(Received for publication, July 3, 1996, and in revised form, October 23, 1996)
From the Laboratoire de Neurobiologie Moléculaire et Cellulaire, Unité CNRS 1857, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France
In transfected COS cells, we analyzed the
formation of heteromeric associations between rat acetylcholinesterase
of type T (AChET) and various constructions derived from
the NH2-terminal region of the collagen tail of asymmetric
forms, QN. Using a series of deletions and point mutations
in QN, we showed that the binding of AChET to
QN does not require the cysteines that normally establish intersubunit disulfide bonds with catalytic subunits and that it
essentially relies on the presence of stretches of successive prolines,
although adjacent residues also contribute to the interaction. We thus
defined a roline-
ich
ttachment
omain or PRAD, which recruits AChET subunits
to form heteromeric associations. Such molecules, consisting of one
PRAD associated with a tetramer of AChET, are exported
efficiently by the cells. Using the proportion of AChET
subunits engaged in heteromeric tetramers, we ranked the interaction
efficiency of various constructions. From these experiments we
evaluated the contribution of different elements of the PRAD to the
quaternary assembly of AChET subunits in the secretory
pathway. The PRAD remained functional when reduced to six residues
followed by a string of 10 prolines
(Glu-Ser-Thr-Gly3-Pro10). We then showed that
synthetic polyproline itself can associate with AChET
subunits, producing well defined tetramers, when added to live
transfected cells or even to cell extracts. This is the first example
of an in vitro assembly of AChE tetramers from monomers and
dimers. These results open the way to a chemical-physical exploration
of the formation of these quaternary associations, both in the
secretory pathway and in vitro.
As shown in the preceding article (1), coexpression in COS cells offers a convenient method to analyze the formation of heteromeric molecules in which tetramers of acetylcholinesterase (AChE,1 EC 3.1.1.7) are associated with small binding proteins derived from the NH2-terminal region of the collagen component of collagen-tailed forms (for review, see Ref. 2). When expressed alone in COS cells, AChE subunits of type T (AChET) produce mostly monomers and dimers, with smaller proportions of tetramers and higher oligomers. In the presence of the binding proteins QN/stop and QN/HC, these subunits are recruited into tetramers that are associated with the binding domain and are efficiently secreted or attached to the cell surface by a glycolipidic anchor (GPI) (1).
In this study we used a series of deletions and point mutations in the QN sequence in an attempt to define the attachment domain that allows its interaction with rat AChET. We show that the cysteine residues are not required for interaction with AChET, and we narrow down the binding domain essentially to a stretch of successive prolines. Moreover, we show that polyproline itself can combine with AChET subunits, inducing their polymerization, mainly into tetramers, when added to transfected cells or to a cell extract.
All reagents were purchased from Prolabo (Paris, France) or from Sigma (St. Louis, MO, U. S. A.). PI-PLC from Bacillus thuringiensis was from Immunotech (Marseille, France). Poly-L-proline with a molecular mass of 1,000-10,000 (mean 8 kDa) and >30,000 (mean 40 kDa) was purchased from Sigma.
Site-directed Mutagenesis and Transfection in COS CellsExpression vectors encoding the binding proteins
QN/HC and QN/stop
(QN/stop551) were described previously (1, 3). The structure of the proteins is illustrated in Fig. 1.
Site-directed mutagenesis was performed with the single strand method
(4). In the case of deletions, we used mutagenic 20-mer
oligonucleotides consisting of 10 nucleotides complementary to each
side of the deleted fragment. In the case of truncations, TGA stop
codons were introduced to terminate the polypeptide chain. Transfection and culture of COS cells were performed as described in the preceding paper (1).
Analysis of AChE Forms
PI-PLC treatments, sedimentation and electrophoretic analyses, were performed as described (1).
Definition of the Proline-rich Attachment Domain (PRAD)
Deletions in the COOH-terminal Part of the QN DomainWe examined the effect of deletions in the COOH-terminal part of the QN domain on the binding of AChET subunits. For this purpose we used two strategies: introduction of stop codons at various positions in the Q sequence, producing QN/stop proteins of various lengths; or deletions of various extents in the chimeric QN/HC protein (Fig. 1).
When coexpressed with rat AChET subunits, QN
molecules truncated at position 87 (QN/stop87) induced a
significant increase in the proportion of cellular
G4na form and more dramatically, a large
increase in the released activity, where
G4na became predominant over
G2a and
G1a (not shown). Thus,
QN/stop87 was able to induce the formation of heteromeric
tetramers, like the QN/stop551 protein, containing the
complete QN domain, which we analyzed in the preceding
paper (1). In contrast, introduction of a stop codon at position 76 totally abolished this capacity. In the case of QN/stop76, the pattern of AChE forms was identical to that of AChET
subunits alone, as illustrated in nondenaturing electrophoresis
(Fig. 2). We performed similar experiments in
cotransfections with Torpedo AChET subunits; the
cells released essentially no activity when Torpedo
AChET was expressed alone or with QN/stop76,
but coexpression with QN/stop87 induced the secretion of
tetramers in the culture medium (not shown).
Deletions in the QN/HC molecule are
schematically shown in Fig. 1B. Deletion of COOH-terminal
segments of the QN domain, extending up to positions 100 or
85 (QN100-110/HC,
QN
85-110/HC) did not abolish the production
of a GPI-G4a form, as shown by the
sedimentation profiles of the cellular enzyme and its sensitivity to
PI-PLC (not shown). However, QN
85-110/HC somewhat weakened this interaction (see below). In contrast, larger deletions extending into the proline-rich sequence that follows the two
vicinal cysteines of QN
(QN
81-110/HC and
QN
76-110/HC) abolished the binding of
AChET.
Together these deletion experiments showed that a large part of the COOH-terminal sequence of QN may be removed without compromising the binding of AChET subunits from rat or from Torpedo. They defined a boundary of the binding domain, between positions 81 (essentially no binding) and 87 (similar to the wild type).
NH2-terminal and COOH-terminal Deletions in the QN DomainTo assess the possible role of the peptidic
sequence that precedes the pair of cysteines
Cys70-Cys71 in the QN domain, we
removed residues 46-69 in deletion 1, leaving only the three
residues that immediately follow the putative cleavage site of the
signal peptide. In deletion
2, we deleted residues 90-110 in the
COOH-terminal part of QN. We introduced these deletions, separately and in combination, in the QN/HC and
QN/stop proteins. All of these deleted molecules were able
to combine with the rat AChET subunit. For example,
QN/stop551 proteins carrying one or both deletions induced
the secretion of G4na rather than
G1a or
G2a (not shown). In the case of
QN/HC, the production of GPI-anchored G4a and the release of
G4na were similar for the three deleted
constructs and for the complete protein, as shown by nondenaturing
electrophoresis (Fig. 3) and by sedimentation analyses
(not shown). Surprisingly, the
1 deletion actually increased
the interaction with AChET, at nonsaturating doses of
DNA encoding QN/HC or QN/stop (see
below).
These observations establish that at least part of the sequence located between residues 70 and 86 plays a critical role in the interaction of QN with the AChET catalytic subunits. The most prominent feature of this short peptidic sequence is the presence of two stretches of five and three prolines, separated by two residues, Met80-Phe81.
Are Disulfide Bonds Necessary for Association of QN with AChET Subunits?The QN domain
contains a pair of vicinal cysteines
(Cys70-Cys71), which form disulfide bonds with
cysteines located at position 4 of the COOH terminus of two
AChET subunits, whereas the same cysteine residues form a
disulfide bond between the other two subunits of the catalytic tetramer
(11). To examine whether the formation of disulfide bonds between
QN and AChET subunits was a necessary
requirement for the assembly or stability of the hetero-oligomeric
structure, we constructed mutants that lacked the vicinal cysteines.
The double mutation C70G/C71S was introduced in the entire Q subunit
and in the QN/HC chimeric protein. In both
cases, the mutated proteins behaved exactly like those containing the
pair of cysteines, producing hetero-oligomers with rat
AChET subunits: collagen-tailed forms in the case of
Q, GPI-anchored and released tetramers in the case of
QN/HC (not shown).
The different QN/HC
constructions, when used at a nonsaturating dose of DNA (1 µg/dish),
varied markedly in the yield of heteromeric tetramers (cellular
G4a and lytic secreted
G4na). We could thus distinguish four
groups, in the following order: inactive
(QN76-110/HC,
QN
81-110/HC), very weak
(QN[Gly77-Pro80-Pro81-Gly83]/HC,
QN[Gly77-Gly83]/HC,
QN[Gly77]/HC), medium
(QN
85-110/HC,
QN[Gly77-Pro80-Pro81]/HC,
QN[Pro80-Pro81-Gly83]/HC),
and good (QN
100-110/HC,
QN[Pro80-Pro81]/HC,
QN[Gly83]/HC,
QN/HC).
It was possible to obtain a more quantitative comparison, using the
proportion of heteromeric AChE to total activity as an index of the
efficiency of interaction, if we assume that the different constructs
were expressed at the same level. (Note that they all contained the
same signal peptide and cleavage site.) We thus show a ladder of the
different QN/stop constructions, according to the
proportion of G4na in the culture medium
(Fig. 4). This shows that QN/stop85 was slightly but significantly less efficient than QN/stop87,
which was itself less efficient than QN/stop551. The
Phe85 and Phe86 residues therefore contribute
to the binding of AChET subunits. The effect of the
2(90-533) deletion also suggests that more distal residues
participate in this interaction. In the case of the
QN/HC constructs, it is possible that the
interaction may depend on a sufficient distance between the binding
domain and the GPI addition signal.
To assess the possible importance of Met80 and Phe81, located between the two proline stretches, in the interaction with AChET subunits, we mutated these residues into prolines. When coexpressed with rat AChET subunits, the QN[Pro80-Pro81]/HC mutant was able to produce GPI-anchored G4a, as well as released G4na, with a slightly reduced efficiency compared to the wild type QN/HC protein (not shown). Similarly, the QN[Pro80-Pro81]/stop85, QN[Pro80-Pro81]/stop87, and QN[Gly72-Gly73-Gly74-Pro80-Pro81]/stop85 mutants produced secreted G4na less efficiently than the corresponding constructions containing Met80-Phe81 (Fig. 4).
We also examined the importance of the proline stretches by replacing the middle residue of each group by a glycine. We found that whereas the P83G mutation had little effect on the yield of GPI-anchored G4a and released G4na, the P77G mutation reduced it markedly, and the double mutation P77G/P83G further weakened the binding. Introducing prolines at positions 80 and 81 partially compensated the effect of P77G but weakened the interaction in the case of P83G or of the double mutation P77G/P83G.
The two cysteines of the attachment domain are separated from the
proline stretches by three residues,
Leu72-Leu73-Thr74. Mutation of
these residues to glycines in complete or partially deleted
QN/HC was found to weaken significantly the
interaction with AChET. In agreement with the effects of
mutations of the Cys70-Cys71 and
Leu72-Leu73-Thr74 residues, a
deletion including the two cysteines (46-71) was as effective as
deletion
1(46-69) in which they were maintained, whereas removal of
the following Leu72-Leu73 residues
significantly reduced the efficiency of interaction (Fig. 4).
These observations indicate that a sufficient stretch of proline
residues is essential for the association with AChET but that hydrophobic residues located before, between, and after the two
proline stretches
(Leu72-Leu73-Thr74,
Met80-Phe81,
Phe85-Phe86) also participate in this
interaction. We constructed a mutant, QN/46-74Pro80-Pro81/stop85, in
which the mature protein only consisted of three residues, EST,
followed by a stretch of 10 prolines, Pro10, as in the
Pro80-Pro81- mutant. We found that this
Glu-Ser-Thr-Pro10 peptide was able to combine with
AChET, decreasing the proportion of
G2a and
G1a and increasing markedly the level of
secreted G4na, although with less
efficiency than other QN/stop constructs (Fig. 4). This
suggested that the polyproline sequence may be sufficient to organize
AChET tetramers in the absence of other residues.
Conversely, QN/HC and QN/stop constructs in which residues 70-86 were deleted had no effect on the molecular forms of AChE, as shown by sedimentation analyses or by immunofluorescence (not shown). They did not induce the production of GPI-anchored G4a at the surface of the cells or the secretion of G4na in the medium.
Interaction of AChET Subunits with Synthetic Polyproline
Exogenous, Synthetic Polyproline Can Combine with AChET in Transfected CellsWe added various concentrations of synthetic
polyproline of about 8 kDa to the culture medium after transfecting
cells with AChET. We observed a dose-dependent
decrease of G2a and
G1a, both in the cell extract (Fig.
5A) and in the culture medium (Fig.
5B), and a concomitant increase of
G4na. These effects could be detected at
5 × 108 M polyproline; at higher
concentrations G4na became the major
secreted form of AChE, as observed with QN/stop constructs.
In addition, a minor 16 S component appeared in the cell extract and in
the medium at higher concentrations of
polyproline.2 The sedimentation coefficient
of this form suggests that it may result from the binding of two
tetramers to a sufficiently long polyproline chain.
Polyproline of higher average molecular mass (40 kDa) also interacted with AChE but induced the formation of ill defined components, sedimenting mainly between 6 and 11 S.
We wondered whether AChET and polyproline could combine
spontaneously or whether the cellular biosynthetic machinery was
required for this interaction. We found that -dipyridyl, which
inhibits hydroxylation of prolines, did not inhibit the production of
G4na by polyproline, even when included
at 10
4 M in the culture medium for 3 days
after transfection. This concentration reduced the production of total
AChE activity to less than 20% of the control but did not alter the
proportions of molecular forms (not shown).
We also examined whether the interaction could occur in
an acellular system. When a cell extract, obtained in the absence of
detergent (high speed supernatant of a low salt-soluble extract), was
incubated with polyproline at 37 °C for 4 h, we observed an important increase of G4na at the
expense of G1a and
G2a (Fig. 6A).
The effect of polyproline on AChE monomers and dimers in a
detergent-free cell extract resembled the effect obtained with living
cells, except for the absence of the 16 S component. The 13.7 S form
was not observed under these conditions, probably because it
disappeared during incubation at 37 °C. The efficiency of
interaction was reduced markedly in the presence of Triton X-100 (Fig.
6B). In addition, the production of
G4na, in the presence of polyproline,
occurred less efficiently or more slowly at lower temperatures, 20 or
4 °C (not shown). The interaction between AChE and polyproline was
therefore dose-dependent, temperature-dependent, and sensitive to the presence of
detergent.
In contrast with cell extracts, we observed only a minimal effect after incubation of the secreted AChE forms with polyproline (less than 5% of G1a and G2a was converted into G4na), and this could in fact reflect the contribution of enzyme forms released by cell lysis. Experiments in which cellular extracts and media from transfected and control COS cells were combined, so that the composition of the mixtures was identical except for the cellular or secreted origin of the AChE molecules, showed that the difference in their capacity to interact with polyproline is an intrinsic property of these monomers and dimers (not shown).
In these experiments, we used a series of deletions and mutations in the NH2-terminal domain of the collagen Q subunit to identify the residues that are involved in the attachment of a tetramer of AChET subunits. These modifications were introduced in the QN/HC and QN/stop proteins and in some cases also in the complete collagenic subunit, Q. Assembly of AChET subunits with Q is less convenient because it produces several collagen-tailed molecules (A12, A8, A4) which aggregate in low salt and cannot be analyzed in nondenaturing electrophoresis. The results were however entirely consistent with those obtained for QN/HC and QN/stop proteins and have not been illustrated here.
Cysteines Are Not Required for Interaction of QN with AChETBy comparing the proportions of heteromeric
tetramers we were able to rank the efficiency of interaction of
different deleted and mutated constructions and thus evaluate the
contribution of various elements of the QN domain. Quite
unexpectedly, deletion of the peptide preceding the cysteines (1)
actually increased the interaction significantly. We also showed that
the vicinal cysteines of QN, Cys70 and
Cys71, are not required, although disulfide bonds probably
stabilize the quaternary association with AChET. The fact
that Cys70 and Cys71 are dispensable is
consistent with our previous observation that intersubunit disulfide
bonds may be reduced without disrupting the structure of
collagen-tailed AChE forms (5). We also showed that sequences following
a polyproline stretch, i.e. beyond position Phe86, could be removed without compromising the binding
capacity.
These experiments showed that the binding domain could be reduced to a short region of 17 residues, starting with cysteines Cys70-Cys71, which we propose to name the polyproline attachment domain or PRAD. This region is remarkably well conserved from Torpedo to higher vertebrates; there are only two replacements in the rat Q subunit3 and none in the chicken.4 The presence of PRAD is sufficient for interaction with AChET subunits, and conversely its deletion abolishes this interaction completely.
The PRAD contains two conserved stretches of five and three consecutive prolines, separated by two residues, Met80-Phe81, preceded and followed by conserved residues, Leu72-Leu73-Thr74 and Phe85-Phe86. By point mutations and deletions we showed that these hydrophobic residues, as well as more distal residues, participate in the interaction with AChET subunits but are not absolutely required.
By analyzing the ratio R of secreted heterotetramers, G4na, to the total activity in the culture medium for nonsaturating doses of QN/stop constructions, as described in the previous paper (1), we obtained an index of the efficiency of interaction, assuming that the binding proteins were expressed at the same level (Fig. 4). This index provides an evaluation of the influence of elements of the binding domain; for example, the M80P/F81P double mutation reduces the value of R to a similar degree in different contexts (QN/stop87, QN/stop85, or QNGly72-Gly73-Gly74/stop85). It seems therefore possible to quantify the contributions of specific residues to quaternary assembly in the secretory pathway.
Interaction of AChE with Synthetic PolyprolineThe critical
feature of the binding domain PRAD appeared to be the presence of a
sufficiently long series of prolines, since a simple
Glu-Ser-Thr-Pro10 peptide remained functional. We showed that, in effect, AChET subunits were able to combine with
synthetic polyproline. When added to cultures of COS cells expressing
AChET, synthetic polyproline of about 8 kDa was able to
mimic the effect of the QN domain, even at a concentration
as low as 109 M, inducing the formation and
the release of tetramers G4na at the
expense of monomers and dimers. It also produced a heavier complex of
16 S, in lesser proportion. Polyproline was probably taken up by
endosomes so that a fraction was able to reach the Golgi apparatus and
the endoplasmic reticulum and thus to interact with AChE during its
biosynthesis and maturation. We showed that
-dipyridyl, which
blocks proline hydroxylation, had no effect on this interaction either
in cells or in vitro, even at concentrations that inhibited
AChE biosynthesis by more than 70%.
In fact, the biosynthetic machinery of the cell was not
absolutely necessary since this interaction could be reproduced in a
soluble extract of transfected COS cells expressing rat
AChET. Polyproline induced the formation of tetramers
G4na from monomers and dimers of rat
AChET and possibly also of dimers from monomers. The effect
was dose-dependent and temperature-dependent and was reduced considerably in the presence of Triton X-100. It is
likely that interaction of the detergent with the COOH-terminal peptide
of AChET, organized as an amphiphilic
-helix5 prevents its interaction with
polyproline. This is the first time that oligomerization of AChE has
been obtained in vitro. In previous studies, molecular forms
of AChE were dissociated, e.g. by limited proteolysis, or
reversibly aggregated, as in the case of collagen-tailed molecules at
low ionic strength in the presence of polyanions (6); but monomers or
dimers were never seen to form stable tetramers. There was, however, a
clear difference between the oligomerization of AChET
subunits obtained in living cells and in acellular extracts, since
heavy polymers, sedimenting around 16 S, were obtained only in the
cells.
It is worth noting that molecules solubilized from cells were
able to interact with polyproline, whereas those secreted in the
culture medium were not. This is consistent with the fact that
post-translational modifications occur during transport and secretion
(1). Cleavage of the COOH-terminal T peptide may affect the ability of
monomers to form oligomers. For example, soluble monomers that had been
purified from bovine brain were found to lack the COOH-terminal part of
the T peptide (7). It should be noted, however, that the secreted
G1a and
G2a forms obtained in our culture media
retained their amphiphilic character, which reveals the presence of the
amphiphilic -helix of the T peptide.
The proline-rich structure of PRAD is reminiscent of the peptide sequences that bind to SH3 domains (8, 9). By analogy, it is therefore tempting to suggest that PRAD is organized as a polyproline II helix. There are marked differences, however, between these proline-rich domains. First, the PRAD exists in the secretory pathway, establishing interactions between externalized proteins, whereas SH3 binding domains are located in the cytosol; such polyproline sequences have not been found previously in extracellular proteins. Second, polyproline in PRAD does not simply provide a scaffold but may actually mimic the interaction. Third, there is no similarity between SH3 domains and the COOH-terminal T peptides of AChET, which are necessary for attachment to the collagen tail. Finally, the stoichiometry of the binding is different, since PRAD simultaneously binds to four AChET subunits. This in fact raises an interesting question about the symmetry of the heteromeric assembly, since polyproline II has an helical repetition of three residues per turn. In any case, it will be interesting to investigate the possible interaction of PRAD with other proteins, such as the 100-kDa protein that is associated with Torpedo asymmetric forms (10, 11). It will also be interesting to examine the possible existence of PRAD in other modular extracellular molecules apart from the Q collagen.
In conclusion, the present study reveals a new type of interaction that occurs intracellularly and withstands extracellular conditions. Such quaternary interactions are important for anchoring AChE and possibly other enzymes and proteins in extracellular matrices. We show that such interactions can induce molecular assembly both in the secretory pathway of living cells and in vitro.
We thank Anne Le Goff and Rizwana Nawaz for expert technical assistance and Jacqueline Pons for typing the manuscript.