Quaternary Associations of Acetylcholinesterase
I. OLIGOMERIC ASSOCIATIONS OF T SUBUNITS WITH AND WITHOUT THE AMINO-TERMINAL DOMAIN OF THE COLLAGEN TAIL*

(Received for publication, July 3, 1996, and in revised form, October 23, 1996)

Suzanne Bon and Jean Massoulié Dagger

From the Laboratoire de Neurobiologie Moléculaire et Cellulaire, Unité CNRS 1857, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

We investigated the production of acetylcholinesterase of type T (AChET) in COS cells during transient transfection. When expressed alone, Torpedo AChET remains essentially intracellular, forming dimers and tetramers; in contrast, rat AChET is secreted and produces mostly amphiphilic monomers (G1a) and dimers (G2a), together with smaller proportions of nonamphiphilic (G4na) tetramers, amphiphilic tetramers (G4a), and an unstable higher polymer (13.7 S). The latter two forms have not been described before. We show that secreted G1a and G2a forms differ from their cellular counterparts and that proteolytic cleavage occurs at the COOH terminus of "flagged" subunits. The binding proteins QN/HC and QN/stop are constructed by associating the NH2-terminal domain of the collagen tail (QN) with a functional or truncated signal for addition of a glycolipidic anchor (glycophosphatidylinositol). Coexpression with QN/stop recruits monomers and dimers to form soluble tetramers (G4na), increasing the yield of secreted rat AChE and allowing secretion of Torpedo AChE. Using antibodies against QN or addition of a flag epitope, we showed that the secreted tetramers contain the attachment domain. Coexpression with QN/HC modifies the distribution of AChET in subcellular compartments and allows the externalization of glycophosphatidylinositol-anchored tetramers at the cell surface.


INTRODUCTION

The collagen-tailed forms of acetylcholinesterase (AChE,1 EC 3.1.1.7) constitute a major component of the enzyme at neuromuscular junctions of higher vertebrates, mammals, and birds (for review, see Ref. 1). Their presence is controlled by innervation, by muscle activity, and by the type of muscle (slow or rapid) in a species-dependent manner. These molecules are hetero-oligomers, composed of AChE catalytic subunits of type T (AChET), associated with collagen subunits (Q). Each strand of the triple helical collagenic tail may be associated with a tetramer of T subunits. The AChET subunits are linked by intersubunit disulfide bonds through a cysteine located at position -4 of their COOH terminus: two "external" subunits are linked together, whereas the two "internal" ones are attached to the Q subunit (2-4).

We previously cloned the Q subunit from collagen-tailed AChE of Torpedo electric organs (5) and showed that it is able to associate with AChET subunits of Torpedo, rat, or human AChE, forming collagen-tailed molecules in transfected COS cells (5-8). The primary sequence deduced from the cDNA encoding Torpedo Q subunits comprises a signal peptide, an NH2-terminal domain (QN) which contains a pair of adjacent cysteines, a central collagen domain flanked by two pairs of cysteine residues, and a COOH-terminal domain (QC). Using antibodies directed against QC, we showed that this COOH-terminal region could be removed by collagenase without disrupting the assembly of catalytic tetramers. This experiment suggested that AChET subunits were linked to the QN domain (8). We further showed that an isolated QN domain was sufficient to bind one AChET tetramer, by constructing a chimeric protein in which QN is fused to the COOH-terminal glycolipid (GPI) addition signal of the H subunit of Torpedo AChE (HC). Coexpression of this QN/HC protein with catalytic T subunits of Torpedo, rat, or human AChE produces GPI-anchored AChE tetramers (8, 9).

We analyzed the enzyme produced by transfected COS cells expressing the rat AChET subunit alone, exploring the effect of various parameters on activity and molecular forms. In agreement with a previous study (6), transfected COS cells expressing the rat AChET subunit produce and secrete amphiphilic monomers and dimers of type II, G1a and G2a, (10-12) and nonamphiphilic tetramers, G4na. We now show that the proportions of the various molecular forms may vary with culture, extraction, or storage conditions. We also show that, in addition to previously characterized molecules, the cells produce an amphiphilic tetramer, G4a, as well as an unstable component of 13.7 S.

We then investigated the effect of cotransfection with vectors encoding the QN/HC protein, in which the attachment domain from AChE-associated collagenic subunits is fused to a GPI addition signal, or the QN/stop protein, which does not possess this signal. Heteromeric associations of AChET with these binding proteins produced tetramers that were attached to the plasma membrane by a GPI anchor (GPI-G4) in the first case and secreted into the medium (G4na) in the second case. We therefore analyzed AChE in both cell extracts and culture media using sedimentation, nondenaturing electrophoresis, and immunofluorescence of the transfected COS cells. Although most experiments were performed with rat AChET, we obtained similar results with Torpedo AChET. We found that the presence of QN induces AChET monomers and dimers to form tetramers, with which the attachment protein remains associated, even after cleavage of the GPI anchor.


EXPERIMENTAL PROCEDURES

Materials

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). The M1 and M2 monoclonal antibodies directed against the "flag" epitope were from Eastman Kodak Co.

Expression Vectors and Site-directed Mutagenesis

The pEF-BOS expression vectors containing the coding sequence of the Torpedo Q subunit or of the chimeric QN/HC protein were described previously (9). In the QN/HC protein, the sequence encoding the QN domain (numbered 1-110) was attached by a linker containing a BamHI site (encoding the residues GI, not numbered) to the sequence encoding the COOH-terminal peptide of Torpedo AChEH, HC (numbered 532-566); the truncated QN/stop protein was obtained by site-directed mutagenesis, inserting a TGA stop codon with the single strand method (13), at position 551 within the sequence encoding HC. The cDNAs encoding Torpedo and rat AChET were also inserted in pEF-BOS. Insertion of a sequence encoding the flag peptidic epitope was performed by mutagenesis in QN/HC and in rat AChET. In the case of QN/stop, the peptidic epitope was added at the end of the QN domain, after the linker G residue.

Transfection of COS Cells

COS cells were transfected by the DEAE-dextran method, as reported (5), using 5 µg of DNA encoding the catalytic subunit AChET and various amounts of DNA encoding the binding protein, as specified. In the case of Torpedo AChE, the cells were allowed to recover for 2 days at 37 °C after transfection and then transferred to 27 °C, to allow production of active enzyme, for 2-3 days. In the case of rat AChE, the cells were maintained at 37 °C and extracted 2-4 days after transfection. The culture medium (7 ml/10-cm dish containing about 5 × 106 cells) was collected after variable periods of time, as indicated, for analysis of released AChE activity. The extracts and culture media were stored at -80 °C.

Sedimentation and Electrophoretic Analyses

Centrifugation in 5-20% sucrose gradients was performed in a Beckman SW41 rotor, generally at 36,000 rpm, for 18 h at 7 °C, as described previously (12). The gradients contained 1% Brij-96 or 0.2% Triton X-100, as indicated. Alkaline phosphatase (6.1 S) and beta -galactosidase (16 S) from Escherichia coli were included as internal sedimentation standards. AChE was assayed by the colorimetric method of Ellman et al. (14).

Electrophoresis in nondenaturing polyacrylamide gels was performed as described previously (11). The gels contained 0.25% Triton X-100 and 0.05% deoxycholate and were electrophoresed for about 2-3 h under 15 volts/cm, with refrigeration at 15 °C. AChE activity was revealed by the histochemical method of Karnovsky and Roots (15).

Production of Anti-QN Antiserum

The rabbit polyclonal antiserum directed against the QN domain was prepared against a recombinant protein produced in E. coli, as described (9).

Treatment with PI-PLC

Samples of extracts (50 µl) were treated with 0.025 IU of PI-PLC for 1 h at 30 °C.

Immunofluorescence

Immunofluorescence was performed with a rabbit antiserum directed against rat AChE (16), as described previously (17), except that the second antibody was an fluorescein isothiocyanate-conjugated F(ab')2 fragment of anti-rabbit IgG (Silenius, Australia). The cells were analyzed either intact or after permeabilization with 0.2% saponin.


RESULTS

Quaternary Structures of Rat AChET Subunits Produced in COS Cells

Expression of the Rat AChET Subunit in Transfected COS Cells: Variable Patterns of AChE Molecular Forms

We studied the production of AChE in COS cells expressing the rat AChET subunit. The activity obtained per culture dish increased with the cell density but plateaued at a maximal value around 106 cells/10-cm dish, and we therefore used this density in all of our experiments. Following transfection, AChE activity was barely detectable in the cells after overnight incubation and then increased steadily for at least 5 days. It appeared slightly later in the culture medium, where its proportion increased from about 30% of the total activity after 30 h to 70% after 4 days. In addition, the yield of cellular activity showed a saturable Michaelis-type increase with the amount of recombinant plasmidic DNA used for transfection, reaching a plateau at about 10-15 µg/dish, with half-saturation around 5 µg of DNA/dish (not shown).

We analyzed the molecular forms of AChE in the cell extracts and in the culture medium, by sedimentation in sucrose gradients (Fig. 1, A and B). The sedimentation profiles did not change with the cell density or with the amount of DNA encoding AChET/dish (not shown). In Fig. 1A, typical sedimentation profiles of cell extracts show the presence of monomers (G1a; 3 S), dimers (G2a; 4.5 S), amphiphilic tetramers (G4a; 8.5 S), nonamphiphilic tetramers (G4na; 10.5 S), and a 13.7 S molecular species. The proportion of oligomers to monomers increased progressively during the first days following transfection, and the sedimentation pattern then remained essentially constant between 3 and 5 days. The sedimentation profiles were reproducible in a given series of experiments, but we observed a rather large variability in the proportions of molecular forms (i.e. in the relative proportion of monomers to dimers and higher oligomers) when comparing experiments performed over a long period of time, possibly because of variation in the batches of cells. The cause of this variability was not investigated systematically. It was less marked in the medium than in cell extracts (Fig. 1B); the medium generally contained about 35% G1a, 45% G2a, 20% G4na, together with small variable proportions of G4a and sometimes of the 13.7 S form. In some cases we observed the presence of a nonamphiphilic monomeric form (G1na), probably generated by a proteolytic process, after prolonged culture, e.g. 4 days (see below).


Fig. 1. Typical sedimentation profiles of AChE produced by COS cells expressing the rat T subunit. Panel A, cell extract; panel B, culture medium. The cells (106 cells/dish) were transfected with 5 µg of vector DNA, and the samples were collected 3 days after transfection. The illustrated profiles correspond approximately to extremes in the proportion of molecular forms observed in different experiments performed over several years; these variations may be related to the batch of cells or to slight variations in the composition of the culture medium, the conditions of culture, or conditions of transfection. The thin lines correspond to extreme cases of sedimentation profiles, obtained in 5-20% sucrose gradients containing 0.2% Brij-96 (open circle , square ). The thicker lines correspond to another sample analyzed in the presence of 1% Brij-96 (triangle ) or 0.2% Triton X-100 (black-triangle). Amphiphilic forms sediment faster in the presence of Triton X-100 than in the presence of Brij-96, whereas nonamphiphilic forms are unaffected. The sedimentation coefficients were deduced by linear interpolation from the position of internal marker proteins, E. coli beta -galactosidase (16 S) and alkaline phosphatase (6.1 S). The AChE activity of the fractions is plotted on an arbitrary scale as a function of the calculated S value, derived from the positions of sedimentation markers.
[View Larger Version of this Image (24K GIF file)]


Fig. 1 illustrates the influence of detergents on the sedimentation of the different molecular forms: amphiphilic forms sediment faster in the presence of Triton X-100 than in the presence of Brij-96, whereas nonamphiphilic forms are unaffected. Both monomers (G1a) and dimers (G2a) are amphiphilic forms of type II (10-12), and the G4a form probably also belongs to this category, as evidenced by the influence of detergents on their electrophoretic migration (not shown) and on their sedimentation in the presence of Triton X-100 and Brij-96, respectively: 4.5 and 3 S (G1a), 6.5 and 4.5 S (G2a), 10 and 8.5 S (G4a).

Sedimentation profiles obtained in the presence of Triton X-100 or Brij-96 or without detergent showed that the 13.7 S component is not amphiphilic (Fig. 1A). Assuming that it is a globular protein composed exclusively of AChE subunits, the ratio of its mass to that of G4na tetramers, which is close to 1.5 ((13.7/10.5)3/2), suggests that it is hexameric. This component is relatively abundant in fresh cellular extracts that had been maintained in the cold, without detergent (Fig. 2). In the absence of detergent it dissociated at 37 °C, mostly into monomers (G1a). In the presence of Triton X-100, dissociation occurred even in the cold and produced amphiphilic tetramers (G4a) as well as monomers (G1a). This instability explains why the proportion of the 13.7 S peak was variable and why this component did not generally appear in the culture medium. We also observed some inactivation of the G1a form at 37 °C, particularly in the presence of Triton X-100; the instability of this form probably contributes to the variability of its proportion in cell extracts, especially when analyzed by nondenaturing gel electrophoresis.


Fig. 2. Stability of cellular and secreted AChE forms. A cellular extract, obtained in 50 mM Tris-HCl, pH 7.5, 40 mM MgCl2, 0.1 mg/ml bacitracin was centrifuged for 15 min at 30 p.s.i. in a Beckman Airfuge (approximately equivalent to 1 h at 100,000 × g). Samples were analyzed in gradients containing Brij-96, after incubation with or without 1% Triton X-100, at 4 or at 37 °C, as indicated (inset). The AChE activity of the fractions is plotted as in Fig. 1.
[View Larger Version of this Image (24K GIF file)]


Distinction between Cellular and Secreted AChE Molecules

Although the cellular and released forms cannot be distinguished readily by their sedimentation, they migrate slightly differently in nondenaturing electrophoresis (Fig. 3), possibly because of differences in glycosylation, or proteolytic cleavage, or other post-translational modification. To investigate the last possibility, we introduced by mutagenesis the peptidic sequence DYKDDDDK (flag epitope) after the normal COOH terminus of the rat AChET subunit. The presence of this peptide at the COOH terminus did not significantly modify the production of active AChE, its distribution in the cellular and secreted fractions, or the proportions of its molecular forms. We found that although the cellular G1a and G2a forms carried the epitope, as shown by the effect of an anti-flag M2 monoclonal antibody on their sedimentation and electrophoretic migration, their secreted counterparts did not (not shown). This shows that proteolytic cleavage removes the flag peptide but not necessarily that it occurs within the COOH-terminal T peptide.


Fig. 3. Migration of cellular and secreted AChE forms in nondenaturing electrophoresis. Samples of cell extracts and culture medium were analyzed in nondenaturing horizontal gels containing 0.25% Triton X-100 and 0.05% deoxycholate. AChE activity was revealed after electrophoresis by the method of Karnovsky and Roots (15). Note the difference in the mobility of cellular and secreted G1a and G2a forms.
[View Larger Version of this Image (54K GIF file)]


In fact, polyclonal antibodies raised against the last 10 residues of the T peptide of Torpedo AChE2 or of rat AChE (18) were able to retard the electrophoretic migration of secreted as well as cellular active molecules (G1a, G2a, G4a, and G4na), in the case of both species (not shown).

Coexpression of Rat and Torpedo AChET Subunits with the Binding Proteins QN/stop and QN/HC

The structure of the binding proteins QN/HC and QN/stop is shown in Fig. 4. In cotransfection experiments, we used a nonsaturating dose of DNA encoding the rat AChET subunit (5 µg/dish) together with various doses of DNA encoding the binding proteins QN/HC or QN/stop (QN/stop551).


Fig. 4. Schematic representation of the binding proteins and heteromeric AChE molecules. Panel A, construction of the QN/stop and QN/HC proteins from the collagenic Q subunit and from the catalytic H subunit from Torpedo, which contains a COOH-terminal GPI addition signal; the peptide sequences of the binding proteins are given in the following article (31). Panel B, quaternary structure of heteromeric associations of AChET tetramers with the binding domain, QN, forming soluble and GPI-anchored membrane-bound molecules.
[View Larger Version of this Image (21K GIF file)]


Coexpression with QN/HC Carries Rat AChET to the Cell Surface

Immunofluorescence of transfected COS cells, with or without permeabilization, showed that AChET was not associated with the cell surface when expressed alone. In contrast, a QN/HC protein that included a flag peptidic epitope at its NH2-terminal extremity (see below) and could thus be visualized with a specific monoclonal antibody (M1), was exposed at the cell surface (not shown). When AChET was expressed together with QN/HC, it was carried with it to the cell membrane (Fig. 5) from where it could be released by PI-PLC (not shown), as in the case of GPI-anchored dimers generated from AChEH subunits3 (19). The distribution of intracellular AChE was quite different in the two cases, showing an accumulation in vesicles in the presence of QN/HC and a reticular pattern in its absence. This suggests that AChET transits differently from the endoplasmic reticulum to the external medium when associated with a GPI-anchored protein.


Fig. 5. Immunofluorescence staining of transfected COS cells expressing rat AChET alone and together with the binding protein QN/HC. Left panels, AChET alone; right panels, AChET + QN/HC; panels a and b, fields; panels c-h, details of individual cells; panels a-d, nonpermeabilized cells, panels e-h, permeabilized cells. Note that AChE is externalized when coexpressed with QN/HC and that the pattern of intracellular staining is markedly different in the case of AChET alone and of AChET + QN/HC. The magnification factor is approximately 50 for panels a and c, 80 for panels b and d, and 200 for panels e-h. The scale bar (panel a) corresponds to about 100 µm in panels a and c (objective × 25), 60 µm in panels b and d (× 40), and 25 µm in panels e-h (× 100). Note that the size of transfected cells was highly variable, i.e. the two cells shown in panel d.
[View Larger Version of this Image (88K GIF file)]


Conditions for Coexpression of Rat AChET and the Binding Proteins QN/stop and QN/HC: Influence of the Levels of Expression Vectors

Fig. 6 illustrates sedimentation profiles of AChE produced at different times in cells expressing rat AChET and QN/HC; these patterns show that tetramers accumulate progressively after transfection, between 1 and 2 days, whereas dimers and monomers increase little during the same period. This is entirely consistent with the fact that the metabolic half-life of monomers and dimers is much shorter than that of higher oligomers (20). To study the assembly of heteromeric tetramers with QN/stop or QN/HC, we analyzed the molecular forms produced after a delay of 2-4 days following transfection.


Fig. 6. Sedimentation patterns of AChE in COS cells expressing AChET and QN/HC: effect of time after transfection. Transfection was preformed with 5 µg of DNA encoding AChET and 9 µg of DNA encoding QN/HC per dish containing about 106 cells. The proportion of GPI-anchored tetramers (G4a) increased markedly in cellular extracts between 1 day (open circle ) and 2 days (triangle ) after transfection.
[View Larger Version of this Image (21K GIF file)]


We studied the effect of varying the amount of DNA encoding the QN/stop or QN/HC binding proteins, while the amount of DNA encoding AChET remained constant (Fig. 7, A and B. In both cases, the cellular activity was increased at low doses of DNA encoding the binding proteins, but the total AChE activity produced per dish, in the cells and in the medium, was considerably reduced at high doses. At 40 µg of DNA/dish, it was reduced to less than 10% of the control activity obtained when cells were only transfected with 5 µg of DNA encoding AChET/dish (not shown). In the case of QN/stop, the proportion of secreted activity was increased significantly at all concentrations of DNA (about 95% of the total activity compared with about 70% in control cells expressing AChET alone), in agreement with the production of soluble heterotetramers, and this was correlated with a much higher total activity/dish (Fig. 7B). In the case of QN/HC, which forms GPI-anchored tetramers attached to cell membrane, the proportion of secreted activity decreased, at least up to 1 µg of QN/HC DNA (Fig. 7A). The activity recovered in the culture medium increased, however, at higher doses of DNA, reflecting the release of nonamphiphilic monomers (G1na), as discussed below.


Fig. 7. Cotransfection of rat AChET with QN/HC or QN/stop: effect of the dose of DNA encoding the binding protein on the production and release of AChE activity, and on the proportion of heteromeric forms. Panel A, QN/HC; panel B, QN/stop. The cells were transfected with 5 µg of DNA encoding AChET and the indicated amount of DNA encoding the binding protein. Cells and culture media were collected 3 days after transfection. bullet , cellular activity; black-square, secreted activity: black-triangle, total activity. The proportion R of heteromeric forms in the cellular (open circle ) and secreted (square ) AChE activity was calculated from the sedimentation profiles shown in Fig. 8. The curves were fitted by equations of the form R = R(0) + Rmaxd/(E + d). In the case of QN/HC, the variation of R in the medium only tends to plateau if we include the lytic nonamphiphilic G1na form (R = (G4a + G4na + G1na)/(total secreted activity), clearly suggesting that G1na was derived from heteromeric GPI-anchored tetramers. The values of E used for fitting the data were 0.7 for both cells and medium in the case of QN/HC, 1 for cells and 0.15 for medium in the case of QN/stop.
[View Larger Version of this Image (19K GIF file)]


Sedimentation profiles of corresponding cell extracts and culture media are shown in Fig. 8. The activity of G1a, G2a, and 13.7 S decreased in the cells and in the medium with the dose of DNA encoding either QN/HC or QN/stop. With increasing doses of QN/stop, the activity of heteromeric tetramers, G4na, increased in the cells and in the medium with a concomitant decrease of G1a and G2a, reaching a maximum around 2-3 µg of DNA encoding the binding protein/dish (Fig. 8, C and D); the decrease observed at higher doses reflected the decrease in total cellular AChE activity. In the case of QN/HC, we observed a similar evolution of GPI-G4a in the cells (Fig. 8A). This GPI-G4a form was accompanied by a smaller proportion of G4na, probably representing a lytic derivative from which the GPI anchor was removed and which was also released in the medium (Fig. 8B). At high doses of DNA encoding QN/HC, we observed the appearance of a nonamphiphilic monomeric form (G1na), both in cell extracts and culture media, which cosedimented with G2a in the presence of Brij-96. This form was abundant in the experiment illustrated in Fig. 8, A and B, but its proportion was variable, and it was not always observed in other batches of COS cells. It probably results from a lytic process in the metabolic pathway of the GPI-anchored molecules, since it did not appear in the case of parallel cotransfections with QN/stop, even at high doses of DNA (Fig. 8, C and D).


Fig. 8. Cotransfection of rat AChET with QN/HC or QN/stop: effect of the dose of DNA encoding the binding protein on the sedimentation profile of AChE activity. Sedimentation profiles of the cell extracts and media correspond to the experiment of Fig. 7. Panel A, QN/HC, cellular extract; panel B, QN/HC, culture medium; panel C, QN/stop, cellular extract; panel D, QN/stop, culture medium. The amount of DNA encoding the binding protein, per dish, was the following: open circle , none; bullet , 0.1 µg; triangle , 1.5 µg; black-triangle, 15 µg (see inset). In each case, the profiles are represented on the same scale so that the activities of the molecular forms obtained at the different doses of vector DNA may be compared directly. Note that the scale of activity is the same in panels A-C but different in panel D because secretion of G4na is increased considerably in the presence of QN/stop. Insets show the total activity of the major AChE forms, which were deduced from the sedimentation profiles. In the case of high doses of QN/HC, we calculated the amount of G1na by assuming that the proportion of G2a to G1a remained constant, as in the case of QN/stop.
[View Larger Version of this Image (53K GIF file)]


Fig. 7 also illustrates the variation of the ratio R of heteromeric forms to total cellular or secreted AChE activity: R varies with the dose of DNA encoding QN/stop (d), as R = R(0) + Rmaxd/(E d), where R(0) is the ratio obtained with AChET alone, and Rmax is the maximal ratio obtained with a saturating dose of DNA. The parameter E thus defines an overall "efficiency" of interaction between the AChET subunits and the binding proteins in the secretory pathway. Under specific experimental conditions, this parameter allows an evaluation of such interactions in living cells. In the case of QN/stop (Fig. 7B), E is smaller for secreted than for cellular AChE, in agreement with the fact that the G4na form is exported more efficiently than the G1a and G2a forms, whereas in the case of QN/HC, the values of E were approximately equal for the cellular and secreted compartments (Fig. 7A).

In the following experiments we generally used 5 µg of DNA encoding AChET and 6 µg of DNA encoding the binding protein under study, unless indicated otherwise, as a compromise between the yield of total AChE activity and the proportion of hetero-oligomers obtained by association of the two types of subunits.

Do Released Tetramers Contain the QN Domain?

Although GPI-anchored tetramers necessarily incorporated a processed (glypiated) QN/HC protein, this was not obvious for the corresponding released G4na molecules obtained in the presence of QN/HC or of QN/stop. To detect the presence of the QN domain, we used a rabbit polyclonal antibody raised against a fusion protein containing this domain. This antibody, anti-QN, increased the sedimentation coefficient of both cell-associated and released tetramers produced by cells expressing Torpedo AChET together with QN/HC by about 1 S unit, whereas the G2 peak was not affected (not shown). The effect of the antiserum was more marked in nondenaturing electrophoresis, probably because dilution was less important in this case (Fig. 9): a fraction of the G4 form, secreted by cells coexpressing rat AChET and either QN/HC or QN/stop, was retarded, indicating that the released tetramers contained the QN binding domain.


Fig. 9. Detection of the QN domain in secreted G4na AChE by the anti-QN antiserum. The culture medium of COS cells expressing rat AChET together with QN/HC or QN/stop was analyzed by nondenaturing electrophoresis, without (-) or with the anti-QN antiserum (+). Whereas nonimmune serum had no effect, the anti-QN antiserum retarded the migration of a fraction of the G4na form. In this experiment, we used QN/stop111, in which a stop codon was added at position 111 within the QN domain (see Ref. 31). Asterisks indicate the position of complexes with the anti-QN antibodies.
[View Larger Version of this Image (51K GIF file)]


To examine whether the complete binding protein remained associated in the hetero-oligomers, we also introduced the flag peptide at the NH2-terminal and at the COOH-terminal extremities of the QN domain.

In the first case, the epitope was placed between residues Ala-42 and Glu-43 of the precursor sequence in the QN/HC chimeric protein ("N-flagged" QN/HC), corresponding to the most likely cleavage site of the signal peptide (21). By coexpression of N-flagged QN/HC with rat AChET, we obtained cell-associated GPI-anchored G4a and released G4na, with the same apparent affinity as with QN/HC, demonstrating that the presence of the flag epitope did not interfere with the heteromeric association of QN and AChET subunits. The sedimentation of these molecules was not affected visibly by the M1 monoclonal antibody, probably because its affinity was not sufficient to withstand dilution in the gradients, but they were both partially retarded by M1 during electrophoretic migration in nondenaturing polyacrylamide gels (Fig. 10A). Only a fraction of the molecules was retarded, as also observed in the case of anti-QN, perhaps because accessibility of the epitope was restricted by the presence of an associated tetramer of catalytic subunits. Alternatively, it is possible that several sites of cleavage coexist or that the flag sequence introduced a new cleavage site, which eliminated the epitope from the mature protein, so that only a fraction of hetero-oligomers possessed a complete epitope.


Fig. 10. Detection of N- and C-flagged QN domains associated with AChE tetramers in nondenaturing electrophoresis. Panel A, detection of N-flagged QN/HC. The cell extract was treated with PI-PLC to accelerate the migration of the G4 form, allowing a better visualization of the effect of the antibodies. The treated extract and the medium were analyzed without or with the M1 antibody, as indicated. Panel B, detection of C-flagged QN/stop. In the cell extract and culture medium, the G4na form is retarded by the M2 antibody. Asterisks indicate the positions of complexes with the anti-flag antibodies.
[View Larger Version of this Image (64K GIF file)]


In any case, this showed that the QN domain was included not only in the GPI-anchored G4a molecules but also in the released G4na. In addition, it indicated that the cleavage of the signal peptide occurred after Ala-42 at least in a fraction of the protein, since the M1 antibody is considered to recognize the flag epitope only in an NH2-terminal position. Glu-43 is therefore the likely NH2-terminal extremity of the mature QN domain that binds an AChET tetramer.

The flag epitope was also added immediately after the QN domain, instead of HC. When this construction (QN/flag) was cotransfected with rat AChET, we obtained the same result as with QN/stop, i.e. a large production and release of G4na, at the expense of G1a and G2a. In the medium the released G4na molecules contained the QN/flag protein, as shown by the M2 antibody, which recognizes the epitope in a COOH-terminal position; it induced a shift in sedimentation (not shown) and retarded migration in nondenaturing electrophoresis (Fig. 10B). The presence of two distinct bands that react with the antibody suggests that at high doses of DNA, QN may also be associated with dimers of AChET subunits, in addition to tetramers.

Thus, the presence of a flag epitope, either in an NH2-terminal or a COOH-terminal position, did not prevent the production of the heteromeric AChE tetramers. The M1 antibody was able to recognize the NH2-terminal flag in the lytic cellular and released soluble tetramers (G4na) derived from GPI-anchored G4a. Soluble tetramers obtained in the presence of QN/stop carrying an NH2- or COOH-terminal flag were recognized by M1 and M2, respectively, showing that they also contained the binding protein.


DISCUSSION

The Fate of AChET Subunits Produced in COS Cells: Quaternary Associations; Secretion

In the first part of this study we analyzed the production of AChE by transfected COS cells expressing rat AChET subunits alone. As shown previously, the cells contained monomers (G1a), dimers G2a, and tetramers (G4) (6). The G1a and G2a forms correspond to amphiphilic molecules of class II, which interact with detergent and lipid micelles, probably through an amphiphilic alpha -helical region in the COOH-terminal T peptide2 (1). The production of AChE increased with the quantity of DNA used for transfection, in a saturable manner, but the proportions of the different forms were not modified. AChE activity increased with time after transfection, with an accumulation of tetramers over monomers and dimers, in agreement with the fact that the metabolic turnover rate of the latter is more rapid (20).

We show here the existence of amphiphilic tetramers (G4a) as well as nonamphiphilic tetramers (G4na). As shown in the case of butyrylcholinesterase (22, 23), the G4na component probably represents homotetramers of AChET subunits, in which the amphiphilic helices of the T peptides interact with each other. In contrast, the structure of G4a might represent another type of quaternary organization in which all or part of the amphiphilic helices is exposed. The fact that this molecular form can be solubilized readily in the absence of detergent indicates that it differs from the membrane-bound G4a AChE of mammalian brain, which contains a 20-kDa hydrophobic anchor (24-26).

COS cells also produced a nonamphiphilic, unstable component sedimenting at 13.7 S, which may be an hexamer of catalytic subunits, unless it contains other subunits. In spite of its instability, the 13.7 S form was sometimes observed in the culture medium, although in a minor proportion, suggesting that it does not incorporate intracellular resident proteins. It was also found in Xenopus oocytes expressing the rat AChET subunit.4 This component readily dissociates at 37 °C, producing mostly monomers (G1a) in the absence of detergent, but also dimers and amphiphilic tetramers (G4a) in the presence of Triton X-100. Interaction with detergent micelles may stabilize a quaternary conformation of tetramers in which hydrophobic surfaces are exposed.

Finally, we illustrate the fact that, although the characteristics of the molecular forms are well defined, their proportions are variable among different experiments, especially over long periods of time, using different batches of COS cells. Such variations may result from differences in the biosynthetic capacity of the cells, in the culture medium, or in the extraction and storage of the enzyme, especially in view of the instability of some of the molecular forms. The fact that the pattern of molecular forms may be modified in a temperature-dependent manner by detergent (Triton X-100) indicates that it does not exactly reflect the state of quaternary associations of AChE in the intact cell.

COS cells expressing Torpedo AChET subunits produce a very small proportion of monomers, but mostly dimers, G2a, together with minor G4a and G4na forms. This suggests that, unlike rat AChET, the Torpedo AChET subunits are unstable in the monomeric state.

The release of Torpedo and rat AChE in the culture medium differed markedly. The culture medium of COS cells expressing rat AChET subunits contained about 30% of the total AChE activity after 2 days and 60% after 3 days following transfection, with similar proportions of amphiphilic dimers (G2a) and monomers (G1a) of type II, together with a smaller proportion of nonamphiphilic tetramers (G4na). Although their sedimentation properties appeared identical, the migration of the cellular and released G1a and G2a forms was clearly different in nondenaturing electrophoresis. This suggests that post-translational modifications accompany the release of rat G1a and G2a AChE, which therefore represents a true secretory process and does not result from cell lysis or other damage. These modifications may include maturation of glycans, proteolysis, or possibly palmitoylation (27).

When a flag peptidic epitope was added by mutagenesis at the COOH terminus of the rat AChET subunit, it did not modify the production, oligomeric assembly, or secretion of the enzyme. The epitope could be detected by a specific monoclonal antibody (M2) in the cellular but not in the secreted molecules, indicating that secretion could be accompanied by proteolytic cleavage. If cleavage does occur within the T peptide, however, only few residues are removed, since antibodies raised against its last 10 amino acids recognize both cellular and secreted molecules. This is consistent with the fact that the secreted monomers and dimers retain their amphiphilic character, which probably depends on the presence of an amphiphilic alpha -helix, constituted by the first 20 amino acids of the T peptide.2 Note, however, that Liao et al. (17) observed that soluble monomers from bovine brain did not react with their anti-COOH-terminal peptide. We also found that media recovered after several days of culture could contain nonamphiphilic monomers (G1na), probably resulting from proteolysis of the T peptide, as already observed by Velan et al. (28), in the case of HEK 293 cells expressing the human AChET subunit. Taken together, these observations suggest the existence of multiple cleavage sites. It will be interesting to investigate whether secreted dimers (G2a) conserve the cysteine residue that is located at position -4 from the COOH terminus and forms an intersubunit disulfide bond in tetramers.

Cells expressing Torpedo AChET subunits produced the G2a form but no G1a form and released very little AChE activity. This difference could not be accounted for by the fact that the cultures were maintained at 37 °C in the case of rat AChE, but transferred to 27 °C to produce Torpedo AChE in an active form. When rat AChE was expressed at 27 °C, the total activity was reduced to about one-third of its value at 37 °C, but the ratio of secreted to cellular enzyme was approximately the same (not shown). Moreover, the heteromeric G4na form of Torpedo AChE, obtained in coexpression with a QN/stop protein, was secreted readily at 27 °C. It seems, therefore, that the Torpedo G2a form cannot be transported efficiently to the membrane and released into the medium.

The structure of AChET is remarkable because these various oligomeric states of the enzyme are not in equilibrium; extracted or secreted molecules may form aggregates under specific conditions but were never observed to assemble into well defined oligomers such as those produced in the cells. The nature of the enzyme has a crucial influence on the proportions of oligomeric and monomeric forms produced in cells; this depends on the alternative COOH-terminal peptides as shown by the difference between rat AChET and AChEH in rat basoleukemia cells (17), but also on the species. Thus, Torpedo AChE preferentially produces dimers, and rat AChE mostly produces monomers. These proportions also depend on the nature and state of differentiation of the cell; for example, it varies during development of the nervous system (for review, see Ref. 1), and AChET subunits are expressed in a tissue-specific manner in Xenopus embryos, even under the control of a viral promoter (29, 30). In culture, the yield of AChE activity was markedly less in rat basoleukemia cells than in COS cells, in parallel transient transfections (17). We show here that, depending on their state, the COS cells themselves produce variable patterns of molecular forms from AChET subunits. In the case of the rat enzyme, the cells may contain almost exclusively G1a, or equal amounts of G1a and G2a, together with significant proportions of G4a, G4na, and 13.7 S oligomers. Thus, the cellular environment somehow controls the formation of oligomers.

Interaction between the QN Domain and AChET in Cotransfected COS Cells: Production of Heteromeric Tetramers

Cotransfection of COS cells with rat or Torpedo AChET subunits, together with an isolated QN domain (QN/stop) or with a QN domain associated with a GPI addition signal (QN/HC), produced heteromeric molecules, G4na or GPI-G4a. In the case of Torpedo AChE, this allowed an active exportation and release of tetramers, whereas dimers were almost entirely retained within the cells. Immunofluorescence of permeabilized cells showed that the enzyme appeared distributed in the reticulum when expressed alone, but concentrated into subcellular bodies, perhaps secretory vesicles, when coexpressed with QN/HC.

At low doses of DNA encoding the binding proteins, we observed an increase in the production of total AChE (cellular and secreted), in agreement with the incorporation of monomeric G1a and dimeric G2a forms, which present a rapid metabolic turnover rate (20) into more stable tetramers. However, the total yield of AChE was decreased at high doses of DNA encoding QN/stop and more markedly QN/HC, probably because of competition between the production of catalytic and structural subunits.

The heteromeric G4na molecules obtained by association of QN/stop with AChET tetramers were soluble and secreted, resulting in a high yield of AChE activity in the culture medium. In contrast, the GPI-anchored molecules obtained by expression of QN/HC alone or in combination with AChET were mostly attached to the cellular surface, as shown by immunofluorescence of intact cells. Thus, the presence of QN/HC induced the assembly of GPI-anchored tetramers of AChET. Coexpression with QN/HC also produced some lytic G4na; in addition, at high doses of DNA encoding QN/HC, and in some batches of COS cells, we also observed the production of G1na molecules, both in cell extracts and in the medium. Since this G1na form was not observed in parallel cotransfections with QN/stop, the production of these lytic molecules possibly occurs in the metabolic pathway of GPI-anchored proteins, e.g. after reinternalization from the cell surface. Note, however, that cells expressing only AChET also produced a G1na form after several days in culture, as also observed in HEK 293 cells (28), but this may result from another type of cleavage.

We considered the possibility that a QN domain might induce the formation of AChET tetramers, without necessarily participating in the final quaternary structure. This hypothesis seemed consistent with the fact that homotetramers are formed at a low level, in the absence of binding proteins, and that the pair of vicinal cysteines of the QN domain could be mutated without affecting the production of G4 molecules (31). To detect the presence of QN in G4 molecules, we used an antiserum raised against this domain. We found that the sedimentation of released G4na was shifted in the presence of this antiserum to the same extent as that of PI-PLC-treated molecules derived from GPI-anchored G4a, indicating that QN was associated with the released G4na tetramers. The presence of QN in the secreted soluble G4na form, as well as in the GPI-anchored cellular G4a form, was confirmed by inserting a flag epitope in NH2-terminal position (N-flagged QN/stop and QN/HC), and in COOH-terminal position (QN/flag). The M1 monoclonal antibody reacted only with a fraction of heteromeric tetramers when a flag epitope was introduced between residues 42 and 43 of the precursor of QN (both in QN/stop and QN/HC), possibly because it could be cleaved during maturation. Although limited, this reaction indicated that cleavage of the signal peptide occurs at the indicated position, that the mature protein probably starts at Glu-43, and that the flag peptide does not prevent interaction with AChET.

In this study we showed that AChET subunits, when expressed in COS cells, are able to generate a number of various more or less stable oligomeric associations. Coexpression with binding proteins derived from the NH2-terminal domain of the collagenic tail induces the preferential formation of heteromeric molecules in which AChET tetramers are assembled with a binding domain. This appears to facilitate the transport of AChET subunits in the secretory pathway and exportation to the cell surface, in the case of GPI-anchored molecules, or their secretion, in the case of soluble molecules. The recruitment of AChET subunits depends on the dose of binding protein, in a saturable manner. It is therefore possible to analyze these protein-protein interactions quantitatively in the secretory pathway of living cells.


FOOTNOTES

*   This research was supported by grants from the CNRS, the Direction des Recherches et Etudes Techniques, the Association Française contre les Myopathies, and the Human Capital and Mobility program of the European Community. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 33-1-44-32-3891; Fax: 33-1-44-32-3887; E-mail: jean.massoulie{at}biologie.ens.fr.
1    The abbreviations used are: AChE, acetylcholinesterase; AChET, AChE catalytic subunit of type T; ACLEH, AChE subunit of type H; Q, collagen subunit; QN, Q subunit NH2-terminal domain; QC, Q subunit COOH-terminal domain; GPI, glycophosphatidylinositol; HC, AChEH subunit COOH-terminal domaion; PI-PLC, phosphatidylinositol-phospholipase C.
2    S. Bon, I. Cornut, J. Dufourcq, J. Grassi, and J. Massoulié, manuscript in preparation.
3    S. Bon, F. Coussen, and J. Massoulié, manuscript in preparation.
4    E. Krejci, personal communication.

Acknowledgments

We thank Anne Le Goff and Rizwana Nawaz for expert technical assistance; Dr. Françoise Coussen and Dr. Jacques Grassi for preparation of the rabbit polyclonal anti-QN antiserum; Prof. Bent Nørgaard-Pedersen, Prof. Urs Brodbeck, and Dr. Nicola Boschetti for the anti-bovine COOH-terminal peptide antiserum; and Jacqueline Pons for typing the manuscript.


REFERENCES

  1. Massoulié, J., Pezzementi, L., Bon, S., Krejci, E., and Vallette, F. M. (1993) Prog. Neurosci. 41, 31-91
  2. Rosenberry, T. L., and Richardson, J. M. (1977) Biochemistry 16, 3550-3558 [Medline] [Order article via Infotrieve]
  3. Anglister, L., and Silman, I. (1978) J. Mol. Biol. 125, 293-311 [Medline] [Order article via Infotrieve]
  4. Lee, S. L., Heinemann, S., and Taylor, P. (1982) J. Biol. Chem. 257, 12282-12291 [Medline] [Order article via Infotrieve]
  5. Krejci, E., Coussen, F., Duval, N., Chatel, J. M., Legay, C., Puype, M., Vandekerckhove, J., Cartaud, J., Bon, S., and Massoulié, J. (1991) EMBO J. 10, 1285-1293 [Abstract]
  6. Legay, C., Bon, S., Vernier, P., Coussen, F., and Massoulié, J. (1993) J. Neurochem. 60, 337-346 [Medline] [Order article via Infotrieve]
  7. Massoulié, J., Sussman, J., Bon, S., and Silman, I. (1993) Prog. Brain Res. 98, 139-146 [Medline] [Order article via Infotrieve]
  8. Camp, S., Bon, S., Li, Y., Getman, D. K., Engel, A. G., Massoulié, J., and Taylor, P. (1995) J. Clin. Invest. 95, 333-340 [Medline] [Order article via Infotrieve]
  9. Duval, N., Krejci, E., Grassi, J., Coussen, F., Massoulié, J., and Bon, S. (1992) EMBO J. 11, 3255-3261 [Abstract]
  10. Bon, S., Toutant, J. P., Méflah, K., and Massoulié, J. (1988) J. Neurochem. 51, 786-794 [Medline] [Order article via Infotrieve]
  11. Bon, S., Toutant, J. P., Méflah, K., and Massoulié, J. (1988) J. Neurochem. 51, 776-785 [Medline] [Order article via Infotrieve]
  12. Bon, S., Rosenberry, T. L., and Massoulié, J. (1991) Cell. Mol. Neurobiol. 11, 157-172 [Medline] [Order article via Infotrieve]
  13. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382 [Medline] [Order article via Infotrieve]
  14. Ellman, G. L., Courtney, K. D., Andres, V., and Featherstone, R. M. (1961) Biochem. Pharmacol. 7, 88-95 [CrossRef][Medline] [Order article via Infotrieve]
  15. Karnovsky, M. J., and Roots, L. (1964) J. Histochem. Cytochem. 12, 219-232 [Medline] [Order article via Infotrieve]
  16. Marsh, D., Grassi, J., Vigny, M., and Massoulié, J. (1984) J. Neurochem. 43, 204-213 [Medline] [Order article via Infotrieve]
  17. Coussen, F., Bonnerot, C., and Massoulié, J. (1995) Eur. J. Cell Biol. 67, 254-260 [Medline] [Order article via Infotrieve]
  18. Liao, J., Boschetti, N., Mortensen, V., Jensen, S. P., Koch, C., Norgaard-Pedersen, B., and Brodbeck, U. (1994) J. Neurochem. 63, 1446-1453 [Medline] [Order article via Infotrieve]
  19. Gibney, G., and Taylor, P. (1990) J. Biol. Chem. 265, 12576-12583 [Abstract/Free Full Text]
  20. Lazar, M., Salmeron, E., Vigny, M., and Massoulié, J. (1984) J. Biol. Chem. 259, 3703-3713 [Abstract/Free Full Text]
  21. von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690 [Abstract]
  22. Lockridge, O., Adkins, S., and La Du, B. N. (1987) J. Biol. Chem. 262, 12945-12952 [Abstract/Free Full Text]
  23. Lockridge, O., Bartels, C. F., Vaughan, T. A., Wong, C. K., Norton, S. E., and Johnson, L. L. (1987) J. Biol. Chem. 262, 549-557 [Abstract/Free Full Text]
  24. Inestrosa, N. C., Roberts, W. L., Marshall, T. L., and Rosenberry, T. L. (1987) J. Biol. Chem. 262, 4441-4444 [Abstract/Free Full Text]
  25. Gennari, K., Brunner, J., and Brodbeck, U. (1987) J. Neurochem. 49, 12-18 [Medline] [Order article via Infotrieve]
  26. Roberts, W. L., Doctor, B. P., Foster, J. D., and Rosenberry, T. L. (1991) J. Biol. Chem. 266, 7481-7487 [Abstract/Free Full Text]
  27. Randall, W. R. (1994) J. Biol. Chem. 269, 12367-12374 [Abstract/Free Full Text]
  28. Velan, B., Grosfeld, H., Kronman, C., Leitner, M., Gozes, Y., Lazar, A., Flashner, Y., Marcus, D., Cohen, S., and Shafferman, A. (1991) J. Biol. Chem. 266, 23977-23984 [Abstract/Free Full Text]
  29. Ben Aziz-Aloya, R., Seidman, S., Timberg, R., Sternfeld, M., Zakut, H., and Soreq, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2471-2475 [Abstract]
  30. Seidman, S., Sternfeld, M., Ben Aziz-Aloya, R., Timberg, R., Kaufer-Nachum, D., and Soreq, H. (1995) Mol. Cell. Biol. 1555, 2993-3002
  31. Bon, S., Coussen, F., and Massoulié, J. (1997) J. Biol. Chem. 272, 3016-3021 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.