The C-terminal t peptide of acetylcholinesterase forms an a helix that supports homomeric and heteromeric interactions Suzanne Bon1, Jean Dufourcq2, Jacqueline Leroy1, Isabelle Cornut2an
Trang 1The C-terminal t peptide of acetylcholinesterase forms an a helix that supports homomeric and heteromeric interactions
Suzanne Bon1, Jean Dufourcq2, Jacqueline Leroy1, Isabelle Cornut2and Jean Massoulie´1
1
Laboratoire de Neurobiologie Cellulaire et Mole´culaire, Ecole Normale Supe´rieure, Paris, France;2Centre de Recherche Paul Pascal, Pessac, France
Acetylcholinesterase subunits of type T (AChET) possess an
alternatively spliced C-terminal peptide (t peptide) which
endows them with amphiphilic properties, the capacity to
form various homo-oligomers and to associate, as a
tetra-mer, with anchoring proteins containing a proline rich
attachment domain (PRAD) The t peptide contains seven
conserved aromatic residues By spectroscopic analyses of
the synthetic peptides covering part or all of the t peptide of
TorpedoAChET, we show that the region containing the
aromatic residues adopts an a helical structure, which is
favored in the presence of lipids and detergent micelles: these
residues therefore form a hydrophobic cluster in a sector of
the helix We also analyzed the formation of disulfide bonds
between two different AChET subunits, and between
AChET subunits and a PRAD-containing protein [the N-terminal fragment of the ColQ protein (QN)] possessing two cysteines upstream or downstream of the PRAD This shows that, in the complex formed by four T subunits with
QN(T4–QN)
4 , the t peptides are not folded on themselves as hairpins but instead are all oriented in the same direction, antiparallel to that of the PRAD
bonds between various pairs of cysteines, introduced by mutagenesis at various positions in the t peptides, indicates that this complex possesses a surprising flexibility
Keywords: acetylcholinesterase; amphiphilic alpha helix; disulfide bonds; proline rich domain
The quaternary associations of acetylcholinesterase (AChE)
and butyrylcholinesterase (BChE) are determined by small
C-terminal domains that are distinct from the catalytic
domain [1,2] In vertebrates, alternatively spliced exons of
the AChE gene
distinguish different types of subunits However, only
subunits of type T (tailed) exist in the BChE and AChEs
of all vertebrates; in mammals they represent the only
AChE variant expressed in the adult nervous system and muscles These subunits possess specific association pro-perties, which depend on their C-terminal t peptide This peptide is strongly conserved in vertebrates, with 75% identity between cartilagenous fishes (Torpedo) and mam-mals; it contains 40 or 41 residues, with a cysteine at)4 from the C-terminus and a series of seven conserved aromatic residues including three tryptophans [3]
Transfected COS cells expressing subunits of type T produce a wide array of catalytically active AChE forms, including monomers, dimers and tetramers [4] The mono-mers, dimers and some tetramers are amphiphilic, as defined
by their interaction with detergent micelles, which modify their sedimentation and their electrophoretic migration in nondenaturing conditions [5] These amphiphilic molecular forms require detergents to be totally solubilized but are also secreted when expressed in transfected COS cells [4] The
t peptide is necessary for the amphiphilic character of AChE and for the formation of tetramers, as deleted subunits that lack this peptide generate only nonamphiphilic monomers [6]
AChE subunits of type T (AChET) can assemble into tetramers with their anchoring proteins ColQ and PRiMA, and these heteromeric associations represent the physio-logically functional species in muscles and brain [7,8] At the neuromuscular junction, collagen-tailed asymmetric forms are inserted in the basal lamina; in these molecules, one AChETtetramer (T4) is attached to the N-terminal region of each of the three strands of the triple helical ColQ collagen
In the mammalian brain, the predominant AChE species
is a tetramer, anchored at the cell surface through the
Correspondence to S Bon, Laboratoire de Neurobiologie Cellulaire
et Mole´culaire, CNRS UMR 8544, Ecole Normale Supe´rieure,
46 rue d’Ulm, 75005 Paris, France.
Fax: + 33 1 44 32 38 87, Tel.: + 33 1 44 32 38 91,
E-mail: jean.massoulie@biologie.ens.fr
Abbreviations: AChE, acetylcholinesterase; AChE H , AChE subunit of
type H; AChE T , AChE subunit of type T (tailed); BChE,
butyryl-cholinesterase; BChE T , BChE subunit of type T (tailed); cmc, critical
micellar concentration; CTAB, cetyltrimethylammonium bromide;
C37, C-terminal cysteine residue at position 37; GPI,
glycophospha-tidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C;
PRAD, proline rich attachment domain; Q N , N-terminal fragment of
the ColQ protein; SMCC, N-succinimidyl-4-(N-maleimidomethyl)
cyclohexane-1 carboxylate; t peptide, the C-terminal peptide of
AChE T subunits; T, AChE T subunits; WAT, tryptophan amphiphilic
tetramerization domain.
Note: In this paper the residues of the t peptides of AChE T from
different species are numbered from 1 to 40 in order to facilitate
comparisons.
(Received 31 July 2003, revised 10 October 2003,
accepted 23 October 2003)
Trang 2transmembrane protein PRiMA (T4–PRiMA) The
N-terminal regions of both ColQ and PRiMA contain a
proline-rich attachment domain (PRAD) [9], which is
responsible for their interaction with AChET or BChET
subunits; in addition, they contain cysteines that form
disulfide bonds with two cholinesterase T subunits in each
tetramer, by means of the cysteines located near their
C-terminus [10–12]
The t peptide is in fact sufficient for association with a
PRAD, as shown by the fact that it can replace a complete
AChETor BChETsubunit in PRAD-associated tetramers,
and can induce the formation of PRAD-linked tetramers
when added at the C-terminus of foreign proteins such as
green fluorescent protein or alkaline phosphatase: it
there-fore constitutes an autonomous interaction domain,
referred to as the WAT [tryptophan amphiphilic
tetra-merization] domain [13] The t peptide also acts as an
enhancer of degradation through the ER-associated
degra-dation pathway [14]
In the present study, we analyse the structural basis for
the hydrophobic and quaternary interactions of the t
pep-tide In particular, we ask whether hydrophobic interactions
result from the structure of the peptide itself or require
post-translational modifications, e.g the addition of lipidic
residues It has been reported that membrane-bound mouse
AChE produced in transfected human embryo kidney 293
cells incorporates palmitic acid, but not mevalonate, in spite
of the resemblance of its C-terminus with an isoprenylaytion
signal [15]
The amphiphilic properties of AChETsubunits suggest
that the t peptide constitutes an amphiphilic a helix, with its
seven aromatic residues located in the same sector, forming
a hydrophobic cluster [1] Here, we present evidence that the
t peptide actually forms an amphiphilic helix and that it is
elongated, rather than folded upon itself in a hairpin as
proposed by Giles [16], in AChETmonomers and dimers as
well as in tetramers associated with an N-terminal fragment
of ColQ (QN) We also show that the four t peptides are
parallel to each other and antiparallel
T4–QNheteromeric complex
Materials and methods
Materials
Egg phosphatidylcholine and its lyso derivative were
prepared as described previously [17] Phosphatidylserine
was obtained from Lipid Products (Nutfield, Surrey, UK)
The detergents used for the spectroscopic studies were from
VWR (Strasbourg, France) and Sigma
recrystal-lized before use A lytic tetrameric form (G4) derived from
collagen-tailed Electrophorus AChE was purified by affinity
chromatography on Sepharose derivatized with
hexylamido-carboxyphenyl-dimethylethylammonium, as described
pre-viously [18]
Peptide synthesis
The t1)32 peptide was synthesized in the laboratory of
J Vandekerckhove (Laboratorium Genetika, Gent,
Bel-gium) It was purified by preparative HPLC and analyzed in
a C-18 Vydac column (The Nest Group, Southborough,
MA, USA): the preparation contained essentially only the monomeric peptide, with less than 10% dimers, spontane-ously formed upon air oxidation and that could be reduced
by dithiothreitol The t1)40 peptide, at 85% purity, was synthesized by Neosystem Laboratoires (Strasbourg, France) The t25)40peptide was synthesized in the laboratory
of J Igolen (Institut Pasteur, Paris, France) and was puri-fied by preparative HPLC Whereas the C-terminal cysteine residue at position 37 of t1)40 (C37) was blocked by an acetamidomethyl group, cysteines were added at the N-ter-minus of t1)32 and t1)40, to allow their linkage to non-amphiphilic AChE tetramers from Electrophorus electric organs, via their N-terminal extremity, as with AChET subunits
Chemical coupling of peptides withElectrophorus G4 AChE
Each of the t1)32, t1)40and t25)40peptides were covalently coupled to the G4 form of Electrophorus AChE by the heterobifunctional reagent N-succinimidyl-4-(N-maleimido-methyl)cyclohexane-1 carboxylate (SMCC) This method involves the reaction of thiol groups from cysteine residues
of the peptides with a maleimido group incorporated into AChE after reaction with SMCC The preparation of AChE–SMCC has been described elsewhere [19]
Subsequently to being dissolved in 0.1M phosphate buffer, pH 6, the thiol content of the peptides was measured
by reaction with 5,5¢-dithiobis(2-nitrobenzoic acid) [20] Coupling between the peptide and the enzyme was obtained
by mixing AChE–SMCC with an excess of thiol groups (the concentration of thiol was 100-fold that of G4) Peptides
t1)32and t1)40were coupled using the added N-terminal cysteine and t25)40was coupled through C37 After 3 h at
30C, the conjugate was purified by molecular sieve chromatography in a Biogel A0.5 column (Bio-Rad Laboratories), as described previously [21] We observed
no significant loss in enzyme activity during the coupling procedure
Production of antibodies against t25)40peptide Anti-(t25)40) polyclonal Ig was raised in rabbit against the
t25)40peptide covalently coupled to BSA The t25)40–BSA conjugate was obtained by reaction with glutaraldehyde, as described previously [22] Immunization followed the pro-cedure described by Vaitukatis [23]
Spectroscopic analyses Circular dichroism spectra were obtained in an AVIV 62DS (AVIV, Zu¨rich, Switzerland) spectrometer at 25 C, using cuvettes of 0.1–1 cm path-length according to the concen-tration of peptide The blank was subtracted in all cases For evaluation of the molar ellipticity per residue (h) expressed
in degÆdmol)1Æcm2, the peptide concentration was calculated
by using an absorbance e280¼ 20 000M–lÆcm–l Fluorescence spectra were obtained with a Fluoromax SPEX spectrophotometer (Jobin et Yvon, Longjumeau, France) at 25C, with an excitation wavelength of 280 nm and a slit width of 1.7 nm The spectra corresponding to an average of at least two or three scans were corrected in
Trang 3emission, and the background fluorescence from buffer and
detergent were subtracted
Mutagenesis and transfections
cDNA encoding rat AChE subunits was inserted in the
pEF-BOS vector, which is under the control of the human
EF-10c promotor; this vector was used for mutagenesis and
expression in COS cells [4] All constructs were identical,
except for the 3¢ sequence encoding the C-terminal peptides
AChET subunits were coexpressed with proteins derived
from QN, containing either the natural PRAD motif with
its two adjacent cysteines upstream of the proline-rich
segment (CC-QN), or a modified PRAD, in which these
cysteines were replaced by serines, and two cysteines were
introduced downstream of the prolines (QN-CC) A QN
construct from which the PRAD was deleted (residues 70–
86) was used in control cultures, to ensure an identical level
of AChETexpression In a number of experiments we used
a construct that contained a C-terminal GPI addition signal
derived from Torpedo type H AChE (AChEH) subunits, so
that the resulting complex, (AChET)4–QN–GPI, could be
recovered from the cell surface by treatment with
phos-phatidylinositol-specific phospholipase C (PI-PLC) For
transfections, DNA was purified on Nucleobond AX
columns (Macherey–Nagel, Hoerdt, France) COS-7 cells
were transfected by the diethylaminoethyl-dextran method,
as described previously [9] The cells were maintained at
37C and were collected after three days
Preparation of extracts and AChE assay
The cells were extracted with TMg buffer [1% (v/v) Triton
X-100; 20 mM Tris/HCl pH 7.5; 10 mM MgCl2] at 4C
when the AChETsubunits were expressed alone or with QN,
and at 20C when they were expressed with a QN–GPI
construct, because the GPI-anchored complex is associated
with sphingolipid/cholesterol microdomains which remain
partially insoluble in Triton X-100 in the cold
The AChE activity was assayed by the colorimetric
method of Ellman [20] Enzyme samples (10 lL) were
added to 0.2 mL of Ellman assay medium and the reaction
kinetics were monitored at 414 nm, at 15 s intervals over a
3 min period, using a Multiskan RC microplate reader
(Labsystems, Helsinki, Finland)
Sucrose gradients and nondenaturing electrophoresis
Aliquots of extracts (typically 200 lL) containing 1% (v/v)
Brij-96 buffer (10 mMMgCl2, 25 mMTris/HCl pH 7) were
loaded on 5–20% (w/v) sucrose gradients in 1% (v/v)
Brij-96 buffer Escherichia coli b-galactosidase (16 S) and
alkaline phosphatase (6.1 S) were included as internal
sedimentation standards The gradients were centrifuged
for 18 h at 36 000 r.p.m at 5C, in a LE80K centrifuge
using an SW-41 rotor (Beckman–Coulter, Villepinte,
France) Fractions of 300 lL were collected and assayed
for AChE, b-galactosidase and alkaline phosphatase
activities Electrophoresis in nondenaturating
polyacryl-amide gels was performed as described previously [24] and
AChE activity was shown by the histochemical method of
Karnovsky and Roots [25]
Metabolic labeling Two days after cotransfection of AChETsubunits with the TorpedoAChEHC-terminal addition signal, the transfected COS cells were preincubated for 45 min in Dulbecco’s modified Eagle’s medium lacking cysteine and methionine, and then labeled with [35S]methionine–cysteine (Amersham Biosciences) for 3 h The cells were then rinsed with NaCl/
Pi, and chased overnight in a medium containing Nu-serum (BD Biosciences, Bedford, MA, USA) The cell surface GPI-anchored AChE was solubilized by treating intact cells for 2 h at 37C with PI-PLC (1 : 600) from Bacillus thuringiensis, kindly provided by I Silman (Weizmann Institute, Rehovot, Israel) Following centrifugation at
10 000 g for 15 min to remove cell debris, the soluble enzyme (secreted and PI-PLC released) was collected for immunoprecipitation
Immunoprecipitation and SDS/PAGE AChE from cell extracts or medium were immunoadsorbed
on protein G immobilized on Sepharose 4B Fast Flow beads (Sigma) The beads were first washed and saturated with 5% (v/v) BSA in a buffer containing 150 mM NaCl,
5 mMEDTA, 50 mMTris/HCl pH 7.4, 0.05% (v/v) NP40 Samples of 1.5 mL of cell extracts or media were incubated with 40 lL of a 10% suspension of beads for 3 h to eliminate nonspecific adsorption and the beads were discarded The samples were incubated with 1 : 500 anti-(rat AChE) serum A63 [26] overnight at 8C, with gentle agitation on a rotating wheel, followed by addition of 80 lL
of a 10% suspension of BSA-saturated washed beads and incubation for 1 h After immunoadsorbtion, the beads were washed and centrifuged three times with 1 mL of buffer containing 1% Triton X-100 and centrifugations at
10 000 g for 5 min All incubations were performed at 8C under mild rotational agitation
For polyacrylamide electrophoresis under denaturing conditions, samples of the washed beads were resuspended
in 30 lL of 0.125MTris/HCl buffer pH 6.8 containing 1% SDS, 0.002% bromophenol blue, 5% 2-mercaptoethanol (v/v/v), heated at 98C for 5 min, and centrifuged at
10 000 g for 5 min at room temperature Aliquots of 10 lL
of the supernatant were submitted to electrophoresis in SDS/polyacrylamide gels, and the resulting bands were revealed with the BAS 1000 Fuji Image analyzer (Fujifilm,
St Quentin-en-Yvelines, France) or by autoradiography, and analyzed with the Fuji ImageGAUGEsoftware Prediction of secondary structure elements The secondary structure of the C-terminal region of the catalytic domain and of the t peptide was predicted according to Rost [27] using PREDICTPROTEIN at http:// maple.bioc.columbia.edu/predictprotein
Results
Modeling of the t peptide as an amphiphilic a helix The primary sequence of the C-terminal region of Torpedo AChE is shown in Fig 1A, including the last 12 residues of
Trang 4the catalytic domain and the t peptide Secondary structure
prediction algorithms show that a large part of this peptide
is expected to assume an a helical structure, extending from
residue five to residue 26 or 28, with a possible interruption
at residues 14–16 that might allow a bend between two
helical segments Giles proposed a similar arrangement, in
which a bend at residues 21–22 would bring together the
aromatic sectors of the two helices [16]; according to this
model, residues located in the N-terminal region of the
t peptide would be in close contact with the C-terminal
cysteine, C37
If we assume an a helical structure for the t peptide, a
lateral view shows that all the aromatic residues are oriented
on the same side (Fig 1B), and a wheel projection [28] shows
that a sector of 100 is totally apolar (Fig 1B) The polar
sector contains five acidic residues (one aspartic and four
glutamic acids) and four basic residues (one lysine, two
arginines and one histidine), which might form internal salt
bridges between residues D4 or E5 and R8, between E7 and
K11, and between E13 and R16, as analyzed in a further
study (S Belbeoc’h, J Leroy, A Ayon, J Massoulie´ &
S Bon, unpublished results) The cluster of hydrophobic side
chains in the apolar sector includes the seven aromatic
residues that are conserved in all known vertebrate AChEs and BChEs, ranging from cartilagenous fishes (Torpedo) to mammals In particular, three tryptophans are evenly spaced
by seven residues and very close to each other in the wheel diagram (Fig 1B) This aromatic cluster could be respon-sible for the hydrophobic interactions of AChETsubunits
Chemical grafting of synthetic peptides confers hydrophobic properties on water-soluble AChE
To characterize the interactions of the t region while excluding possible effects of putative post-translational modifications, we used chemically synthesized peptides, as shown in Fig 1C Peptide t1)40corresponds to the whole Torpedot peptide; peptide t1)32corresponds to its first 32 aminoacids and contains all seven conserved aromatic residues
The peptides were grafted onto a water-soluble tetrameric form (G4) of Electrophorus electricus AChE, obtained by tryptic digestion of collagen-tailed forms from the electric organ [29,30] We used this enzyme preparation because we could obtain it in a highly purified form [18] and because
it was very stable, totally nonamphiphilic and could be
Fig 1 Sequence and putative organization of the C-terminal t peptide from AChE T (A) Primary structure of the last 12 residues of the catalytic domain and of the t peptide.
A comparison of the Torpedo and rat sequences shows the high degree of conserva-tion, particularly of the seven aromatic resi-dues, throughout vertebrates The N-terminal region of the human amyloid Ab peptide is shown to indicate a 12 residue segment which presents some homology with the t peptide (underlined) (B) Proposed helical structure of the N-terminal region of the t peptide: in the side view, the distance of each residue from the helix axis corresponds to the vertical dimen-sion, with the central residue of the aromatic cluster (W17) at the top The position along the axis corresponds to the horizontal dimen-sion (arbitrary scales) The wheel representa-tion corresponds to a faceview along the helix axis of the segment of the t peptide containing the aromatic residues (C) Synthetic peptides corresponding to different parts of the
t peptide The underlined residues have been substituted from the wildtype sequence of the Torpedo marmorata t peptide.
Trang 5analyzed by the same methods used for the amphiphilic
AChE species Chemical coupling of the synthetic peptides
to exposed lysine residues occurred randomly and did not
affect enzymic activity
We deduced the mean number of peptides added per
tetramer from the apparent increase in molecular mass: the
modified Electrophorus G4AChE molecules obtained after
coupling of the peptides sedimented as fairly homogenous
peaks, as illustrated in Fig 2A The sedimentation
coeffi-cient of G4-t1)32 and of G4-t1)40 was about 12.8 S, as
compared to 11.8 S for the original G4 form (Fig 2B)
Assuming that the mass of this globular protein is
propor-tional to S3/2, we estimate that the mass of the tetramer
increased from 320 kDa to 360 kDa, i.e 10 kDa per
subunit, which corresponds to an average of three grafted
peptides per AChE subunit In the case of G4-t1)40and
G4-t25)40, the formation of complexes with antibodies raised
against t25)40confirmed that essentially all the Electrophorus
G4AChE molecules had been modified (not shown) The
G4-t1)32derivative did not bind the antibodies, indicating
that the t1)32peptide did not contain the necessary epitopes
The G4-t25)40derivative, like the original Electrophorus
G4enzyme, was not amphiphilic: its sedimentation coeffi-cient (12.9 S) was not influenced by the presence of detergent in the gradients By contrast, the G4-t1)32 and
G4-t1)40 derivatives were clearly amphiphilic, as they sedimented more slowly in the presence of Triton X-100 and even more slowly in the presence of Brij-96 (Fig 2A,B) This amphiphilic character was confirmed by charge-shift electrophoresis under nondenaturing conditions The t-peptide–AChE conjugates migrated in opposite directions
in the presence of the negatively and positively charged detergents, cetyltrimethylammonium bromide (CTAB) and
Na+deoxycholate (not shown)
The fact that the short t1)32peptide and the long t1)40 peptide confer amphiphilic properties to Electrophorus AChE tetramers, whereas the t25)40 peptide does not suggests that the 1–32 region, containing an a helix with seven aromatic residues, is sufficient to support hydropho-bic interactions
Characterization of t peptide–lipid interactions
by use of circular dichroism Figure 3 shows the CD spectrum in the far UV of the t1)32 peptide under various conditions In organic solvents, such
as methanol, the spectrum presents the characteristic features of an a helical structure, with double minima at
210 nm and 222 nm The h222value of)31 600 degÆdmol)1Æ
cm2indicates that about 85% of the polypeptide is a helical
We obtained a similar proportion of a helical structure by reconstituting the whole spectrum as a sum of the contri-butions of different secondary structures, derived from a set of known proteins [31] This high a helical content
is comparable to that of amphiphilic peptides of similar length, which have been characterized by various methods
as monomeric 20-residue a helical rods [32,33] When the peptide was dissolved in an aqueous buffer, the minima at
210 nm and 222 nm displayed ellipticities of only
h¼)12 210 degÆdmol)1Æcm2and h¼)9770 degÆdmol–lÆcm2 respectively, indicating a much lower a helical content of
35%
Fig 2 Effect of detergentson the sedimentation of Electrophorus AChE
tetramers, chemically coupled with the t 1)40 peptide (A) Sedimentation
patterns of a conjugate of Electrophorus AChE G 4 species with the
t1)40peptide, obtained in sucrose gradients containing no detergent;
0.1% Triton X-100 or 0.1% Brij-96 (B) Sedimentation coefficients
obtained in these different conditions for G 4 AChE and its conjugates.
The conjugated enzymes containing peptides t1)32 and t1)40
sedi-mented faster without detergent than in the presence of Triton X-100
or Brij-96, indicating that they bind detergent micelles, in contrast with
conjugated enzyme containing peptide t25)40and the nonconjugated
enzyme, which sedimented in the same way under all three conditions.
Fig 3 Far UV dichroic spectrum of peptide t 1-32 Peptide (5 l M ) in
1 m M Tris/HCl buffer, pH 7.5, using a 1 cm path-length cuvette (dotted line); the same solution after addition of lysolecithin micelles, with a lipid/peptide molar ratio of 20 (thin line); 50 l M peptide in methanol, using a 0.1 path-length cuvette (bold line).
Trang 6The CD spectrum was markedly modified by addition of
lysolecithin micelles It approached that observed in
meth-anol when the lysolipid/peptide molar ratio was about 10,
and was not modified further at higher micelle
concentra-tions (Fig 3) Under these condiconcentra-tions, the helical content
was about 68%, corresponding to 18–22 residues per
peptide organized into an a helix Thus, lipid micelles can
induce an a helical conformation in the t peptide
Intrinsic fluorescence of the t peptide
The t1)32peptide displays intrinsic fluorescence due to the
fact that it contains three tryptophans; W10, W17 and W24,
and two tyrosines, Y20 and Y31 When dissolved in aqueous
buffer and excited at 280 nm, its emission spectrum was
centered at 345 nm The shape of the emission spectrum was
identical when excitation was at 295 nm, a wavelength at
which tyrosine residues do not absorb Thus the fluorescence
of the peptide is totally due to tryptophan residues: Y20 and
Y31 are either totally quenched or very efficiently transfer
their energy to tryptophan residues in their neighbourhood
In aqueous solution, the fluorescence of the tryptophan
residues showed a blue shift of 6 nm relative to
N-acetyltryptophanylamide, indicating that they are only
slightly buried The blue shift was increased by about 2 nm
when dithiothreitol was omitted Addition of methanol,
which decreased the polarity of the medium, prevented
aggregation and increased the a helical content; this
pro-duced an increase in quantum yield and a slight shift of the
maximum emission wavelength, indicating that the
trypto-phan residues were more exposed to the solvent
We obtained similar results with the t1)40peptide, except
that it was more aggregated in aqueous solution; the t1)32
peptide also aggregated above 1 lM, as indicated by an
increase in the light scattering On the contrary, reducing the
concentration below 0.2 lMinduced a progressive red shift
of the emission kmaxfor both peptides; however, this never
reached 350 nm, which would correspond to total exposure
of tryptophan residues
Interaction of peptides with detergents and
phospholipids as followed by fluorescence
We followed changes of the intrinsic tryptophan
fluores-cence by addition of phospholipids (Fig 4) and detergents
(Fig 5) The induced blue shifts in the kmaxof emission and
intensity changes were similar for t1)32and t1)40
Figure 4 shows that addition of lipid vesicles to an
aqueous solution of the t1)32 peptide at pH 7.5 (5 lM)
produced changes both in intensity and wavelength of
fluorescence For the zwitterionic egg lecithin vesicles, the
changes did not reach a plateau even at Ri values greater
than 150, indicating a low affinity of the peptides for the
lecithin–water interface In contrast, we observed a stronger
blue shift and more pronounced quenching upon addition
of negatively charged phosphatidylserine vesicles, and both
effects reached a plateau below an Ri value of 100 The
emission maximum at the plateau, 327 nm, indicates that
the tryptophan residues were in a very hydrophobic
environment
Figure 5 shows that addition of 32 lM lysolecithin to
peptide t1)32(3.2 lM) shifted the emission maximum close
to 330 nm, and increased the intensity twofold The affinity
of the peptide was much higher for lysolecithin micelles than for lecithin vesicles, indicating that insertion of the peptide is easier in the more fluid and dynamic lysolecithin micelles than in the bilayer of lecithin vesicles, as observed for other amphiphilic peptides [34]
At lower concentrations of the peptide (0.5 lM), lysolecithin induced a similar shift in kmax but a more complex variation of the intensity, which first decreased, reaching a minimum at a lipid : peptide molar ratio (Ri)
of 30–40 and then increased again (not shown) Such biphasic curves were previously observed for lipid–peptide interactions occurring in the concentration range of the critical micellar concentration (cmc) [17] For lysolecithin, the cmc is 20 lM, corresponding to Ri values of 5–6 and 30–40, for peptide concentrations of 3.2 and 0.5 lM respectively These observations show that the t1)32 peptide interacts with lysolecithin both below and above the cmc
CTAB is a positively charged detergent with a cmc of 0.2–0.3 mM, and SDS is negatively charged and has a cmc
of 1–2 mM [35] At neutral pH, addition of CTAB to 3.2 lM peptide shifted kmaxdown to 334 nm, reaching a plateau for Ri¼ 20, and induced a large increase in the intensity at 334 nm, attaining 260% for Ri values above
100, i.e above the cmc that corresponds to Ri values of 60–90 (Fig 5) In contrast, SDS did not induce any significant change in fluorescence up to Ri¼ 150; above this value, we noted a gradual shift of kmaxdown to 330 nm for Ri¼ 300–400, i.e above the cmc of the detergent We obtained similar results at pH 5.7, in spite of a reduction in
Fig 4 Effects of phospholipid vesicles on the intrinsic fluorescence of peptide t 1)32 The peptide concentration was 5 l M , in 20 m M Tris/ acetate buffer pH 7.5 containing 5 m M dithiothreitol to avoid the formation of disulfide bonds, under a nitrogen stream, at 25 C (A) Variation of the wavelength of maximum emission (k max ) as a function
of the molar ratio of lipids to peptide (Ri) (B) Relative variation of emission intensity at 333 nm (DI/I 0 ) as a function of Ri (m) egg lecithin vesicles; (s) phosphatidylserine vesicles.
Trang 7the negative charge of the peptide Thus, the zwitterionic
and positively charged detergents readily interact with the
peptides even below the cmc, while the negatively charged
detergent interacts only when approaching the cmc
Formation of disulfide bonds in homomeric oligomers
with cysteines at various positions in the C-terminal
region of rat AChETsubunits
The preceding studies were performed on isolated peptides
or on conjugates in which peptides were chemically coupled
at the surface of a protein However, the t peptide is
normally linked to the C-terminus of the catalytic domain of
AChET subunits and it contains a cysteine (C37) which
allows their dimerization through an intersubunit disulfide
bond The crystallographic structure of AChE dimers
[36,37] or monomers [38] shows that the catalytic domain
terminates with an a helix (helix a10) constituted by residues
)18 to )1 Secondary structure predictions suggest that this
helix is separated from the a helical portion of the t peptide
by a short loop (around residues)1 to 2), and may present a
break around residues 15–16 (Fig 1A) An interrupted helix
could form a hairpin, as proposed by Giles [16], who
suggested that the aromatic-rich sectors of two a helical
segments would constitute a compact aromatic cluster
According to this model, a bend at residues 21 and 22 would
bring the N-terminal and C-terminal ends into close proximity
To obtain information on the articulation between the catalytic domain and the t peptide, we analyzed the formation of intercatenary disulfide bonds by cysteine residues located at the end of the catalytic domain of rat AChETor at the beginning of its t peptide, in the)5 to 6 interval; in these mutants, the original cysteine was either retained or replaced by a serine (C37S) We also introduced cysteine residues near the middle of the t peptide, in the predicted a helical region containing aromatic residues (at positions 19 and 21), and in its C-terminal region, which is not predicted to be a helical, at positions 34 to 36 The AChET cysteine mutants were expressed in transi-ently transfected COS cells In the absence of any cysteine in the C-terminal region of rat AChE, we obtained mainly monomers, with a small proportion of tetramers, as reported previously in the case of human AChE [39] and rat AChE [40] Therefore, the presence of dimers, as observed in the case of the other mutants, indicates the formation of an intercatenary disulfide bond
In the hypothesis of a hairpin structure, an intracatenary disulfide bond might be formed in mutants containing the original cysteine or another C-terminal cysteine, together with a cysteine in the N-terminal region of the t peptide; this would preclude the formation of dimers, which requires an intercatenary disulfide bond However, we did not observe this in any combination of N-terminal and C-terminal cysteines (not shown) Therefore, the t peptide almost certainly adopts an elongated conformation in AChET monomers and dimers
When the original cysteine was mutated to serine (C37S), all mutants containing a single cysteine at positions)5 to 6,
19, 21, or 34 to 36, produced active AChE which was secreted at variable levels (Fig 6A) The cellular and secreted enzymes contained different proportions of dimers, sometimes with a small amount of tetramers, as indicated by nondenaturing electrophoresis (Fig 6B)
Sedimentation patterns illustrating the amounts of mono-mers, dimers and tetramers are shown in Fig 7 for cysteines
in the)5 to 6 interval The proportion of dimers produced was very low with cysteines in the)5 to )3 interval, a region which is predicted to be a helical The distances between pairs of alpha carbons corresponding to residues)5 to )2 can be determined from the crystallographic structure of a catalytic dimer [36]: they are 8.6, 13, 15 and 8.9 A˚ respectively A small proportion of AChETsubunits were dimerized with cysteines at position)5 and )2, for which the distance is smallest but still appears too high for establishment of a disulfide bond, which is normally < 6 A˚ This indicates that, in AChETsubunits, the distal part of the catalytic domain is sufficiently flexible to allow the forma-tion of a disulfide bond in this segment, between the two subunits in a dimer The production of dimers was higher than for the wildtype with cysteines at positions )2 to 3, suggesting that this region, which is predicted to form a coil, constitutes a flexible hinge between the catalytic domain and the amphiphilic helix of the t peptide; it was lower at positions 4 and 5 and increased again at position 6 As these three residues are probably included in the N-terminal region of the helix, the observed variations in the efficiency of dimerization may be due to their orientation relative to the
Fig 5 Effectsof zwitterionic and charged detergentson the intrinsic
fluorescence of peptide t 1-32 The peptide concentration was 3.2 l M , in
20 m M Tris/HCl buffer containing 5 m M dithiothreitol to avoid the
formation of disulfide bonds (A) Variation of the wavelength of
maximum emission (k max ) as a function of the molar ratio of detergent
to peptide (Ri) (B) Relative variation of emission intensity at 333 nm
(DI/I 0 ) as a function of Ri (h, j) SDS; (s, d)
cetyl-trimethyl-ammonium bromide (CTAB); (n, m) lysolecithin Filled symbols
(j, d, m), pH 7.5; open symbols (h, s, n), pH 5.7.
Trang 8aromatic sector: residue 6 is in the aromatic sector, while
residues 4 and 5 are on the opposite side
We also studied the production of dimers with cysteine
residues located at positions 19 and 21, in the center of the
predicted amphiphilic a helical region but in opposite
sectors With a cysteine at 19, the cellular enzyme contained
dimers but their secretion was very low (Fig 6B), suggesting
that the presence of a disulfide bond at this position induced
their degradation In contrast, a cysteine at position 21,
within the sector containing aromatic residues, appeared
much more favorable for dimerization and secretion In
contrast with dimers containing disulfide bonds in the
N-terminal or C-terminal regions of the t peptide, the
M21C/C37S dimers did not interact with detergent micelles
(not shown), indicating that the two aromatic clusters
occluded each other
Dimers were as efficiently produced and secreted with
cysteines located at positions 34, 35 or 36 as with the original
cysteine (at position 37) suggesting that this C-terminal
region of the t peptide is flexible It is noteworthy that the
level of cellular activity was markedly higher with a cysteine
at 35, corresponding to an increased amount of monomers; the presence of a cysteine instead of an aspartic acid at this position seems to increase the retention or decrease the degradation of monomers
Figures 6B and 7 show that the production of tetramers varied with the position of the cysteine and was not proportional to that of dimers Tetramer production was systematically higher with C-terminal cysteines (34–37) than with cysteines in the N-terminal region of the t peptide ()2
to 3) This suggests that the relative organization of the
t peptides and of the catalytic domains is more favorable for tetramerization when dimers are joined through a C-terminal disulfide bond
Hetero-oligomerization: orientation of the PRAD and t peptides in the T4–QNcomplex
The QN protein possesses two adjacent cysteine residues (C70 and C71) located immediately upstream of the
proline-Fig 6 Effect of cysteines at various positions
in the C-terminal region of rat AChE T subunits Cysteines were introduced into rat AChE T
subunits at various positions at the junction of the catalytic domain and the t peptide ( )5 to 6), in the middle of the t peptide (19 or 21), and in the C-terminal part of the t peptide (34
to 36); in these mutants, the original cysteine (C37) was replaced by a serine, so that all mutants possessed a single cysteine (A) Cel-lular and secreted AChE activities: all mutants produced and secreted active AChE when expressed with or without Q N When cysteines were present in the )5 to 6 interval, we used a modified Q N (Q N -CC) with cysteines down-stream of the proline-rich region; with the other mutants, we used the Q N protein con-taining cysteines upstream of the proline-rich region Activities are expressed as percentage
of the wildtype; the bars indicate the standard errors of two to three independent experi-ments The shaded and hatched rectangles correspond to mutants expressed without and with Q N , respectively (B) Nondenaturing electrophoresis of AChE oligomers produced
by rat AChE T subunits containing a single cysteine at different positions, expressed without Q N
Trang 9rich motif (Fig 8A,B) such that the disulfide linkage of two
AChETsubunits with one QNprotein produces a heavy
dimer that can be distinguished by SDS/PAGE under
nonreducing conditions from light dimers consisting of
only two disulfide linked AChETsubunits [12] (Fig 8C) In
order to study the formation of these disulfide bonds,
AChET mutants were coexpressed with the natural QN
protein possessing two adjacent cysteines C70 and C71
upstream of the PRAD (CC-QN), and with a QNmutant in
which the original cysteines were mutated to serines and two
cysteines were introduced downstream of the PRAD, at
positions 87 and 88 (QN-CC), as shown in Fig 8A The
C37S mutant that formed no intercatenary disulfide bonds
but was recruited into T4–QN complexes, served as a
control
Figure 8Ca illustrates the fact that CC-QN formed
disulfide bonds with AChE mutants that contained a
cysteine in the C-terminal region of the t peptide (as expected, given that this corresponds to the wildtype situation), but not with AChET mutants containing an upstream cysteine (Fig 8Cb); in the latter case, all AChET subunits were disulfide-linked in homodimers Recipro-cally, disulfide bonds could be formed, although less efficiently, between QN-CC and some of the AChET mutants that contained a cysteine in the N-terminal region
of the t peptide (Fig 8Cd) but not in the C-terminal region (Fig 8Cc) This indicates that the N- and C-terminal extremities of the t peptides are distant in the complex, eliminating the possibility that the peptides would be folded in hairpins as suggested above for the free t peptides; the same reasoning shows that the PRAD
is also elongated
Taking into consideration that both the t peptides and the PRAD are elongated in the heteromeric complexes, we
Fig 7 Formation of homo-oligomersof rat
AChE T subunitswith cysteinesat
variousposi-tionsin their C-terminal region Sedimentation
patterns in sucrose gradients, for mutants of
the )5 to 6 interval The patterns obtained for
the wildtype (solid line) and for the C37S
mutant with no C-terminal cysteine (C37S)
(dotted line) are shown for comparison The
monomers, dimers and tetramers are indicated
(T 1 , T 2 , T 4 respectively) The areas under the
sedimentation profiles are proportional to the
cellular and secreted activities, so that the
areas of the peaks represent the relative
amounts of the corresponding molecular
forms.
Trang 10can then question their respective orientations: the
forma-tion of intercatenary disulfide bonds shows that the four
t peptides are all parallel, and are oriented in the opposite
direction to the PRAD, as the N-terminal extremity of the
PRAD can only be disulfide linked to the C-terminal region
of two t peptides, and vice versa
Exploring the association of t peptides and PRAD
in the T4–QNcomplex by the formation of heterophilic intercatenary disulfide bonds
Figure 9A shows an analysis of the complexes formed between the various AChET cysteine mutants and QN, in
Fig 8 Disulfide bonds between Q N and two AChE T subunits, in the T 4 –Q N complex (A) Schematic representation of the constructs used The T 4 –Q N
complex was formed when AChE T subunits possessing a cysteine near the N- or C-terminus of the t peptide were expressed with Q N constructs containing pairs of cysteines located either upstream or downstream of the PRAD The arrows indicate the N-terminal to C-terminal orientation (B) Schematic representation of the different combinations of cysteine mutants; the PRAD is shown as a thick central line and the t peptides as zigzags; the cysteines are indicated by circles and the disulfide bonds by thick lines Scheme a corresponds to the wildtype
an association of wildtype t peptides with Q N -CC; c and d correspond to associations of t peptides containing an upstream cysteine (L3C/C375) with CC-Q N and Q N -CC, respectively (C) Analysis of disulfide-linked species by SDS/PAGE after metabolic labeling Lanes a, b, c, d correspond
to the four diagrams in panel
45 (B) Heavy dimers (composed of one Q N protein linked to two AChE T subunits) were produced only when cysteines were at opposite ends of the t peptide and PRAD.
Fig 9 Effect of the position of cysteines in the C-terminal region of AChE T on the formation of hetero-oligomers(T 4 -Q N ) (A) Nondenaturing electrophoresis of AChE oligomers secreted by cells expressing AChE T subunits with the appropriate Q N construct (Q N -CC for AChE T subunits containing a cysteine in the )5 to 6 interval, CC-Q N for AChE T subunits containing a cysteine at positions 19, 21 and in the 34 to 37 interval) (B) Analysis of disulfide bonds between AChE T subunits and Q N (heavy dimers), by nonreducing denaturing electrophoresis after metabolic labeling There were no heavy dimers with cysteines at 19 or 21, with either the CC-Q or Q -CC construct (not shown).