavra1,2and Socrates J Tzartos1,2
1 Department of Biochemistry, Hellenic Pasteur Institute, Athens, Greece
2 Department of Pharmacy, University of Patras, Greece
The nicotinic acetylcholine receptor (AChR) is a
mem-ber of the superfamily of ligand-gated ion channels,
which also includes the glycine, c-aminobutyric acid A,
and 5-HT3 receptors [1] Its physiological role is to
mediate the fast chemical transmission of electrical
signals in response to acetylcholine released from the nerve terminal to the end-plate
The muscle AChR is a transmembrane glycoprotein ( 290 kDa) located on the postsynaptic membrane of the neuromuscular junction and is composed of five
Keywords
acetylcholine receptor; extracellular domain;
myasthenia gravis; protein expression
Correspondence
S J Tzartos, Department of Biochemistry,
Hellenic Pasteur Institute, GR11521 Athens,
Greece
Fax: +30 210 6478842
Tel: +30 210 6478844 or +30 2610 969955
E-mail: tzartos@mail.pasteur.gr,
tzartos@upatras.gr
(Received 29 March 2006, revised 25 May
2006, accepted 7 June 2006)
doi:10.1111/j.1742-4658.2006.05363.x
The nicotinic acetylcholine receptor (AChR) is a ligand-gated ion channel found in muscles and neurons Muscle AChR, formed by five homologous subunits (a2bcd or a2bce), is the major antigen in the autoimmune disease, myasthenia gravis (MG), in which pathogenic autoantibodies bind to, and inactivate, the AChR The extracellular domain (ECD) of the human mus-cle a subunit has been heterologously expressed and extensively studied Our aim was to obtain satisfactory amounts of the ECDs of the non-a sub-units of human muscle AChR for use as starting material for the determin-ation of the 3D structure of the receptor ECDs and for the characterizdetermin-ation
of the specificities of antibodies in sera from patients with MG We expressed the N-terminal ECDs of the b (amino acids 1–221; b1–221), c (amino acids 1–218; c1–218), and e (amino acids 1–219; e1–219) subunits of human muscle AChR in the yeast, Pichia pastoris b1–221 was expressed at
2 mgÆL)1 culture, whereas c1–218 and e1–219 were expressed at 0.3– 0.8 mgÆL)1 culture All three recombinant polypeptides were glycosylated and soluble; b1–221 was mainly in an apparently dimeric form, whereas c1–218 and e1–219 formed soluble oligomers CD studies of b1–221 sugges-ted that it has considerable b-sheet secondary structure with a proportion
of a-helix Conformation-dependent mAbs against the ECDs of the b or c subunits specifically recognized b1–221 or c1–218, respectively, and poly-clonal rabbit antiserum raised against purified b1–221 bound to125I-labeled a-bungarotoxin-labeled human AChR Moreover, immobilization of each ECD on Sepharose beads and incubation of the ECD–Sepharose matrices with MG sera caused a significant reduction in the concentrations of auto-antibodies in the sera, showing specific binding to the recombinant ECDs These results suggest that the expressed proteins present some near-native conformational features and are thus suitable for our purposes
Abbreviations
AChR, nicotinic acetylcholine receptor; ECD, extracellular domain; MG, myasthenia gravis; b1–221, amino acids 1–221 of the human AChR b subunit; c1–218, amino acids 1–218 of the human AChR c subunit; e1–219, amino acids 1–219 of the human AChR e subunit.
Trang 2homologous subunits in the stoichiometry a2bcd
(embryonic muscle) or a2bed (adult muscle), with the
subunits arranged around a central ion pore [2,3]
Each mature subunit (after cleavage of the signal
peptide) consists of three domains, an extracellular
domain (ECD) (210–220 residues), a
membrane-span-ning domain, and an intracellular domain [3] The
N-terminal ECD of each of the two a subunits
con-tains the major part of the binding site for the
cho-linergic ligands The two sites are nonequivalent, one
being formed at the interface between one a subunit
and the c⁄ e subunits and the other between the second
a subunit and the d subunit [4] The c⁄ e and d subunits
play a major role in shaping the ligand-binding sites
and also in maintaining cooperative interactions
between the a subunits [5–7] The b subunit is an
important determinant in receptor localization, as
shown by studies on the properties of hybrid muscle
AChRs, in which the muscle b subunit was replaced
by its neuronal counterpart [8]
In addition to its physiological function, the muscle
AChR is involved in the pathology of the autoimmune
disease, myasthenia gravis (MG), being the main
anti-gen against which MG autoantibodies are produced
These autoantibodies bind to AChR molecules at the
neuromuscular junction, leading to their loss and
the weakness and fatigability of the voluntary muscles,
the main symptoms of MG [9] A proportion of
patients lacking autoantibodies against the AChR
har-bors antibodies against the muscle-specific kinase,
MuSK [10]
The pathophysiological importance of the AChR
necessitates the solution of its 3D structure Current
knowledge of its structure is mainly based on data
from electron images of the AChR found in large
amounts in the electric organ of the marine ray,
Torpedo californica[3] The acquisition of the
crystallo-graphic structure of the mollusc acetylcholine-binding
protein [11] has provided an insight into the
ligand-binding domain of nicotinic receptors However, the
fact that this protein is most closely related to the a7
subunit of the neuronal AChR (24% identity of amino
acids) than each of the muscle AChR subunits (22%
on average) necessitates the solution of the structure of
the mammalian AChR molecule A prerequisite for
this is the availability of large amounts of native,
sol-uble AChR molecules, a target that can be partially
achieved by expression of the ECDs of the AChR
sub-units in heterologous expression systems Several
stud-ies have been carried out on the expression of the
muscle-type a subunit ECD in bacterial systems, in
which the protein is expressed in large amounts, but is
unglycosylated and forms inclusion bodies, requiring
refolding to allow partial renaturation [12–14] Other studies involved the expression of different subunits (whole subunits or ECDs) in mammalian systems, in which the protein has the correct structure, but is only produced in limited amounts because of the inherent difficulty in scaling up expression in cell culture or oocytes [15,16]
In this report, we present the expression and charac-terization of the ECDs of the b, c and e subunits of the human muscle AChR We describe their expression
in a soluble, glycosylated form and in satisfactory amounts using the yeast Pichia pastoris expression sys-tem, which combines the speed of bacterial systems with the advantages of eukaryotic expression systems (e.g post-translational modification) and which had been successfully used in the past by our group to express the ECDs of human muscle a1 [17] and human neuronal a7 [18] CD analysis of amino acids 1–221 of the human AChR b subunit (b1–221) showed that the protein has a b-structure with a contribution from a-helices Two conformation-dependent mAbs (one anti-b and one anti-c) specifically bound to their cog-nate ECDs, whereas autoantibodies in MG sera, the binding of which is highly conformation-dependent [19,20], bound to all three ECDs
As all three ECDs were expressed in satisfactory amounts and were recognized by human MG autoanti-bodies, they may be suitable as starting material for preliminary biophysical and structural studies and for the study of MG
Results
Rationale for the construction and testing
of AChR ECD variants N-Terminal addition of the FLAG peptide (DY-KDDDDK) or addition of the first transmembrane amino acid of the mouse muscle a subunit, a proline, which is conserved in human AChR subunits, results in higher expression of the mouse muscle a ECD [21] To test the effect of these additional epitopes⁄ tags on the yield of the present proteins, we constructed a set of eight human c ECD variants (c, amino acids 1–218) with or without a proline at position 219 and⁄ or the FLAG epitope and⁄ or a 6-His tag (6-HIS) (Fig 1A)
We then performed small-scale cultures for each pro-tein and quantified the amounts of expressed propro-tein in the culture supernatant using dot-blots and a series of supernatant dilutions Expression varied depending on the presence of the different modifications (Fig 1B) The yield of amino acids 1–218 of the human AChR c subunit (c1–218) without additional tags was taken as
Trang 3the 100% reference ( 0.3 mgÆL)1, see below)
Addi-tion of the proline residue had no significant effect on
expression (less than 10%) The presence of the FLAG
tag increased expression of c1–218 by almost 20%, but
did not improve expression of c1–219 The presence of
the HIS tag alone reduced the expression of both
con-structs by 30–40% (Fig 1B, bars 2 and 6), and further
addition of FLAG to c1–218HIS gave 100% expression
(construct FLAG⁄ c1–218HIS, bar 4) Strangely, when
both epitopes were present on c1–219, no expression
was observed (Fig 1B, lane 8) We purified two c ECD
variants, FLAG⁄ c1–218HIS and c1–219HIS from 1-L
cultures, obtaining 0.3 mgÆL)1 and 0.2 mgÆL)1
pro-tein, respectively We then constructed b1–221HIS and
FLAG⁄ b1–221HIS and expressed, purified and
quanti-fied them using 2-L cultures The results showed that
expression was increased threefold when the protein
carried the FLAG tag (2 mgÆL)1 protein instead of
0.7 mgÆL)1)
Expression and purification of the b, c and e ECDs
As (a) the presence of the HIS tail on the constructs
greatly facilitates purification, (b) its negative effect on
the yield of c1–128 was considerably counteracted by the addition of the FLAG epitope, and (c) the pres-ence of the proline residue did not improve expression,
we proceeded to large-scale expression of the b, c and
e ECDs using constructs carrying both the FLAG and 6-HIS tags and no additional proline (i.e FLAG⁄ b1– 221HIS, FLAG⁄ c1–218HIS and FLAG ⁄ e1–219HIS) (Fig 2A) The yields ranged from 2 mgÆL)1culture for FLAG⁄ b1–221HIS to 0.3–0.8 mgÆL)1for both FLAG⁄ c1–218HIS and FLAG⁄ e1–219HIS The ECDs were purified using Ni2+⁄ nitrilotriacetate affinity chroma-tography under native conditions Typically, the pro-teins were eluted with 150 mm imidazole, although some protein was eluted at 100 mm (less than 10% of the total) Each protein migrated on SDS⁄ PAGE with
an apparent molecular mass of 35 kDa compared with the estimated molecular mass of 29 kDa, which was apparently due to the glycosylation of the product
in the yeast cell (see below) The proteins were 90% pure, based on quantification of the protein bands on Coomassie Brilliant Blue-stained SDS⁄ polyacrylamide gel (Fig 2B)
Deglycosylation of b1–221, c1–218 and e1–219 Each recombinant protein carries at least one Asn-X-Ser motif (glycosylation pattern for eukaryotes), b1–221 at Asn141, c1–218 at Asn30 and Asn141, and amino acids 1–219 of the human AChR e subunit (e1– 219) at Asn66 and Asn141 To verify that the recom-binant proteins were glycosylated in the yeast cell (as suggested by the observed difference in the molecular mass of the purified proteins on SDS⁄ PAGE), each protein was deglycosylated with peptide–N-glycosidase
F For each of the three proteins, this resulted in the appearance of a band migrating at the expected mass
of 29 kDa (Fig 2C), confirming that the proteins were glycosylated
Gel-filtration analysis of polypeptides
To examine the solubility and oligomerization state of the recombinant polypeptides, we performed FPLC analysis in detergent-free solution (50 mm phosphate buffer, 300 mm NaCl, pH 8.0) in the presence of trace amounts of 125I-labeled soluble 66-kDa and 29-kDa protein markers To verify that the observed peaks on the FPLC coincided with the presence of our proteins, dot-blots were performed using anti-b (mAb 73) or anti-c (mAb 67) [22] (Fig 3) As the expected molecu-lar mass of a monomer of each of the three ECD pro-teins was 30–32 kDa, the results showed that b1–221 was probably eluted as a dimer with an apparent
A
B
His His
1-219
1-219
His
1-219
His
1-219
His
1-218
1-218
1-218
His
1-218
0
20
40
60
80
100
120
140
1-218 1-218
HIS
FLAG 1-218 FLAG 1-218HIS 1-219 1-219 HIS FLAG 1-219 FLAG 1-219HIS
Recombinant polypeptide
Fig 1 Expression of the c ECD variants (A) Schematic
representa-tion of the various c ECD constructs The drawings depict the
poly-peptides with their tags ⁄ epitopes; the additional amino acid,
proline, is shown as a black bar at the C-terminus of some c
ECD(s) (B) Relative yields of the different c ECD constructs All
yields were expressed as a percentage of the yield of the
non-tagged c1–218 construct, measured as the pixels for the positive
dot-blots of the culture expressing c1–218 The results shown are
the mean from five experiments.
Trang 4A γ γ 1-218 His
β
C
6 3
6 3
+ -e s a G
5 5
B
5 5
5 5
5 5
Fig 2 Purification and deglycosylation of the AChR ECDs (A) Schematic representation of the constructs used for expression of the b1–221, c1–218 and e1–219 ECDs of the human AChR in yeast P pastoris The arrowhead indicates the cleavage site of the a-factor peptide after secretion, and the circles indicate putative glycosylation sites (Asn-X-Ser motif) (B) SDS ⁄ PAGE of the proteins purified by Ni 2+ ⁄ nitrilotri-acetate metal affinity chromatography stained with Coomassie Brilliant Blue; the left lane in each panel contains molecular mass markers, and the right lane the test protein (C) Deglycosylation of the b, c, and e ECDs using N-glycosidase F Purified proteins (1 lg) were incubated for 3 h at 37 C in the absence (lane 1) or presence (lane 2) of N-glycosidase F, then the mixture was analyzed by SDS ⁄ PAGE (12% gel) and western blotting using anti-FLAG mAb M2 The arrows indicate the bands corresponding to the glycosylated (upper) and deglycosylated (lower) forms of each protein.
66kDa
29kDa 158kDa
0
500
1000
1500
2000
0
500
1000
1500
2000
ml
β1-221
A
B
–3 )
66kDa
29kDa 158kDa
20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2
1
0
20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2
1
0
ml
γ
γ 1-218 Fig 3 Gel filtration analysis of the
polypep-tides (A) 2.0 mg b1–221 or (B) 2.0 mg c1–218 protein was run on a Superose-12 column (Amersham-Pharmacia) at a flow rate of 0.5 mLÆmin)1, together with 125
I-labeled protein markers of known mole-cular mass (66 and 29 kDa) The fractions were screened for ECD protein by dot-blots using anti-b (mAb 73) or anti-c (mAb 67) The position of the 158-kDa (aldolase) marker is also shown (nonradioactive, obtained from a separate run).
Trang 5molecular mass of 60–65 kDa (Fig 3A), whereas
c1–218 was mainly present as an oligomer (possibly
trimers-pentamers) (Fig 3B) e1–219 displayed a
sim-ilar pattern to c1–218 (data not shown), indicative of
an oligomeric state Although these elution patterns
are typical of the proteins produced, occasional
prepa-rations showed a considerable percentage (10–20) of
higher aggregates
CD spectra
When the b subunit ECD was subjected to far-UV CD
analysis to examine its secondary structure, the CD
spectrum in 50 mm phosphate buffer containing
0.15 m NaCl, pH 8.0, was characterized by a positive
Cotton effect in the 190–200 nm region (peak
196 nm) and a negative effect in the 200–240 nm
region (Fig 4), suggesting a major contribution from a
b-sheet structure However, the quite high negative
dichroism intensity over a relatively wide region
215 nm is indicative of the presence of bands at 208
and 222 nm, characteristic of a contribution of
a-heli-cal regions [23]
Binding of mAbs to the ECDs using ELISA
ELISAs were performed using the
conformation-dependent mAbs 73 (binds to an epitope on the
extra-cellular side of the b subunit) [22] and 67 (binds to an
epitope on the extracellular side of the c subunit) [22]
and the nonconformation-dependent mAb M2
(anti-FLAG) As a negative control, mAb 25 [24] was used,
which recognizes an epitope on Electrophorus electricus
AChR, but not on mammalian AChR Figure 5 shows that mAbs 73 and 67 specifically recognized their cog-nate proteins, whereas mAb 25 did not bind to any of the three polypeptides, as expected The strong and specific binding of the mAbs to the appropriate ECD suggested the correct folding of at least b1–221 and c1–218 Owing to the unavailability of a conforma-tion-dependent e subunit mAb, only binding of anti-FLAG mAb was tested
Binding of the rabbit anti-b serum to recombinant b1–221 and human AChR
Purified b1–221 was used to raise a rabbit anti-b ECD serum After three immunizations, the antiserum was tested for its ability to bind to the antigen (b1–221) using ELISA The results (Table 1) showed strong and specific binding to b1–221 ( 1.8 absorbance units), with relatively weak cross-reactivity with either a1–210
or yeast proteins ( 0.4 absorbance units) The anti-serum was then tested for its ability to bind to native human TE671 AChR [25] in RIA experiments The high titer of the anti-(b ECD) serum for native AChR (870 nm, Fig 6) further suggests that recombinant b1–221 retains some native-like conformational features
Binding of human MG antibodies to recombinant ECDs
To further examine the structure of the ECDs pro-duced and their potential as tools for MG studies, we tested their capacity to bind the highly conformation-dependent AChR antibodies present in MG sera We had previously identified MG patient sera in which the antibodies are mainly directed against the a subunit
195 200 205 210 215 220 225 230 235 240 245 250
-14
-12
-10
-8
-6
-4
-2
0
2
4
6
8
10
Wavelength (nm)
Fig 4 Far-UV CD spectrum of b1–221.
0 0,5 1 1,5 2 2,5
β1-221 γ1-218 ε1-219 BSA
Recombinant ECD
A450
n
A ti-β n
A ti-γ n
t a
Fig 5 mAb binding to b, c, and e ECDs using ELISA ELISA plates were coated with one of the three ECDs or BSA as a control, and the binding of mAbs tested by ELISA as described in Experimental procedures (duplicate samples) mAb 73, checker-board bars; mAb
67, dark gray bars; FLAG mAb, light gray bars mAb 25 (black bars) was used as the negative control.
Trang 6(anti-a sera) and others with a very small proportion
of antibodies against a (nonanti-a sera) [26] We
incu-bated five nonanti-a and one anti-a (82% antibodies
against a) sera with a single ECD (b, c or
e)–Seph-arose or BSA–Sephe)–Seph-arose resin, then measured the
nonbound AChR antibodies in the initial and final
samples If the AChR antibodies in the MG serum
recognized and bound to the recombinant,
immobi-lized proteins, there would be a reduction in the
amount of antibodies in the sample incubated with the
ECD–Sepharose, and this reduction should be
propor-tional to the percentage of subunit-specific antibodies
in each serum The anti-a serum should display little
or no reduction when incubated with any of the test
ECDs Table 2 shows that incubation of each of the five nonanti-a sera (samples 1–5) with different ECD– Sepharose resins resulted in different percentage reduc-tions in AChR antibody titers In contrast, the anti-a serum (sample 6) did not show any significant reduc-tion in titer when incubated with any of the non-a ECDS, as expected, but 82% loss of antibodies when incubated with a ECD–Sepharose Similar results were obtained even when much higher serum quantities were incubated with the ECD–Sepharose resins (data not shown), which suggests that the immunoadsor-bents in this experiment adsorbed all corresponding subunit antibodies As the binding to the AChR of AChR antibodies in MG patient sera is highly confor-mation-dependent, our findings support the presence
of native-like conformational features on all three recombinant ECDs
Discussion
In this paper, we describe the expression of soluble forms of the ECDs of non-a subunits of the human muscle AChR, using the yeast P pastoris system We have successfully used this system for the human muscle a ECD (a1–210) [17] and human neuronal type a7 subunit (a7 1–208) [18] Based on this experi-ence, we embarked on the expression of three of the four non-a ECDs, namely b, c and e The expression
of the d subunit ECD, which is currently under pro-gress, presents major difficulties, which require further investigation
Aiming to improve expression yields, we designed, constructed and tested different variants of the c ECD with and without a 6-HIS tail and⁄ or the FLAG
Table 1 Binding of the rabbit anti-(b ECD) serum to purified b1–
221 in ELISA tests Results shown are the mean from two
experi-ments Recombinant human a ECD (a1–210) was used to test for
nonspecific binding of the rabbit anti-(b ECD) serum to a protein
related to b1–221, rather than to a totally unrelated protein, such as
BSA The purified b1–221 used for immunization was purified from
a P pastoris yeast culture and possibly contained traces of yeast
culture components (e.g peptides originating from yeast protein
degradation and other metabolic by-products) To eliminate the
pos-sibility that rabbit antibodies raised against such components could
lead to spurious ELISA results, a control yeast supernatant sample
was prepared as described in Experimental procedures BSA was
used as a negative control.
Rabbit anti-(b ECD) serum (A450)
Normal rabbit serum (A450)
0
200
400
600
800
1000
1200
Serum Volume (µl)
Fig 6 Binding of the rabbit anti-(b ECD) serum to 125
I-a-bungaro-toxin-labeled native human AChR Various amounts of the rabbit
anti-(b ECD) serum were incubated with 14 fmol intact125
I-a-bung-arotoxin-labeled human AChR, then bound receptor was
precipita-ted with sheep anti-rabbit IgG, and radioactivity was measured.
Samples were processed in duplicate, and the results shown are
the mean of those of three experiments The titer of b antibodies
in the serum was calculated to be 870 n M
Table 2 Adsorption of AChR antibodies from human MG sera by immobilized ECDs AChR antibody titer given in parentheses in n M The reduction in total AChR antibodies present in MG sera observed after incubation of sera with b1–221, c1–218, or e1–219 immobilized on CNBr–Sepharose beads was measured by RIA using 125 I-a-bungarotoxin-labeled native AChR.
Serum
Reduction (%) in AChR antibodies in MG serum after incubation with immobilized ECDs
anti-a serum a
Data from [26].
Trang 7epitope and⁄ or the first transmembrane amino acid of
the AChR c subunit (proline), which is found at this
position in all human muscle subunits and has been
shown to positively affect expression of the mouse
AChR a subunit [21] Our results showed that the
addition of a Pro residue and the presence of common
epitopes⁄ tags used for purification influenced the
expression yield When the widely used 6-HIS tail was
added to the C-terminus of c1–218, it reduced
expres-sion almost twofold, whereas an N-terminal FLAG, a
peptide sequence rich in charged residues, improved
expression of c1–218 by 20% and that of c1–218HIS
by 40% (Fig 1B) The effect of the hydrophilic FLAG
epitope was most dramatically seen with the b1–221
ECD, its addition leading to an almost threefold
increase in expression Although Yao et al [21]
showed that addition of a Pro to the mouse a ECD
increased expression fourfold, no such effect was seen
with the non-a human ECDs in our system Aiming
simultaneously at easy purification (achievable using
the 6-HIS tail) and high yields, we used the
N-FLA-G⁄ ECD ⁄ HIS-C constructs for the large-scale
expres-sion of the proteins and found that b1–221 was
consistently expressed at a concentration of 2 mgÆL)1
of culture and the c and e ECDs at a concentration of
0.3–0.8 mgÆL)1 These yields are an improvement over
the previous 0.1–0.2 mgÆL)1 expression of the a1–210
protein [17], which, however, was in the monomeric
form, in contrast with the three recombinant proteins
described here Gel-filtration analysis showed that b1–
221 existed mainly as a dimer, whereas both c1–218
and e1–219 were mainly present as oligomers, possibly
trimers–pentamers (Fig 3) The state of the proteins
was confirmed by dynamic light scattering experiments
(data not shown); the proteins appeared polydisperse
with an estimated diameter of 7.5–9.8 nm (b ECD)
and 12.0–13.8 nm (c ECD), suggesting, respectively, a
dimeric or an oligomeric structure and confirming the
FPLC data, considering that the ‘height’ of the ECD
of the AChR is 6 nm [3] This difference in solubility
between the c–e and the b ECDs might be attributed
to the primary structure of the protein: in addition to
the ‘standard’ cysteine pair (residues 128 and 142) [27],
present in all AChR subunit ECDs, both c and e carry
extra cysteine residues at residues 61, 105, and 115 (c)
and 190 (e), which could be involved in the formation
of intramolecular or intermolecular bonds, leading to
oligomer formation However, if ‘free’ cysteines were
the only factors responsible for multimer formation,
then the b ECD should exist as a monomer; as this
was not the case, exposed hydrophobic regions, which
are presumably present in the b ECD, may also
con-tribute to intermolecular association of monomers
The results from a range of experiments suggested that the recombinant polypeptides are, at least to some extent, properly folded Firstly, they were glycosylated, like native AChR [28] (Fig 2C) Even though we lack direct evidence about the site and structure of the glycosylation sites on the ECDs, indirect evidence of correct glycosylation of our ECDs comes from our previous studies on the a ECD [17]: deglycosylation abolished a-bungarotoxin activity, strongly suggesting that glycosylation was at the right site and possibly of correct structure Secondly, the CD spectrum of the b ECD indicated a folded protein consisting mainly of b-sheet (Fig 4) The solved crystallographic structures
of the molluscan Lymnaea stagnalis [11] and Bulli-nus truncatus [29] acetylcholine-binding proteins, which provide the prototypes for the AChR ligand-binding domain, show a predominance of b-sheet, and the CD spectra for these proteins largely resemble our spectra [29] and are also similar to those for mouse a1 expressed in mammalian cells [16] or yeast [21] and the Torpedo a ECD expressed in Escherichia coli [13] These results suggest that the acetylcholine-binding proteins and the b ECD have similar structures and that the secondary structures of a non-a ECD (b) and the a ECD resemble one another, being largely com-posed of b-structure Thirdly, conformation-dependent anti-b and anti-c (mAbs 73 and 67, respectively) bound specifically to their cognate ECD (Fig 5), and
a polyclonal serum raised against the b1–221 polypep-tide specifically bound to native AChR in RIA experi-ments (Fig 6) Finally, AChR antibodies in different
MG sera were specifically adsorbed by matrix-immobi-lized ECDs, with variable concentrations of AChR antibodies being retained on each ECD matrix (up to 89% of b antibodies for MG serum 3; Table 2) The presence of antibodies against several AChR subunits
in a single serum (e.g MG serum 1; Table 2) is inter-esting, although it was not unexpected because of pre-vious indirect information (e.g from competition experiments between mAbs against different subunits and MG sera) [22] The actual autoantigen in anti-AChR-mediated MG is still uncertain It may be intact AChR, AChR subunit(s) or fragments, or an AChR cross-reactive molecule The polyspecificity of the sera may either mean that the autoantigen is an intact AChR or that epitope spreading occurred after initial induction by a single AChR subunit or a cross-reactive molecule The adsorption results also indicated the presence of a considerable percentage (29–39%) of c antibodies in three of the five tested nonanti-a MG sera (e.g MG sera 1, 4 and 5; Table 2) The c subunit, present in the fetal isoform of the AChR, is replaced
by the e subunit in adult muscle; however, this fetal
Trang 8isoform is expressed in myoid cells in the thymus
[30,31], and c expression persists into adulthood in
mouse and bovine ocular fibers [32,33], justifying the
presence of c antibodies in adult MG sera The
major-ity of AChR antibodies in MG sera are directed
against various nonlinear, conformation-dependent
epitopes on the extracellular part of the AChR
mole-cule, a fact that has prevented their characterization
using synthetic peptides or denatured recombinant
polypeptides obtained using prokaryotic expression
systems [20,34] In addition, the immune responses
against the non-a AChR subunits have not been
exam-ined as carefully as those against the a subunit, even
though the differential expression of the different
sub-units may be highly significant in the pathogenesis of
MG [35]
All four ECDs of the Torpedo AChRs have
previ-ously been expressed as soluble proteins using
baculo-virus-infected insect cells [36], and the proteins showed
proper folding, but the amounts produced were
insuffi-cient for crystallization trials Moreover, the a ECDs
of Torpedo and human AChR, which have been
pro-duced as inclusion bodies in bacteria in quantities
suf-ficient for structural studies [13,14], require denaturing
conditions for solubilization of the protein and
refold-ing and also do not undergo post-translational
modifi-cations In the present study, we obtained stable
Pichia clones expressing satisfactory amounts of three
non-a ECDs (b1–221, c1–218 and e1–219) which were
in a soluble, secreted form and probably correctly
folded, a fact that may permit preliminary
crystalliza-tion trials Crystallizacrystalliza-tion trials require protein
sam-ples of concentration 10 mgÆmL)1, purity of at least
95%, and monodispersity Based on the yields of our
yeast cultures (0.5–2 mgÆL)1), a medium-scale
expres-sion would suffice to provide material that, after
puri-fication and gel filtration, should be sufficiently
concentrated The risk in this case would be the
puta-tive formation of aggregates that would render the
sample unusable for downstream processing, especially
for the recombinant c1–218 and e1–219, which were
already in the form of oligomers; this approach,
how-ever, could possibly be applicable to b1–221, which is
dimeric, stable on concentration (data not shown),
and exhibits the highest expression yield For the c
and e ECDS, improvement in their solubility is
required before attempts at structural trials We are
working towards this by constructing mutant forms of
the proteins Nevertheless, these polypeptides, together
with the already produced a1–210 [17], were all
specif-ically recognized by human AChR antibodies in MG
sera, allowing their immediate use for the detailed
study of the specificities of the antibodies in MG sera
and the development of antigen-specific therapeutic approaches
Experimental procedures
Bacterial and yeast strains, growth conditions, plasmids and DNA manipulations
The E coli K-12 strain TOP10F¢ (Invitrogen, San Diego,
CA, USA) was used for replication of plasmid DNA Clo-ning of the ORFs encoding the b1–221, c1–218 and e1–219 ECDs was performed by standard techniques [37] Luria– Bertani broth and agar were used for amplification of transformed bacteria Ampicillin (100 lgÆmL)1) was used in liquid or solid media
The vector pPIC9 (Invitrogen) was used to clone the ORFs in-frame with a leader sequence allowing secretion
of the produced protein after cleavage of the secretion signal An oligonucleotide, 5¢-GTAGATTACAAGGATG ACGATGACAAAG-3¢ encoding the FLAG sequence, DYKDDDDK, was introduced into the vector between the unique SnaBI and EcoRI sites This allowed the subsequent in-frame cloning of our PCR products with a 5¢-EcoRI site
in such a way that the cloned ORF was expressed as a polypeptide carrying the FLAG peptide at its N-terminus The resulting plasmid was named pPIC9⁄ FLAG
Cloning using PCR
We used PCR to amplify the extracellular region of each of the b, c and e subunits, using the plasmid templates, pcDNA3.1⁄ Beta, pcDNA3.1⁄ Gamma and pcDNA3.1⁄ Epsilon (cDNA clones of the human b, c and e AChR sub-units in pcDNA3.1 respectively; all kindly provided by D Beeson, University of Oxford, UK) [38] PCR was per-formed on the appropriate template for each subunit on a Perkin-Elmer (Boston, MA, USA) thermal cycler; 5 min denaturation at 94C was followed by 25 cycles of 94 C for 20 s, 58C for 30 s, and 72 C for 90 s, and a final 5-min extension step at 72C The reaction mix consisted
of 10 ng template, 50 mm each dNTP, 20 pmol each pri-mer, and 1 U Taq DNA polymerase in a volume of 50 lL 10-fold diluted reaction buffer (Promega, Madison, WI, USA) For b1–221, the forward primer was 5¢-GCGGA ATTCTCGGAGGCGGAGGGTCGAC-3¢ and the reverse primer 5¢-ATAGTTTAGCGGCCGCTCAATGGTGATGG TGATGGTGCTTGCGGCGGATGATGAG-3¢ For the c1–218 variants (some with an additional C-terminal Pro giving c1–219), the forward primer 5¢-GGTGTAGA ATTCCGGAACCAGGAGGAGCGC-3¢ was used in all cases, together with the reverse primer (a) 5¢-ATA GTTTAGCGGCCGCTTACTTGCGCTGGATGATGAG CAGG-3¢ for c1–218, (b) 5¢-ATAGTTTAGCGGCCGC TTAGTGATGGTGATGGTGATGCTTGCGCTGGATG
Trang 9AGCAGG-3¢ for c1–218HIS (c1–218 with a 6-HIS tag
at its 3¢ end to facilitate purification), (c) 5¢-ATAG
TTTAGCGGCCGCTTAGGGCTTGCGCTGGATGAGCA
GG-3¢ for c1–219, or (d) 5¢-ATAGTTTAGCGGCC
GCTTAGTGATGGTGATGGTGATGGGGCTTGCGCT
GGATGAGCAGG-3¢ for c1–219HIS For the e1–219
variants (e1–220 with additional Pro), the forward
pri-mer 5¢-GGTGTAGAATTCAAGAACGAGGAACTGCG-3¢
was combined with (a) 5¢-ATAGTTTAGCGGCCG
CTTACTTCCGGCGGATGATGAGCGAG-3¢ for e1–219,
(b) 5¢-ATAGTTTAGCGGCCGCTTAGTGATGGTGATG
GTGATGCTTCCGGCGGATGATGAGCGAG-3¢ for e1–
219HIS, (c) 5¢-ATAGTTTAGCGGCCGCTTACGGCTT
CCGGCGGATGATGAGCGAG-3¢ for e1–220, or (d)
5¢-ATAGTTTAGCGGCCGCTTAGTGATGGTGATGGTGA
TGCGGCTTCCGGCG-GATGATGAGCGAG-3¢ for e1–
220HIS (underlined EcoRI and NotI) The PCR products
were purified (Qiagen PCR clean-up kit; Qiagen, Hilden,
Germany), EcoRI–NotI digested, repurified, and cloned
into the EcoRI–NotI-digested pPIC9 or pPIC9⁄ FLAG
plas-mid Each PCR product was cloned into both plasmids
Sequencing was used to verify the identity of the inserts
Yeast transformation and dot-blot screening
of positive clones
Plasmids (10 lg) encoding the b1–221 ECD (with or
with-out the FLAG epitope) and the various c1–218 and e1–219
ECDs were linearized using SacI (for b1–221) or SalI or
SacI (for c1–218 and e1–219) and electroporated into
freshly made competent GS115 P pastoris cells Selection of
positive transformants (cells able to grow in the absence of
histidine) was achieved by plating on regeneration dextrose
plates (1 m sorbitol, 2% dextrose, 1.34% yeast nitrogen
base, 4· 10)5% biotin, 0.005% l-glutamic acid, l-lysine,
l-methionine, l-leucine, and l-isoleucine, 2% agar) without
histidine Small-scale cultures of single colonies were tested
after growth overnight in 3 mL BMGY medium (1% yeast
extract, 2% peptone, 100 mm potassium phosphate, pH 6.0,
1.34% yeast nitrogen base, 4· 10)5% biotin, 1% glycerol)
and resuspension of the cells in 3 mL BMMY medium to
induce expression (BMMY medium is identical with
BMGY, but contains 0.5% methanol instead of glycerol)
(day 0) Methanol was added to 0.5% every 24 h to
main-tain induction, and 0.75 mL liquid medium was removed
every 24 h after day 0 to test for the expression and
secre-tion of the produced protein The cleared supernatant was
tested on dot-blots using mAb 73, mAb 67, or anti-FLAG
mAb M2 (Sigma, St Louis, MO, USA) to test for the
expression of b, c or all ECDs, respectively After the initial
screening, phosphate buffers with a pH of 6.5 or 7.0 were
also tested, and the pH 7.0 buffer was finally adopted for
large-scale expression Expression levels of the different c or
e variants were estimated by quantification of the positive
signal on dot-blots of culture supernatant (at serial dilu-tions) using imagej software (http.//rsb.info.nih.gov/ij/)
Large-scale expression and purification of proteins
The best expressing clone was selected for each protein A 0.1-mL sample of a small overnight culture of 20 mL BMGY medium was used to inoculate 1 L fresh BMGY medium After growing to an A600 of 3 ( 18–20 h), the cells were spun down, washed, and resuspended in 3 L BMMY medium to induce expression On day two, the cul-tures were cleared of cells by centrifugation for 20 min at
2500 g (Jouan 11175372 M4 rotor), and the supernatant concentrated using a Millipore (Bedford, MA, USA) ultra-filtration system (filter cut-off 10 kDa); these steps and all subsequent steps were performed at 4C The concentrate was dialyzed overnight against 50 mm phosphate buffer,
2 m NaCl, pH 8.0, for the c and e ECDs or 50 mm phos-phate buffer, 0.5 m NaCl, pH 8.0, for the b ECD before binding of the protein to 1.5 mL pre-equilibrated Ni2+⁄ ni-trilotriacetate⁄ agarose (Qiagen) The protein was purified under native conditions following the manufacturer’s instructions Eluates were analyzed by SDS⁄ PAGE (12% gel) and Coomassie blue staining or western blotting using mAb 73 (for the b ECD) or anti-(FLAG M2) (Sigma) The purity of the protein was estimated from Coomassie Brilli-ant Blue-stained gels and quBrilli-antification of the bands using imagejsoftware, and protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA, USA)
In vitro deglycosylation
A sample (1 lg) of purified protein was deglycosylated by incubation for 3 h at 37C with 1000 U N-glycosidase F (New England Biolabs, Frankfurt, Germany) in a final vol-ume of 50 lL under the conditions recommended by the manufacturer for a nondenatured protein The protein was then precipitated by the addition of 200 lL methanol⁄ acet-one (1 : 1, v⁄ v), incubation at)20 C for 20 min, centrifu-gation for 15 min, and resuspension in 15 lL distilled water The samples were analyzed by SDS⁄ PAGE and western blotting using FLAG mAb M2
FPLC analysis of polypeptides
To determine the size of b1–221, c1–218 or e1–219, FPLC analysis on a Superose-12 column (Amersham-Pharmacia, Munich, Germany) was performed in 50 mm sodium phos-phate buffer⁄ 300 mm NaCl, pH 8.0, at a flow rate of 0.5 mLÆmin)1 Samples of each fraction (normally 1 and
10 lL of each 0.5-mL fraction) were tested for the presence
of the specific protein by dot-blotting with FLAG mAb M2
Trang 10Radioactive labeling of protein markers and
a-bungarotoxin
a-Bungarotoxin (24 lg) or 2 lg either bovine erythrocyte
carbonic anhydrase ( 29 000 Da) or BSA ( 66 200 Da)
(both from Fluka; Sigma-Aldrich, Athens, Greece) was
labeled, respectively, with 2 mCi or 0.1 mCi 125I, using the
chloramine T method [39], loaded on to a G50-Fine column
(Amersham-Pharmacia), and the labeled protein collected
and stored at )20 C Approximately 100 000 c.p.m of
each of the 125I-labeled protein markers was loaded on
every FPLC run as size markers
Preparation of rabbit anti-(b subunit) serum
An 8-week-old female New Zealand White rabbit was
injec-ted subcutaneously with 0.5 mg purified b1–221 protein
in 50% (v⁄ v) complete Freund’s adjuvant, followed by
three injections at monthly intervals in 50% incomplete
Freund’s adjuvant One week after the last injection,
anti-serum was collected, aliquoted, and stored at)20 C in the
presence of 0.05% sodium azide Use of experimental
ani-mals abides by law 2015/27-2-1992 of the Greek Republic
and Presidential Decree 160/3-5-1991 in accordance with
directive 86/609EOK of the Council of Europe for
protec-tion of vertebrates/animals used for experimental or other
research purposes
CD spectra
CD spectra were measured at 20C using a Jasco model
J-715 spectropolarimeter (located at NCSR, Demokritos,
Athens, Greece) in semi-automatic slit adjustment mode
The scan speed was set at 50 nmÆmin)1, the response time
at 2 s, and the scan range at 180–260 nm Optical activity
was expressed as the mean residue ellipticity (Q), in
degree-sÆcm2Ædmol)1, based on a mean residue weight of 115 for
the b ECD polypeptide The derived spectrum represents
the mean of eight scans and was corrected for light
scatter-ing by buffer subtraction The protein concentration was
optimized as 0.2 mgÆmL)1, and the quartz cell path length
was 1 mm All samples were optically homogeneous
ELISA
ELISA plates (Maxi-Sorb; Nun Roskilde, Denmark) were
coated, as described previously [14], using 0.25 lg purified
recombinant protein (b1–221, c1–218 or e1–219) per well
Control wells were coated with BSA (0.25 lg) Additional
control wells were coated with 0.25 lg a ECD (a1–210) or
100 lL yeast culture supernatant prepared as follows:
100 mL of a culture of P pastoris GS115 strain was spun,
and the supernatant filtered, concentrated 40-fold, and
dia-lyzed against 50 mm phosphate buffer, pH 8.0
The plates were washed with phosphate-buffered saline,
pH 7.5 (NaCl⁄ Pi) and blocked for 30 min at 37C with blocking solution (5% nonfat milk in NaCl⁄ Pi), then incu-bated for 1 h at 25C with primary antibody in blocking solution; mAbs were used at a 1 : 100 dilution (the concen-tration of the undiluted mAb ‘stock solution’ was 0.1– 0.5 mgÆmL)1), and the rabbit antiserum was used at dilu-tions of 1 : 100–1 : 10 000 After three washes with block-ing solution, the plates were incubated for 1 h at 25C with secondary antibody [horseradish peroxidase-conju-gated rabbit anti-rat IgG (Dako, Glostrup, Denmark) in the case of the mAbs and sheep anti-rabbit IgG (Dako)] at
a 1 : 500 dilution in blocking solution No secondary anti-body was used when the FLAG mAb M2 was used, as the antibody was supplied in its horseradish peroxidase-conju-gated form (Sigma) The ELISA plate was developed using 3,3¢,5,5¢-tetramethylbenzidine ready-to-use substrate (MBI-Fermentas, St Leon-Rot, Germany), stopping the reaction with 0.2 m H2SO4 The plate was read at 450 nm on a microtiter plate reader
Preparation of ECD–Sepharose beads ECD (0.25 mg) mixed with BSA (1.25 mg, as carrier) were bound to 0.25 g CNBr-activated Sepharose beads (Pharma-cia, Munich, Germany) according to the manufacturer’s protocol as described previously [26] The beads were then diluted in NaCl⁄ Pi⁄ 2% BSA ⁄ 0.05% NaN3 so that 120 lL
of the mixture contained 1 lg recombinant protein Control beads were prepared using 1.5 mg BSA
Use of the ECD–Sepharose matrix for binding AChR antibodies in MG sera
Depending on the AChR antibody titer, different dilutions
of sera were prepared: the MG sera were diluted 1 : 10 (for serum titer 5 nm) to 1 : 500 (for titer 290 nm) supplemented with normal human serum to a final serum dilution of
1 : 10 This guaranteed that the amount present in the untreated sample would immunoprecipitate 50% of the labeled AChR A 40-lL portion of the dilution was incuba-ted for 2 h at 4C with 120 lL Sepharose–ECD or Seph-arose–BSA matrix (final volume 160 lL), and then duplicate 40-lL samples of supernatant (containing 1 lL serum) were tested in the RIA described below
RIA for MG sera or rabbit anti-(b1–221) serum
We tested the ability of MG sera to precipitate a-bungaro-toxin-labeled human AChR prepared from either TE671 cells or a mixture of CN21⁄ TE671 cells (CN21 cells express the e and c AChR subunits at a ratio of approximately
2 : 1 [40], whereas TE671 cells express only the c subunit AChR [25]) The TE671-derived AChR was used when sera