Elements of the C-terminal t peptide of acetylcholinesterase that determine amphiphilicity, homomeric and heteromeric associations, secretion and degradation Ste´phanie Belbeoc’h, Cinzia
Trang 1Elements of the C-terminal t peptide of acetylcholinesterase that determine amphiphilicity, homomeric and heteromeric associations, secretion and degradation
Ste´phanie Belbeoc’h, Cinzia Falasca, Jacqueline Leroy, Annick Ayon, Jean Massoulie´ and Suzanne Bon Laboratoire de Neurobiologie Cellulaire et Mole´culaire, CNRS UMR 8544, Ecole Normale Supe´rieure, Paris, France
The C-terminal t peptide (40 residues) of vertebrate
acetyl-cholinesterase (AChE) T subunits possesses a series of seven
conserved aromatic residues and forms an amphiphilic
a-helix; it allows the formation of homo-oligomers
(mono-mers, dimers and tetramers) and heteromeric associations
with the anchoringproteins, ColQ and PRiMA, which
contain a proline-rich motif (PRAD) We analyzed the
influence of mutations in the t peptide of Torpedo AChETon
oligomerization and secretion Charged residues influenced
the distribution of homo-oligomers but had little effect on
the heteromeric association with QN, a PRAD-containing
N-terminal fragment of ColQ The formation of
homo-tetramers and QN-linked tetramers required a central core of
four aromatic residues and a peptide segment extending to
residue 31; the last nine residues (32–40) were not necessary,
although the formation of disulfide bonds by cysteine C37
stabilized T4and T4–QNtetramers The last two residues of the t peptide (EL) induced a partial intracellular retention; replacement of the C-terminal CAEL tetrapeptide by KDEL did not prevent tetramerization and heteromeric association with QN, indicatingthat these associations take place in the endoplasmic reticulum Mutations that disorganize the a-helical structure of the t peptide were found to enhance degradation Co-expression with QN generally increased secretion, mostly as T4–QNcomplexes, but reduced it for some mutants Thus, mutations in this small, autonomous interaction domain bringinformation on the features that determine oligomeric associations of AChETsubunits and the choice between secretion and degradation
Keywords: acetylcholinesterase; degradation; disulfide bonds; oligomerization; secretion
In vertebrates, the acetylcholinesterase (AChE) gene
gener-ates several types of catalytic subunits through alternative
splicingin the 3¢ region of the transcripts [1–3] These
subunits possess the same common catalytic domain,
followed by distinct C-terminal peptides (r, h and t),
characterizingthe AChER, AChEH and AChET variants
[4–6] In mammals, AChERsubunits seem to be expressed
mostly duringembryogenesis and in the brain after stress
[7,8]; they correspond to a soluble, monomeric enzyme
species AChEHsubunits possess one or two cysteines and a
GPI-addition signal in their C-terminal peptide: they
generate GPI-anchored, disulfide-linked dimers, which
represent a major fraction of AChE in Torpedo electric
organs and muscles, and are expressed on the surface of blood cells in mammals [9–12] AChET subunits are expressed in muscles and in the nervous system of higher vertebrates and therefore represent the functional cholinest-erase species in the cholinergic system [3,13,14]
The C-terminal t peptide confers several characteristic properties to AChET subunits, allowingthem to form a series of homo-oligomers (monomers, dimers, tetramers and higher oligomers) when expressed in transfected COS cells [13,15]; some of these molecules are amphiphilic, i.e interact with detergent micelles [16,17] AChETsubunits also form hetero-oligomers with the collagen, ColQ, or with the transmembrane protein, PRiMA [18,19]; in mammals, these structural proteins anchor the major functional species
of cholinesterases in the basal lamina of the neuromuscular junction and in neuronal cell membranes, respectively [20,21]
In the collagen-tailed and hydrophobic-tailed forms, four catalytic AChE subunits are associated, through their C-terminal t peptides, with proline-rich attachment domains (PRAD) localized in the N-terminal regions of ColQ or PRiMA [19,22,23]
The t peptide of AChE consists of 40 residues, with a series
of seven strictly conserved aromatic residues, includingthree evenly spaced tryptophans, as well as acidic and basic residues that are conserved or semiconserved in most vertebrates [5] This peptide is necessary for the amphiphilic properties which characterize AChET subunits and some
of their oligomers (Ta, Ta, Ta), for the formation of nonamphiphilic homotetramers (Tna), as well as for 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 R , AChE subunit of type R; AChE T , AChE subunit
of type T; ERAD, endoplasmic reticulum associated degradation;
PRAD, proline-rich attachment domain; r, h, t, alternative C-terminal
peptides of AChE; WAT, tryptophan (W) amphiphilic tetramerization
domain.
Enzymes: acetylcholinesterase (E.C 3.1.1.7).
(Received 8 January 2004, revised 20 February 2004,
accepted 24 February 2004)
Trang 2heteromeric association of AChET subunits with QN, an
N-terminal fragment of collagen ColQ that contains a
proline-rich motif, thus producingT4–QNcomplexes [23,24]
The t peptide constitutes an autonomous interaction
domain, called the WAT [tryptophan (W) amphiphilic
tetramerization] domain, because it can associate with a
PRAD, even in the absence of the catalytic domain;
moreover, addition of a t peptide at the C-terminus of
foreign proteins, green fluorescent protein and alkaline
phosphatase, endowed them with amphiphilic properties
and enabled them to form PRAD-associated tetramers [25]
We also found that the simultaneous presence of the t
peptide and of mutations at the interface of AChE dimers –
the four helix bundle [26] – prevented the secretion of
AChET subunits [27] We recently showed that the t
peptide induces intracellular degradation through the
endoplasmic reticulum associated degradation (ERAD)/
proteasome pathway, to different extents, dependingon the
protein to which it is attached, and that aromatic residues
are necessary for this effect [28]
Recent spectroscopic studies showed that the t peptide is
organized as an amphiphilic a helix, in which aromatic
residues form a hydrophobic sector [29,30] In addition,
an analysis of intercatenary disulfide bonds in the T4–QN
complex also demonstrated that the four t peptides are
parallel and oriented in the same direction, opposite to that
of the PRAD [30] The structure of a complex formed
between synthetic peptides (four t peptides with one PRAD)
confirmed this orientation (M Harel et al., manuscript in
preparation)
In the present study, we mutated aromatic and charged
residues, suppressed the C-terminal cysteine or introduced
cysteines at other positions, and deleted more or less extended
C-terminal segments of the t peptide, in Torpedo AChET
subunits, to determine the structural basis of the
character-istic properties that the t peptide confers to AChETsubunits
Materials and methods
AChE constructs and site-directed mutagenesis
Mutagenesis was performed according to the method of
Kunkel et al [31] cDNAs encodingwild-type and mutated
Torpedo AChET, as well as the previously described
Torpedo QN protein [23], intact or deleted of its PRAD
motif (residues 70–86), were inserted into the pEFBos
vector Throughout this article, the residues of the t peptide
are numbered from 1 to 40, correspondingto positions
536–575 in the Torpedo AChETsubunit, so that the Torpedo
mutants are indicated by the modified residues, e.g W17P
Transfection of COS cells
COS cells were transfected by the DEAE-dextran method,
as described previously [24], using4 lgof DNA encoding
the AChE catalytic subunit and 4 lgof DNA encodingQN
or PRAD-deleted QN, per 100 mm dish Because Torpedo
AChE folds into its active conformation at 27C, but not at
37C, the cells were incubated for 2 days at 37 C after
transfection, then transferred to 27C and maintained at
this temperature for 3–4 days, in a medium containing10%
Nuserum (Inotech, Dottikon, Switzerland), which had been
pretreated with 10)6Msoman to inactivate serum cholin-esterases
To analyze its heteromeric interaction with an associated structural protein, AChET was coexpressed with QN[23]
By usingQN, rather than full-length ColQ, we avoid the complexity caused by the formation of the triple helical collagen and by the low salt aggregation of collagen-tailed AChE forms [32] We added a flagepitope (DYKDDDDK) at the C-terminus of QN, so that complexes containingthis protein could be characterized with the anti-flagimmunoglobulin, M2 (Kodak), as described previously [24] The effect of QNon the level of cellular and secreted activity was analysed by comparing the coexpression of AChETwith full-length QNand with a PRAD-deleted QN, to compensate for competition between the two transfected vectors
Cell extracts The cells were extracted at 20C with TMgbuffer (1% Triton X-100, 50 mM Tris/HCl, pH 7.5, 10 mM MgCl2), and then centrifuged at 10 000 g for 30 min Media were also centrifuged at 10 000 g for 30 min to remove cell debris before analysis
Enzyme assays AChE activity was determined accordingto the colorimetric method of Ellman et al [33] at room temperature As the monomeric Torpedo AChE forms produced by some mutants were inactivated by 5,5¢-dithiobis(2-nitrobenzoic acid) [34], the enzyme samples were incubated for variable periods of time, dependingon their activity, with a reaction medium containingacetylthiocholine iodide in phosphate buffer, pH 7; 5,5¢-dithiobis(2-nitrobenzoic acid) was then added and the absorbance at 414 nm was determined using
a Labsystems (Helsinki, Finland) Multiskan RC automatic plate reader Alkaline phosphatase and b-galactosidase from Escherichia coli were assayed with the chromo-genic substrates p-nitrophenyl phosphate and o-nitrophenyl galactoside, respectively
Sedimentation and electrophoretic analyses Centrifugation was performed in 5–20% sucrose gradients (50 mM Tris/HCl, pH 7.5, 50 mM MgCl2, either in the presence of 0.2% Brij-97 or in the presence of 0.2% Triton X-100) in a Beckman SW41 rotor, at 36 000 r.p.m., for 18 h
at 6C The gradients contained E coli b-galactosidase (16 S) and alkaline phosphatase (6.1 S) as internal sedi-mentation standards [24] Amphiphilic molecules generally sediment faster in the presence of Triton X-100 than in the presence of Brij-97, providingan indication of their amphiphilic character
Electrophoresis in nondenaturatingpolyacrylamide gels was performed as described by Bon et al [16], and AChE activity was revealed by the histochemical method of Karnovsky & Roots [35] In charge shift electrophoresis, the electrophoretic migration of amphiphilic molecules was accelerated in the presence of sodium deoxycholate, when compared to migration in the presence of the neutral detergent, Triton X-100, alone As an index of the degree of
Trang 3amphiphilicity, we used the ratio between migration in the
presence of DOC to migration in Triton X-100 alone, after
normalizingthese migrations to that of a nonamphiphilic
species, the wild-type tetramers T4naor T4–QN
Both sedimentation and nondenaturingelectrophoresis
provide semiquantitative information on the interaction of
AChE molecules with micelles, and are generally in
complete agreement However, in the present study, we
found that some mutations in the t peptide perturb
amphiphilic interactions in such a way that sedimentation
became essentially identical in the presence of Triton X-100
and Brij-97, while charge shift electrophoresis still showed a
marked influence of the detergent: this was the case for
dimers of aromatic mutants such as W17H or W17A In
addition, the T4–QNcomplexes formed by mutants W17F
and W17A showed an unusual retardation in sedimentation
in the presence of Triton X-100, compared with Brij-97
Results
Analyses of AChE activity and molecular forms
Figure 1 shows the sequence of the t peptide of Torpedo
AChETsubunits, and schematically illustrates its proposed
a helical structure, its association with the PRAD of
ColQ, and the various oligomers of AChETsubunits that
result from its interactions
We analyzed how mutations in the t peptide affect the
levels of cellular and secreted activity of Torpedo AChE in
transfected COS cells The activities were normalized to
those obtained for wild-type AChETin parallel transfections
Immunofluorescence of the protein produced at early stages
after transfection indicated that all mutants were expressed
in a similar manner After 2 days at 27C, a temperature
which allows the correct foldingof active Torpedo AChE
(see the Materials and methods), the level of cellular activity
reached a plateau and the rate of secretion remained
constant Maximal secretion was obtained for a truncated
mutant (I3C/stop4), which retained only the first two
residues of the t peptide, followed by a cysteine at position
3; this cysteine allowed the formation of dimers, which
lacked the aromatic residues and were therefore
nonamphi-philic The secretion of active wild-type AChET subunits
was less than 10% of the truncated mutant, showingthat a
large fraction is degraded intracellularly [27,28]
The molecular forms of AChE were identified by
electrophoresis in nondenaturingpolyacrylamide gels and
their amphiphilic character was evaluated by charge shift
electrophoresis in the presence or absence of sodium
deoxycholate [16] As Torpedo AChET monomers are
rapidly inactivated under the conditions of electrophoretic
migration, the distribution of AChE molecular forms was
analyzed by sedimentation in sucrose gradients
To analyse the capacity of Torpedo AChETsubunits to
associate with a PRAD, we coexpressed them with protein
QN(Fig 1D) This QNprotein organizes wild-type AChET
subunits into tetramers (T4–QN) that are nonamphiphilic
and efficiently secreted [24], reaching 40% of the secretion
observed with the truncated I3C/stop4 mutant The
forma-tion of QN-linked oligomers therefore rescued an important
fraction of the wild-type catalytic Torpedo AChETsubunits
from intracellular degradation
Fig 1 Structure of the t peptide and oligomeric associations of acetylcholinesterase type T subunits (AChE T ) (A) primary sequence
of the t peptide from Torpedo AChE T subunits The residues of the
t peptide, encoded by an alternatively spliced 3¢ exon, are numbered from 1 to 40 and correspond to residues 536–575 of the mature Torpedo AChE T subunit; cysteine C37, which is responsible for intercatenary disulfide bonds, is circled (B) Side view of the t peptide, with its 1–32 segment organized as an a helix The con-served aromatic residues are located in the upper sector of the helix (C) Wheel representation of the entire t peptide, putatively organ-ized as an a helix Aromatic residues, shown in shaded circles, are located in the upper sector; charged residues are in double circles (white for basic residues, grey for acidic residues) and possible salt bridges are marked by hatched bars; cysteine C37 is in a double, grey circle; arrowheads indicate residues that have been mutated to cysteines (D) Primary sequence of the proline-rich attachment domain (PRAD) motif from Torpedo ColQ The PRAD residues are shown in bold text (from cysteines 70 and 71 to phenylalanine 86), and a few adjacent residues are shown in non-bold text (E) Schematic representation of a complex between four t peptides and a PRAD The N- and C-terminal extremities (indicated N and C) and arrows show the orientations of the t peptides (black zig-zags) running opposite to the PRAD (grey line); cysteines are indicated by circles, joined by lines representingdisulfide bonds (F) Major types of homomeric and heteromeric associations analyzed in this study: T a , Ta and Ta, amphiphilic monomer, dimer and tetramer of AChE T subunits; T na
4 , nonamphiphilic tetr-amer; T 4 –Q N , tetramer associated with the N-terminal Q N fragment
of ColQ, containingthe PRAD motif The schemes of heteromeric complexes are derived from recent studies (M Harel, H Dvir,
S Bon, W Q Liu, M Vidal, C Garbay, J L Sussman,
J Massoulie´ & I Silman, unpublished results) [30].
Trang 4Mutation of charged residues of the t peptide
The t peptide contains seven acidic (D, E) and eight basic
(H, K, R) residues, which may form intracatenary salt
bridges in the helical conformation (Fig 1C) and perhaps
intercatenary salt bridges in oligomeric assemblies; we
mutated these residues to alanines, individually or in groups
Mutations D4A/E5A, E7A/R8A, K11A, E13A, R16A,
K25A, or D29A did not markedly modify the levels of
cellular and secreted activities However, other mutations
had a stronger effect, as shown in Fig 2A Both cellular and
secreted activities were increased by the point mutation,
E1A, but decreased by replacement of the first four acidic
residues (E1, D4, E5, E7) by alanines Mutation H15A
enhanced the efficiency of secretion, because it decreased the
cellular activity but increased the secreted activity
Like the wild-type AChETsubunits, all mutants produced
amphiphilic dimers (T2a) and nonamphiphilic tetramers
(T4na) However, their proportions varied, as illustrated by
the sedimentation profiles of four mutants (Fig 2B) These
profiles did not change with time after transfection They
characterize each mutant and are not simply related to the
intracellular concentration of the enzyme, as shown by the fact that the D4A/E5A and R16A mutants produced appro-ximately the same levels of cellular activity with the same proportions of molecular forms as the wild type, but differed
in the activity and molecular forms of the secreted enzyme Conversely, the secreted enzyme was quantitatively and qualitatively similar for mutants K11A and D29A, although the patterns of cellular molecular forms were different
In all cases, coexpression with QNincreased the level of secretion and produced T4–QNcomplexes, as for the wild type
Mutation of aromatic residues The three tryptophans (W10, W17, W24) and Y31 were mutated to alanines, and all seven aromatic residues were mutated to prolines These mutations had little effect on the cellular activity; the secreted activity was reduced by about half by most mutations, but significantly increased by Y31A (data not shown) and Y31P (Fig 3A) The major molecular
Fig 2 Mutations of charged residues in the t peptide (A)
Acetylcho-linesterase (AChE) activities in cell extracts and secreted into the
cul-ture medium are shown for the wild type and four mutants Grey bars
and hatched bars correspond to the AChE activities of mutants
expressed without or with Q N , respectively (Materials and methods);
the activities are normalized to those obtained for the wild type (100%)
both in the cell extracts and in the medium; the standard errors were
obtained from five independent experiments For other individual
mutations (K11A, E13A, R16A, K25A and D29A), the cellular
activities ranged from 77% to 100%, and the secreted activities
between 68% and 136% (B) Sedimentation patterns of cellular and
secreted AChE, in sucrose gradients containing 0.2% Triton X-100.
The shaded areas, as well as the total areas under the sedimentation
profiles, are proportional to the relative activities of the mutants, so
that the surface of each peak represents the actual activity of the
correspondingmolecular form: monomers (T 1 ), dimers (T 2 ) and
tetramers (T ).
Fig 3 Mutations of aromatic residues (A) Secreted activities, nor-malized to that of the wild type, for mutants of aromatic residues to prolines: the bars represent secreted activities obtained when acetyl-cholinesterase type T subunits (AChE T ) were expressed without Q N
(g rey bars) and with Q N (hatched bars); the indicated values are the means of at least three independent experiments The cellular activities ranged from 86 to 114% without Q N and from 65 to 117% with Q N (B) Amphiphilic character of dimers produced by each mutant, indicated by charge shift electrophoresis RDOC/TX is the ratio of electrophoretic migrations in the presence of Triton X-100 with sodium deoxycholate and Triton X-100 alone, normalized to those of a nonamphiphilic species (T na
4 ); the indicated values represent the means of three to six independent experiments (C) Existence of Q N -linked dimers with mu-tant W10P: electrophoretic patterns, in the presence of Triton X-100 and sodium deoxycholate; the third lane shows that a fraction of dimers and tetramers was retarded by the M2 antibody (x), indicatingthat they were associated with the Q N -flagprotein Note that the coexpression with Q N increased the secretion of dimers as well as tetramers (D) AChE molecular forms secreted by mutants of aromatic residues to prolines, expressed with and without Q N ; electrophoretic analysis in the presence of Triton X-100 and deoxycholate s, AChE T dimers; d,
Q N -linked dimers; h, tetramers; j, T 4 –Q N complexes (i.e tetramer associated with the N-terminal Q N fragment of ColQ, containing the proline-rich attachment domain motif); the origin of migration is shown
by a thin line.
Trang 5forms produced by these mutants were T2 dimers, as
illustrated in electrophoretic patterns (Fig 3D); the
pro-duction of tetramers was strongly reduced or abolished,
again with the exception of Y31 mutants The amphiphilic
character of T2 dimers was retained when individual
aromatic residues were replaced with alanines, but it was
reduced when the central aromatic residues were mutated to
prolines, in a position-dependent manner (Fig 3B)
The production of T4–QN complexes, resultingin an
increased secretion, was not affected by mutation of Y31,
but was reduced by mutations of W10 and F28, and was
essentially suppressed by mutations of F14, W17, Y20
and W24, either to alanine (not shown) or to proline
(Fig 3A,D) As illustrated in Fig 3C, we found that when
the W10P mutant was coexpressed with the flagged QN
protein, the anti-flagM2 immunoglobulin reacted with a
fraction of dimers as well as with tetramers, indicatingthe
presence of T2–QN complexes, in addition to T4–QN
complexes, in the culture medium
We replaced the central tryptophan (W17) with a
hydrophobic aromatic residue (F), an aliphatic aromatic
residue (L), a heterocyclic residue (H), as well as a proline
(P) and an alanine (A) As shown in Fig 4, these mutations
did not strongly modify the cellular activity, which remained
within the range of 83–127% of the wild type, but reduced
or suppressed the formation of homotetramers; the
amphi-philic character of the resultingdimers was similar to that of
the wild type with F, L or A, it was significantly reduced
with H and it was essentially abolished with P
Figure 4 also shows that there was no interaction with
QNin the case of W17L and W17P, a very small production
of T4–QNin the case of W17A, and a significant production
of this complex in the case of W17F and W17H For these
two mutants, coexpression with QNincreased the level of
secreted activity to about 60% of that obtained in the wild
type; in addition to the T4–QNcomplex, this coexpression
markedly increased the secretion of dimers, particularly for
W17H, but unlike those formed with the W10P mutant,
these dimers did not seem to be associated with QN, as they did not react with the M2 antibody Whereas the sedimentation of the wild-type T4–QNcomplex was abso-lutely unaffected by the presence of Triton X-100 or Brij-97
in the gradient, the sedimentation of T4–QN complexes formed with W17F and W17A was reproducibly retarded in the presence of Triton X-100 (compared to Brij-97), showingan opposite effect to that normally observed for amphiphilic enzyme species, such as T1a, T2a or T4a (see Fig 6A); the electrophoretic migration of these complexes was also slower than that of the wild-type complex This may reveal an interaction with Triton X-100 micelles, but not with Brij-97 micelles, perhaps because of an unusual exposure of the aromatic groups in these complexes
It is noteworthy that, in contrast to the wild-type Tna
4 and
T4–QN, the tetramers formed with the W17F, W17H or W17A mutants were only observed in the medium, but not
in the cell extract This indicates a significant difference in the cellular traffickingof the wild-type and mutant complexes
Perturbation of the helical organization of an aromatic cluster
To perturb the a helical organization of the aromatic-rich segment of the t peptide, we deleted residues T12 and M21, located, respectively, in its N-terminal region and near its centre (Fig 1A) Mutation M21W introduced an additional aromatic residue, which might create a steric disturbance in oligomers or in heteromeric complexes with QN
These mutations had moderate effects on the level of cellular activity, and decreased secretion to 50% of the wild type The three mutants produced mostly amphiphilic
T2a dimers in the cell extracts; in the case of M21D, the medium only contained T4natetramers (Fig 5A), in contrast
to the wild type, in which these molecular forms are present both in the cell extracts and in the medium
Figure 5A also shows that coexpression with QN increased secretion for T12D and M21W (to about 35% and 50% of the wild type, respectively), but not for M21D;
T4–QNcomplexes of T12D and M21W were characterized
in the medium by reaction with the M2 antibody, but were undetectable or barely detectable in the cell extracts, in contrast to the wild-type T4–QNcomplex
Effect of a cysteine at various positions in the t peptide The formation of intercatenary disulfide bonds between wild-type AChET subunits depends on the free cysteine residue located near the C-terminus of the t peptide, C37 Mutation of this cysteine to a serine reduced both cellular and secreted activities; it suppressed the formation of dimers and reduced cellular and secreted tetramers (Fig 6A); in the presence of QN, the secretion of T4–QN complexes was reduced to 75% of that of the wild type Thus, the presence of an intercatenary disulfide bond appears to be necessary for dimerization, but not for tetramerization, particularly in the presence of QN
To determine whether cysteines at other positions could allow dimerization and further oligomerization, we replaced residues I3, A6, T12, S19, M21, M22 or H34 with a cysteine, with or without mutation of C37 (C37S)
Fig 4 Molecular forms produced by W17 mutants; interaction with
Q N Sedimentation patterns of cellular and secreted molecular forms;
the areas under the profiles are proportional to the corresponding
activities; the top of the wild-type T 4 –Q N (tetramer associated with the
N-terminal Q N fragment of ColQ, containing the proline-rich
attach-ment domain motif) peak exceeds the frame and is shifted downwards.
Molecular forms expressed without Q N (––s––) and with Q N (- - -j- - -)
were analyzed in the presence of Triton X-100; sedimentation was also
performed in the presence of Brij-97 (Bj) for molecular forms secreted by
mutants W17F, W17H and W17A (ÆÆÆÆÆÆ) Note an unusual retardation
by Triton X-100 (Tx) for W17F and W17A.
Trang 6The relative levels of cellular and secreted activities, as
well as the distribution of molecular forms, are illustrated
in Fig 6A,B Unlike C37S, none of these mutants
produced monomers without dimers; therefore, when
two cysteines were present, they were not engaged in an
intracatenary disulfide bond, but could form intercatenary
bonds in dimers
Mutation I3C (with or without C37S) considerably
increased the cellular activity, mostly as amphiphilic dimers;
secreted activity was also increased, but to a much lesser
degree The presence of the N-terminal cysteine thus
appears to facilitate dimerization and to reduce
degrada-tion This mutation also increased the cellular activity
obtained with the W17P mutant, without restoringits
capacity to interact with QN
Mutation A6C somewhat decreased the cellular activity,
but strongly increased secretion The A6C mutant mainly
produced a nonamphiphilic 14 S species, possibly
corres-pondingto octamers This unusual oligomer dissociated
duringstorage, particularly in the presence of detergent
(Triton X-100), transiently producingamphiphilic tetramers
(T4a) (which are not usually observed in the wild type) and
amphiphilic dimers (T2a) When C37 was absent (A6C/
C37S), the 14 S species was observed in the cell extracts, but
seemed to be less stable, beingalmost entirely converted to
T2adimers in the medium
The T12C mutation introduced a cysteine in the
N-terminal part of the aromatic-rich segment (not shown)
Fig 6 Oligomeric forms obtained with cysteines at different positions in the t peptide (A) Sedimentation patterns of cellular and secreted molecular forms in gradients containing Triton X-100 (––) and Brij-97 (- - -); the shaded areas and the areas under the profiles are propor-tional to the correspondingactivities Note that dimers containing cysteines at position 21 did not sediment faster with Triton X-100 than with Brij-97, in contrast to the amphiphilic dimers (wild type, or with cysteines at positions 3, 6 or 34) Mutants A6C and A6C/C37S pro-duced a 14 S species that was progressively dissociated into amphi-philic tetramers (T a ) and ultimately amphiphilic dimers (T a ), as shown for cell extracts in an upper profile (B) Interaction of cysteine mutants with Q N , as shown by electrophoretic analysis of secreted molecular forms, in the presence of Triton X-100 and sodium deoxy-cholate (compare with Fig 5A) Note that mutant M21C (with cysteine C37) produced complexes with Q N , retarded by M2, whereas mutant M21C/C37S (without cysteine C37) did not.
Fig 5 Mutations in the aromatic-rich region; mutations of methionines
M21 and M22 (A) Electrophoretic patterns of cellular and secreted
molecular forms, in the presence of Triton X-100, with and without
deoxycholate (DOC); complexes with Q N that were retarded by M2
are indicated by x; the symbols are as in Fig 4 The secretion of
mutant M21D was not increased by coexpression with Q N , and no
complex reactingwith M2 was detected In the case of T12D and
M21W, T 4 –Q N (tetramer associated with the N-terminal Q N fragment
of ColQ, containingthe proline-rich attachment domain motif)
com-plexes were secreted, but undetectable or barely detectable in cell
extracts (B) Cellular and secreted activities (represented as in Fig 2A)
for mutants containingcysteine C37 or not (C37S) Coexpression with
Q N increased the secretion of mutants of methionine M22 and mutants
of methionine M21 which possessed cysteine C37, but not of mutants
M21A/C37S and M21S/C37S (data not shown), lackingboth M21
and C37.
Trang 7In the absence of cysteine C37, this allowed the formation
of amphiphilic dimers, which were secreted together with
nonamphiphilic tetramers and a 14 S species This species
was, in fact, predominant in the secreted enzyme and
appeared to be much more stable than that formed with
A6C, as it was not dissociated after secretion In the
presence of cysteine C37, the T12C mutant produced mainly
nonamphiphilic tetramers, which represented the only
secreted form This suggests that tetramers may be stabilized
when disulfide bonds were formed at the two positions,
12 and 37
A cysteine at position 19, in the aromatic-rich segment
but opposite to the aromatic cluster, had very different
effects, dependingon the presence of cysteine C37 Without
C37, mutant S19C/C37S produced very low levels of
cellular or secreted activity In contrast, mutant S19C
(containingtwo cysteines at positions 19 and 37) showed a
high level of secretion, mostly as nonamphiphilic tetramers,
as observed for T12C
Mutations M21C and M22C, with or without cysteine
C37, had little effect on cellular activity (compared to the
wild-type and C37S mutant, respectively), but increased
secretion to various degrees M21C and M21C/C37S
secreted both tetramers and nonamphiphilic dimers, while
M22C and M22C/C37S secreted mostly tetramers The
dimers formed with a cysteine at position 21 appeared
nonamphiphilic, suggesting that the aromatic clusters may
be masked by an intercatenary disulfide bond in the
aromatic-rich segment
Finally, the mutants containinga cysteine at position 34
(H34C, H34C/C37S) behaved essentially like the wild type,
suggesting that the C-terminal segment of the t peptide is
flexible
Figure 6B shows that the various cysteine mutants
formed T4–QNcomplexes (reactingwith the anti-flagM2
immunoglobulin), except M21C/C37S Thus, mutation
M21C suppressed the heteromeric complex when cysteine
C37 was absent, but not when it was present: this illustrates
the importance of the C-terminal cysteine for the assembly
and/or stabilization of the T4–QN complex, in agreement
with the formation of intercaternary disulfide bonds
between the t peptide and QNcysteines
Coexpression with QNgenerally increased secretion when
T4–QNcomplexes were produced, although this effect was
marginal or absent for mutants that showed a high level of
secretion without QN(A6C, A6C/C37S, S19C) However,
coexpression induced a decrease, of 40%, in the secretion
of mutant M21C/C37S, for which complexes could not be
detected (not shown) This suggests that QN did interact
with the mutant AChET subunits, but induced their
degradation rather than the assembly of a stable, secretable
hetero-oligomer
The role of methionine 21
The fact that M21C/C37S did not associate with QN,
whereas M22C/C37S formed a T4–QN complex, may be
related to the orientation of the two adjacent methionines
relative to the aromatic cluster, and to a possible structural
role of methionine M21: this residue is conserved in
vertebrate t peptides, while M22 is replaced with other
residues in some species To examine this possibility, we
mutated M21 and M22 to alanines or serines (with or without C37S) Figure 5B shows that coexpression with QN increased the level of secretion, indicatingthe formation of
T4–QNcomplexes, for all mutants except M21A/C37S (and M21S/C37S) Thus, the presence of a methionine at position
22 is dispensable, but a methionine at position 21 contri-butes to the stability of the complex, especially when cysteine 37 is absent
The C-terminal region of the t peptide: a retention motif? The last four residues of the t peptide, CAEL, contain the cysteine involved in intercatenary disulfide bonds and also resemble the classical ER-retention signal, KDEL To determine whether its presence might induce a partial retention of AChET subunits, we introduced various mutations in this motif (Fig 7A)
It should first be noted that mutation C37S (where the cysteine was removed) did not increase secretion, but rather decreased both cellular and secreted activities; this effect may result from the fact that suppression of the cysteine prevented dimerization and reduced the level of secreted tetramers, as discussed above
Fig 7 Effects of the C-terminal cysteine (C37), of C-terminal segments and of a KDEL motif on acetylcholinesterase (AChE) molecular forms (A) Sedimentation patterns of cellular (upper row) and secreted (lower row) enzyme, in gradients containing Triton X-100 The shaded areas and the areas under the sedimentation profiles are proportional to the activities In mutant T KDEL , the C-terminal tetrapeptide (CAEL) was replaced with the canonical ER retention motif (KDEL) All mutants lackinga cysteine produced and secreted amphiphilic monomers with variable proportions of nonamphiphilic tetramers; these tetramers were secreted at a higher level for T KDEL than for C37S (SAEL) (B) Cellular and secreted activities obtained with and without Q N The cellular activities decreased with the extent of C-terminal deletions; coexpression with Q N increased secretion to a level comparable to that
of the wild type for mutants C37S, T KDEL , stop34 and stop32, but had essentially no effect for stop29.
Trang 8Dimerization was also suppressed when the CAEL motif
was replaced with KDEL (TKDEL) because this mutation
removed the cysteine The presence of a C-terminal KDEL
tetrapeptide increased the level of cellular activity by about
threefold relative to C37S, mostly correspondingto T1a; this
increase in cellular enzyme appeared to facilitate
tetra-merization, as tetramers (T4na) were secreted at a higher
level than with C37S (Fig 7A)
We also deleted the last two residues, EL (stop39): the
mutant in which the C-terminal motif was reduced to CA
produced dimers and secreted 1.7-times more activity than
the wild type More extensive deletions, which removed the
cysteine, suppressed dimerization, so that monomers were
predominant in the cells and in the medium, and the levels
of activity were reduced in both compartments; the secretion
of homotetramers was reduced in stop34, compared to
C37S, and tetramerization was abolished in stop32
Like C37S, the TKDEL, stop39, stop34 and stop32
mutants formed T4–QNcomplexes, so that their secretion
was increased in the presence of QN(Fig 7B) In contrast,
the shorter mutant, stop29, showed no interaction with QN,
as indicated by the fact that coexpression did not affect
either the secreted activity or the molecular forms,
charac-terized by sedimentation To determine whether the
differ-ence between stop32 and stop29 could be ascribed to one of
the three residues D29, Q30 or Y31, we mutated each of
them to alanine: the level of T4–QNcomplexes was similar to
that of stop32 for Y31A/stop32 and Q30A/stop32, but
considerably reduced for D29A/stop32, suggesting a specific
influence of residue D29; the strongeffect observed by
mutation of this charged residue contrasts with the result
obtained when it was mutated in the full-length t peptide
(see above)
Progressive deletions from the C-terminus
of the I3C mutant
To analyze the effect of C-terminal deletions without
preventingdimerization, we used the I3C/C37S mutant,
which produced mostly amphiphilic dimers (Fig 6A) and
formed T4–QN complexes when coexpressed with QN
(Fig 8A) As in the wild type, the replacement of the
C-terminal tetrapeptide by KDEL increased the cellular
activity (mainly Ta) and reduced its secretion, but did not
abolish association with QN, producingT4–QNcomplexes
which were secreted Deletion of the last nine residues
(I3C/stop32) did not abolish association with QN, but
deletion of the last 12 residues (I3C/stop29) suppressed it
completely Thus, the presence of a cysteine at position 3
did not modify the requirement of residues 29–31 for
interaction with QN
The effect of deletions on the cellular and secreted
activities is illustrated in Fig 8B The cellular activity
remained approximately constant for all deletions, about
50% of the value observed with the full-length t peptide
Removal of the last two residues (EL) increased secretion, as
in the case of the wild type More extensive deletions in the
C-terminal region reduced secretion, compared to that of
I3C/C37S/stop39, but deletions within the aromatic region
progressively increased it, reaching a plateau when all
aro-matic residues were removed The dimers were amphiphilic
when they contained at least 29 residues of the t peptide
(stop29 and longer), but not if they contained 24 residues
or fewer (stop24 and shorter), i.e when they lacked some of the core aromatic residues
Fig 8 Effects of C-terminal segments and of a KDEL motif, in mutants containing an N-terminal cysteine (I3C) (A) Interaction with
Q N , indicated by electrophoretic patterns of cellular (top) and secreted (bottom) molecular forms obtained with and without Q N , in the presence of Triton X-100 and sodium deoxycholate Note that mu-tants I3C/C37S, I3C/KDEL and I3C/stop37 produced homomeric
T 2 dimers (s), homomeric T na
4 tetramers (h) and T 4 –Q N (tetramer associated with the N-terminal Q N fragment of ColQ, containing the proline-rich attachment domain motif) complexes (j), whereas I3C/ stop29 did not Homomeric tetramers of the I3C/KDEL mutant appeared to be partially retained intracellularly (B) Effect of C-terminal deletions on cellular and secreted activities; progressive deletions were made from the C-terminus of mutants containingan N-terminal cysteine (I3C) which allows an efficient dimerization; in the two longer mutants, the C-terminal cysteine was replaced with a serine (C37S); mutated residues are underlined in the sequence, shown alongthe horizontal axis Cellular (upper frame) and secreted (lower frame) activities, expressed as percentage of the wild type, are shown
as a function of the remainingleng th of the t peptide Asterisks indicate mutants that produced amphiphilic dimers; mutants stop34 and shorter produced nonamphiphilic dimers.
Trang 9The t peptide: an elongated amphiphilic a helix
with a cluster of aromatic residues
Previous studies suggested that the amphiphilic properties
of the t peptide reflect the formation of a cluster of aromatic
residues in its a helical conformation [30] The present
mutations confirm the role of aromatic residues, but show
that they differ considerably in their importance
Amphiphilicity was not affected by mutations of charged
residues, or by deletions of the C-terminal region which
removed up to 11 residues, i.e maintained all the aromatic
residues, except Y31 The amphiphilic character was
reduced to various extents when the central residues (F14,
W17, Y20, W24, F28) were replaced with prolines, but was
not affected by mutation of the first and last residues (W10,
Y31) Replacement of the most critical residue, W17, by
other, different, amino acids (F, L, A, H, P) showed that
amphiphilicity was indifferent to their aromatic nature, that
it did not strongly depend on their hydrophobicity, but was
very sensitive to the a helical structure Thus, the
amphi-philic properties of the t peptide appear to depend
predominantly on the spatial organization of a cluster of
hydrophobic residues
In agreement with a previous study [30], we obtained no
evidence that two cysteines, introduced at several positions
in the N- and C-terminal regions of the mutated t peptides,
could form an intracaternary disulfide bond The present
results thus confirm that the t peptide forms an elongated
amphiphilic helix, rather than foldingback on itself as a
hairpin in which the N- and C-terminal ends might be joined
by a disulfide bond
Homomeric associations of AChETsubunits
In contrast to the wild-type AChET subunits, the C37S
mutant did not form stable dimers, showingthat an
intercaternary disulfide bond is necessary The position of
this bond appeared very flexible, as dimers were formed
when cysteines were introduced at various positions along
the t peptide (with or without cysteine C37): this did not
seem to depend on the orientation of the residue relative
to the helical axis, although some positions produced
higher proportions of dimers than others However,
although most dimers were amphiphilic, those formed in
the presence of a cysteine at position 21 were
nonamphi-philic, indicatingthat, in this case, the hydrophobic
patches occluded each other because of the formation of a
disulfide bond joiningthe aromatic clusters near their
centers
Dependingon the position of an added cysteine, AChET
subunits could form predominantly dimers or tetramers – or
even higher oligomers sedimenting at 14 S, possibly
octa-mers This shows that the interactions between the t peptides
may present different geometries Thus, the t peptides can
form different homomeric assemblies, which may be
stabilized by intercaternary disulfide bonds and are
influ-enced by the positions of these linkages
In contrast to dimers, tetramers can be formed in the
absence of cysteine in the t peptide [27,36], although at a
lower level than for the wild type Tetramers are generally
nonamphiphilic (T4na), but some tetramers may also be amphiphilic (T4a), particularly those resultingfrom the dissociation of the nonamphiphilic 14 S species Thus, aromatic clusters may be either occluded or at least partly exposed in tetrameric assemblies, indicatingthat they correspond to distinct quaternary organizations
Heteromeric associations with the PRAD-containing protein, QN
The major physiological role of the t peptide is clearly to allow the functional localization of AChET tetramers through their association with PRAD-containing proteins, ColQ and PRiMA In the present study, we focused our attention on the formation of quaternary associations with
an N-terminal fragment of ColQ, QN This protein assem-bles with wild-type AChET subunits to form QN-linked tetramers (T4–QN), which are nonamphiphilic Previous studies showed that in these hetero-oligomers, two catalytic subunits are disulfide-linked with QN, while the other two are disulfide-linked together However, in the absence of cysteine C37, this association still occurs, indicatingthat it does not require the formation of intercaternary disulfide bonds
The complex was formed when the t peptide carried an additional cysteine at positions 3, 6, 12, 19, 22 or 34, with or without the original cysteine C37 It was not formed with a cysteine at position 21, except when C37 was present: a cysteine instead of a methionine at position 21 therefore appears unfavorable This may be partly because of the formation of nonamphiphilic disulfide-linked dimers in which the aromatic clusters are not available for interaction with the proline-rich domain of QN(PRAD) However, the mutation of methionine 21 to an alanine or a serine also weakened the formation of the complex, which again required the presence of cysteine C37: mutants M21A/C37S and M21S/C37S did not associate with QN In contrast, similar mutations of methionine M22 did not prevent the formation of T4–QNcomplexes This demonstrates that the complex was stabilized by disulfide bonds through cysteine
37, and by the presence of methonine 21, in agreement with stronginteractions of this methionine with the PRAD in a complex of isolated peptides (WAT)4 PRAD (M Harel
et al., manuscript in preparation) Nevertheless, the fact that M21 could be replaced with a tryptophan suggests that the complex can accommodate the steric constraint owingto a more bulky residue
The role of aromatic residues in the formation of the QN -linked complex has been established previously [4,28,37] Usingdeletions of single residues, we show here that the structure of the cluster is crucial for this quaternary interaction: it was abolished by deletion of residue 21, in the middle of the aromatic-rich segment The fact that deletion of residue 12, near the N-terminal end of the aromatic cluster, had no such effect, indicates that the orientation of the aromatic cluster relative to the catalytic domain is not crucial This is consistent with the notion of a flexible junction between the catalytic domain and the amphiphilic helical region of the t peptide [30] In fact, addition of a variable number of residues between the catalytic domain and the t peptide did not prevent association with Q (N Morel & S Bon, unpublished)
Trang 10In the present study, we assessed the importance of
aromatic residues, individually, by point mutations We
observed that mutation of the central residues (F14, W17,
Y20, W24) to proline or alanine had a much stronger effect
than mutation of W10 and F28, and that Y31 had no effect
Although not identical, the impacts of these mutations were
similar to those observed on the amphiphilic character of
AChETsubunits However, mutations of W17 to different
amino acids clearly dissociated the two properties, as
mutation W17L maintained the amphiphilic character,
but totally abolished the association with QN, like mutation
W17P This association was reduced when W17 was
replaced with a phenylalanine or a histidine, and even more
strongly when it was replaced with an alanine, emphasizing
the importance of an aromatic side-chain
With some mutants, we obtained evidence that QN
induced the formation of AChETdimers: coexpression with
QNstrongly increased the secretion of dimers that were not
covalently associated with QN(as in the case of W17F and
W17H), or were at least partially disulfide-linked with it (as
in the case of W10P) Such dimers may represent an
intermediate stage in the assembly of QN-linked tetramers,
or result from the dissociation of unstable tetramers
QN-linked tetramers are usually nonamphiphilic,
indica-tingthat the clusters of aromatic residues are occluded when
the t peptides are associated with the PRAD, in agreement
with their stronginvolvement in these quaternary
interac-tions and with the crystallographic structure of a complex of
synthetic peptides (M Harel, H Dvir, S Bon, W Q Liu,
M Vidal, C Garbay, J L Sussman, J Massoulie´ &
I Silman, unpublished results) However, the QN-linked
tetramers, found with the W17F and W17A mutants, showed
some interaction with detergents, suggesting that they were
less compact than the wild-type complexes In fact, the
formation of QN-linked complexes in the presence of cysteines
within the t peptides reveals a considerable flexibility, because
disulfide bonds between these residues do not seem to be
compatible with the distances between pairs of homologous
residues in a complex of wild-type synthetic peptides [30]
The heteromeric assocation with QNwas not suppressed
by removal of the last nine residues (followingY31),
showingthat it depends primarily on the aromatic-rich
segment and does not require the C-terminal part of the
t peptide However, removal of three additional residues
(D29, Q30, Y31) abolished the interaction, and point
mutations showed that this was mostly caused by the
deletion of D29: although mutations of charged residues in
the full-length t peptide had little effect on association with
QN, this suggests that a salt bridge contributed significantly
to its stability when the complex was weakened by a
C-terminal deletion
Similarity between nonamphiphilic homomeric
and QN-linked tetramers
We observed that the formation of homomeric T4na
tetramers was suppressed by all mutations which affected
the heterometic complex, T4–QN, suggesting that both
quaternary assemblies depend on the same interactions and
possess a similar organization, in agreement with the fact
that they are both nonamphiphilic In fact, except for
M21C/C37S, mutations that affected Q -linked tetramers
appeared to reduce homomeric tetramers more severely, indicatingthat tetramers are generally stabilized by the presence of a PRAD In T4–QNcomplexes, the four a helical
t peptides are organized as a super-helix, forming a hollow tube lined by aromatic side-chains, which is occupied by the PRAD This central space is unlikely to be filled with water molecules or to remain empty in T4natetramers; it may be reduced by a change in the pitch of the super-helix
Homo- and hetero-oligomerization occur in the ER, subcellular trafficking
The presence of an ER-retention motif (KDEL) at the C-terminus of the t peptide blocked secretion, as expected, but did not prevent dimerization when a cysteine was introduced at position 3, in the N-terminal region of the
t peptide The KDEL motif actually increased the formation
of homotetramers (T4na) in the TKDELmutant, compared to the C37S mutant which also lacked the C-terminal cysteine and was terminated by the tetrapeptide, SAEL Similarly, a C-terminal KDEL did not block the formation of hetero-meric complexes T4–QN; furthermore, the KDEL motif was sterically masked in the complexes, as the complexes were efficiently secreted The fact that retention of AChET subunits in the ER did not prevent homomeric or hetero-meric associations indicates that they occur in this com-partment
We found that the last two residues of the t peptide, EL, which are also present in the ER-retention tetrapeptide, KDEL, exert a weaker, but significant, retention effect, as their deletion increased secretion It is possible that these residues help to retain isolated AChETsubunits in the ER, and thus facilitate their physiological association with the anchoringproteins, ColQ and PRiMA
Structural differences certainly explain that complexes formed with the T12D and some aromatic mutants followed
a different cellular traffickingthan the wild type: these mutant complexes were secreted, but not detectable in cellular extracts, showingthat they did not accumulate in the secretory compartment, but were either rapidly secreted
or degraded We made a similar observation for homomeric tetramers in the case of mutant M21D
Oligomerization, secretion and degradation
In steady-state cultures, we may assume that all mutants were produced at the same rate, as they only differ in the short C-terminal t peptide, so that the rates of secretion of different mutants represent the difference between the common rate of synthesis and the rate of degradation In
a previous study, we established that the presence of the
t peptide induces a partial degradation of AChETsubunits through the ERAD pathway and that this effect depends on the presence of aromatic residues [28] Occlusion of these residues may explain that oligomerization generally facili-tates secretion: for example, the relative proportions of monomers, dimers and tetramers in cellular extracts and in the medium indicate that the secretion of wild-type AChET subunits increases with their degree of oligomerization This explains why the introduction of cysteines at certain positions increased secretion, by facilitatingthe formation
of dimers (I3C), tetramers (M22C) or 14 S oligomers (A6C)