©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Determinants Conferring -Toxin Resistance in Recombinant DNA-derived Acetylcholine Receptors (*)

(Received for publication, November 7, 1994)

Steven H. Keller Hans-Jürgen Kreienkamp Chiaki Kawanishi (§) Palmer Taylor

From the Department of Pharmacology, 0636, University of California, La Jolla, California 92093

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Sequences of the alpha-subunits of the nicotinic acetylcholine receptor from the snake and mongoose contain several differences in the region between amino acids 183 and 200. Receptors from both of these species reveal resistance to the snake alpha-toxins presumably arising as a protective evolutionary mechanism. Sequence differences include the added glycosylation signals at residue 187 in the mongoose and at residues 189 and 111 in snake. Although previous observations with peptides and fusion proteins either synthesized chemically or in a bacterial expression system indicate that certain amino acid residues may contribute to the resistance, our findings with the intact receptor in an eukaryotic expression system indicate the major role for glycosylation. In this study, we show that addition of glycosylation signals gives rise to virtually complete glycosylation at the added sites, although heterogeneity of oligosaccharide processing is evident. By analysis of combinations of mutants, we document that glycosylation exerts the predominant influence on alpha-toxin binding. Substitutions at other residues are largely without influence as single mutations but appear to decrease affinity further in multiple mutants, particularly where the receptor is glycosylated at the 187 and 189 positions. Glycosylation exerts a major influence on the dissociation as well as the association rates of the alpha-toxin-receptor complex, suggesting that the decrease for alpha-toxin affinity is not simply a consequence of restricted diffusional access, rather glycosylation affects the conformation and stability of the bound complex.


INTRODUCTION

The nicotinic acetylcholine receptor (nAChR) (^1)at the neuromuscular junction is a ligand-gated cation channel displaying a pentameric configuration of four subunits (alpha, beta, , and ) in the stoichiometric ratio of alpha(2)beta (cf.Unwin, 1993). Ligand-binding domains are positioned at the alpha- and alpha- interfaces (cf. Karlin, 1993). The nAChRs in mouse and humans have a high affinity for snake neurotoxins, such as alpha-bungarotoxin, a competitive but slowly dissociating antagonist at the receptor. Regions of ligand recognition on the intact receptor have been identified by affinity labeling (Dennis et al., 1988; Kao et al., 1984; Kao and Karlin, 1986; Abramson et al., 1989; Galzi et al., 1990, 1991; Cohen et al., 1991; Middleton and Cohen, 1991) and site-directed mutagenesis (Mishina et al., 1986; Tomaselli et al., 1991; O'Leary and White, 1992; Aylwin and White, 1994; Sine et al., 1994), and are believed to reside primarily between amino acid residues 185 and 199 on the alpha-subunit (Griesmann et al., 1990) and on adjoining interfaces of the and subunits (reviewed in Changeux et al., 1992; Karlin, 1993).

A comparison of amino acid sequences with characteristics of receptor specificity provides an additional means of detailing residues contributing to ligand recognition. The unique features of the sequences of the alpha-subunit of the nAChR of the cobra (Naja naja), and its predator, the mongoose, are of particular interest. Receptors from these species have a low affinity for alpha-bungarotoxin, and cDNA sequences of their alpha-subunits encode unique residues in the presumed toxin-binding domain. These substitutions include the addition of glycosylation signals (Asn-X-Ser/Thr) on asparagine 189 in cobra and asparagine 187 in mongoose, and the substitution of proline 194 in human and mouse to leucine 194 in cobra and mongoose (Neumann et al., 1989; Barchan et al., 1992).

The contribution of amino acid substitutions in the toxin-binding domain to alpha-toxin resistance was investigated using chemically synthesized peptides and by mutagenesis and expression of fusion proteins in bacteria of an approximately 20 amino acid sequence of the Torpedo alpha-subunit known to bind alpha-bungarotoxin (Conti-Tronconi et al., 1991; Barchan et al., 1992; Chaturvedi et al., 1992, 1993). Substitutions in the toxin-sensitive sequence that abolished toxin binding included changes from tyrosine 189 in Torpedo to asparagine 189 present in cobra or to threonine 189 present in mongoose, and a substitution of proline 194 in mouse to leucine 194 in cobra (Ohana, 1991; Chaturvedi et al., 1992, 1993). Other studies indicated that the presence of tryptophan at residue 187 in mouse contributes to toxin affinity (Neumann et al., 1986). Since these studies assayed ligand binding affinity to peptide sequences expressed in bacteria, the roles played by glycosylation and by subunit interfacial contacts in influencing the three-dimensional configuration of the intact receptor could not be considered.

The influence of glycosylation signals and additional single amino acid changes unique to the mongoose and cobra were also investigated by using site-directed mutagenesis and transfection in human embryonic kidney cells of the mouse wild-type and mutant alpha-subunits along with the beta-, -, and -subunits (Kreienkamp et al., 1994). This study demonstrates that the mutation F189N in the alpha-subunit, which introduced one of the glycosylation signals present in the cobra, lowers alpha-bungarotoxin affinity 140-fold. Likewise, expression of the mongoose glycosylation signal W187N,F189T reduces alpha-bungarotoxin affinity by 600-fold. Additional mutations introduced separately in the mouse cDNA template were also investigated, including the mutation P194L, which results in no significant diminishment of toxin affinity, a finding in conflict with results from experiments with the bacterial expressed proteins (Chaturvedi et al., 1992, 1993).

In the study, we have extended these initial observations to single and multiple site mutations in order to examine interactions between sites and quantitate the free energy contributions of the additional substitutions. We also use mobility shifts on SDS-polyacrylamide gel electrophoresis before and after glycosidase treatments to ascertain the extent of oligosaccharide conjugation and its heterogeneity at the introduced glycosylation sites.


EXPERIMENTAL PROCEDURES

Materials

I-alpha-Bungarotoxin (specific activity of 8-16 µCi/µg) was purchased from DuPont NEN. Monoclonal antibodies directed to the alpha-subunit of the acetylcholine receptor were obtained from Dr. Darwin Berg, University of California, San Diego and Dr. Jon Lindstrom, University of Pennsylvania (mAbs 35 and 210). Peroxidase-conjugated antimouse antibody was from Life Technologies, Inc. and the ECL Western blot chemiluminescence detection system was manufactured by Amersham Corp. UltraLink Protein-G resin (Pierce) was used for immunoprecipitation. Recombinant glycosidases PNGase-F and Endo-H were purchased from New England Biolabs.

Site-directed Mutagenesis and Subcloning

Double-stranded plasmids containing the cDNA sequences of the four receptor subunits used for mutagenesis and expression were prepared as described in Kreienkamp et al.(1994). To prepare single-stranded plasmids for mutagenesis, plasmids containing a cDNA encoding the mouse muscle alpha-subunit were transfected into competent CJ236 bacteria and selected in ampicillin and chloramphenicol. Helper phage stock was added, further selection was in kanamycin, and single-stranded plasmids were isolated following polyethylene glycol precipitation and extraction with chloroform-phenol. Mutagenesis was performed using the above single stranded templates (Kunkel et al., 1987). Following a synthesis reaction, double-stranded plasmids were transfected and raised in DH1-alpha bacterial cells and isolated following a large scale plasmid preparation by CsCl density centrifugation. The DNA sequence changes were verified by dideoxy-DNA sequencing. The mutations introducing F189N, W187N,F189T (Kreienkamp et al., 1994) and D111N were introduced into a wild-type alpha-subunit cDNA sequence. The mutation P194L was introduced into plasmids previously mutated to contain either F189N or W187N,F189T. The mutant D111N was added to plasmids containing the other mutants by subcloning at PstI sites. Fig. 1lists the mutations introduced into the wild-type mouse sequence and the sequence differences between species.


Figure 1: Amino acid sequences of the nicotinic acetylcholine receptor alpha-subunits in regions flanking the glycosylation sites found in snake (Naja naja) and mongoose. The glycosylated asparagines at 187, 189, and 111 and the leucine to proline substitution are shown in bold type. The human sequence is from Noda et al.(1983), snake and mouse are from Neumann et al.(1989), and mongoose is from Barchan et al.(1992). The cobra sequence is believed to be identical to the alpha-toxin-resistant water snake Natrix in the region near amino acid residue Asn, as described in Neumann et al.(1989). The mongoose amino acid sequence in the vicinity of amino acid residue Asn has not been published.



Expression of Receptors

For cell surface expression used in the binding assays and Western blots, plasmids encoding each of the four receptor subunits were transfected in human kidney embryonic cells (HEK-293) using calcium phosphate precipitation with 15 µg of alpha-subunit DNA and 7.5 µg of the beta-, -, and -subunits for each 10-cm culture dish (Kreienkamp et al., 1994). For receptor binding assays, cells were harvested by treating plates with phosphate-buffered saline (PBS) containing 5 mM EDTA. The cells were gently sedimented and resuspended in K Ringers buffer and the specified concentrations of ligands were added. For the Western blots, after the culture media was removed, the plates were washed three times in PBS. The cells were harvested with PBS-EDTA, sedimented, and resuspended in an appropriate buffer.

Binding Assays

Association and dissociation rates were measured as described in Kreienkamp et al.(1994). Briefly, for association measurements, I-alpha-bungarotoxin (18 nM) was added to cells, and the reaction was stopped at appropriate times by placing an aliquot of cells into an excess of carbamylcholine. Unbound toxin was washed away and the cells were sedimented. Nonspecific toxin binding was measured by prior incubation of the cells in 10 mM carbamylcholine and then determining bound alpha-toxin at the initial and late time points. The measurements for nonspecific toxin binding were subtracted from the appropriate values of total toxin binding in the samples to estimate the specific toxin binding used to calculate the association rate constant. For dissociation rate measurements, cells were incubated in 20-40 nMI-alpha-bungarotoxin for at least 2 h. The cells were aliquoted into a large volume of K Ringers buffer, toxin was allowed to dissociate, and cells were washed free of toxin at specified times. To measure nonspecific binding, cells were incubated with alpha-toxin in the presence of 10 mM carbamylcholine for 10 min, and the dissociation was assayed from the initial and later times. Values of specific toxin binding were used to estimate the dissociation rate constant. Dissociation constants were calculated by assuming a bimolecular association and unimolecular dissociation process.

Solubilization of the Receptor for Western Blots

For Western blots of whole cell extracts, cells were harvested with PBS-EDTA, washed three times in PBS and solubilized in 1% Triton X-100, 10 mM EDTA, 150 mM NaCl, 50 mM HEPES, pH 7.4 for 4 h on ice. Debris were removed by sedimentation, the supernatant was added to Laemmli sample buffer, and agitated at room temperature for 1 h.

To immunoprecipitate selectively receptors from the cell surface, cells were washed three times in an excess of PBS then resuspended with PBS containing 1 µg/ml each of the protease inhibitors, benzamidine, pepstatin A, aprotinin and leupeptin, and 10 mM EDTA, then incubated with the monoclonal antibody 35 directed against the nAChR for 2 h at 4 °C. Cells were washed five times in the PBS-protease inhibitor solution to remove unbound antibody, then the cell membranes were solubilized in 50 mM sodium acetate, pH 5.0, 500 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.02% sodium azide, 10 mM EDTA, and with the same protease inhibitors as above for 3 h on ice. Cell debris were removed by light sedimentation, the supernatant was collected, and Immobilized Protein G (UltraLink Protein G; Pierce) was added for 2 h at room temperature. The immunoprecipitation beads were sedimented, washed, and sample buffer was added. The beads were agitated at room temperature for 1 h, then the solution was heated at 65 °C for 5 min. To verify that antibodies were not incorporated intracellularly, cells were transfected with the exclusion of the beta-subunit, which limits the expression of the alpha-subunit to intracellular locations of the cell (Blount and Merlie, 1988); intact cells were incubated with mAb 35 as above and the immunoprecipitation protocol was performed as detailed above.

Glycosidase Treatments

One µl of mercaptoethanol was added to 30 µl of solubilized receptor, the solution was agitated vigorously, and 1 µl of PNGase-F (New England Biolabs) or 1 µl of Endo-H (New England Biolabs) was added. This solution was incubated for 5 h at room temperature, then 70 µl of Laemmli sample buffer was added.

Electrophoresis and Western Blots

The number of plates of cells immunoprecipitated or extracted was adjusted to the expression levels of the mutant alpha-subunits to load approximately equivalent amounts of receptor protein in each gel lane. Since receptors with alpha-subunits encoding the F189N substitution expressed at approximately 15% the level of wild-type (Kreienkamp et al., 1994), unless stated otherwise, approximately six times more cellular material was used for this mutant. Equivalent amounts of cellular materials were used for alpha-subunits encoding the Asn glycosylation site as wild-type alpha-subunits. Each gel lane for the wild-type alpha-subunit was derived from approximately one 10-cm plate of confluent cells with an expression of approximately 200 fmol of I-alpha-bungarotoxin-binding sites/plate. Receptors from detergent extracts or immunoprecipitation were solubilized in Laemmli sample buffer and separated in a 22-cm 17.5% polyacrylamide gel. Prestained molecular weight standards were used to trace the positions of the alpha-subunits. To amplify migration differences, current was applied until the prestained 45 kDa marker migrated approximately 5 cm from the end of the gel. The gel section between the prestained 45 and 29 kDa molecular mass markers was blotted onto nitrocellulose for 1 h at 500 mA (Towbin et al., 1979). Blots were incubated with a monoclonal antibody directed against the alpha-subunit (mAb 210) at a dilution of 1:1000. The secondary antibody was conjugated to horseradish peroxidase, and visualization was by chemiluminescent detection (ECL, Amersham).


RESULTS

Wild-type and Mutant Receptor Glycosylation Patterns

Fig. 1diagrams the residue substitutions investigated in this study. To assess the extent of glycosylation of alpha-subunits expressed on the cell surface, cells were transfected with plasmids encoding the alpha-, beta-, -, and -subunits (except in Fig. 2lanes B and F), alpha-subunits were immunoprecipitated from intact cells, and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting (Fig. 2, lanes A-D). Lane A on Fig. 2displays wild-type alpha-subunits expressed with the beta-, -, and -subunits: lane B excluded the beta-subunit used in lane A and demonstrates that detectable alpha-subunits do not reach the cell surface. The importance of the beta-subunit for assembly was previously shown by Blount and Merlie(1988). Lane C displays the mutant alpha-subunit expressing an additional glycosylation site encoded by the substitution F189N: it predominantly migrates slower on gels than the wild-type alpha-subunit. Lane D diagrams alpha-subunits expressing two additional glycosylation sites encoded by the substitutions F189N and D111N. Addition of two glycosylation signals results in a further reduction in the migration of the alpha-subunit. Lanes E-H show the antibody blots of the cell extracts after removal of the cell surface receptor by prior incubation of the intact cells with antibody and removal of the antibody complex. Since a Western blot of the detergent extract of the sample where the beta-subunit was excluded in the transfection detects the alpha-subunit intracellularly (lane F) as determined by an alignment of a protein band with the alpha-subunit detected in lane A, the alpha-subunits in lanes A, C, and D must be situated on the cell surface. Fig. 2demonstrates the encoding of glycosylation signals at Asn and Asn results in a decrease in migration of alpha-subunits on the cell surface, indicating additional oligosaccharides are present. Diffuse alpha-subunit banding patterns for the mutants in Fig. 2also indicate that oligosaccharide processing is heterogeneous at these sites.


Figure 2: Western blots of wild-type and mutant alpha-subunits containing the substitutions F189N, P194L, and D111N. Except for lanes B and F, cells were transfected with DNA coding for the alpha-, beta-, -, and -subunits; beta-subunit DNA was excluded in the transfections in lanes B and F. Lanes A-D, receptors immunoprecipitated from the cell surface using mAb 35 for immunoprecipitation and mAb 210 for Western blot detection. Approximately 200 fmol of alpha-subunit protein was loaded in each gel lane of immunoprecipitated receptors. Lanes E-H, Western blots of residual whole cell extracts after removal of receptors immunoprecipitated from the cell surface. Each lane was derived from approximately one-third of a 10-cm plate of confluent cells. mAb 210 was used for Western blot detection. Lane A, wild-type alpha-subunit immunoprecipitated from the cell surface; lane B, wild-type alpha-subunit with the exclusion of the beta-subunit in the transfection, which prevents receptor export to the cell surface; lane C, mutant alpha-subunit with the substitutions F189N which introduces a glycosylation site and P194L; lane D, mutant alpha-subunit with the substitutions F189N, P194L, and D111N. D111N also introduces another glycosylation site. Lane E, whole cell extract of cells transfected with wild-type alpha-subunit DNA; lane F excludes the beta-subunit in the transfection; lane G, the mutant alpha-subunit with the substitutions F189N and P194L; lane H, mutant alpha-subunit with the substitutions F189N, P194L, and D111N.



Detergent extracts of cells transfected with receptor DNA were subjected to PNGase-F treatment to assess the role of glycosylation on gel mobility shifts (Fig. 3). Lanes A-C display Western blots of detergent extracts of whole cells for wild-type (lane A), and mutants alpha-subunits with the substitutions F189N and F189N,D111N (lanes B and C, respectively), and lanes D-F display samples treated with PNGase-F (lane F, wild-type; lane E, F189N; lane D, F189N,D111N). The region between arrows points to the location of bands of the alpha-subunit, as determined by their presence at similar migration distances on gels displaying alpha-subunits immunoprecipitated from the cell surface (Fig. 2, lanes A, C, and D). Dark staining protein bands do not appear at these locations in blots of cells not transfected with receptor DNA (data not shown). The number of plates of cell solubilized in these detergent extractions was normalized to the expression efficiency of the various alpha-subunits in order to load approximately equivalent amounts of receptor protein in each gel lane. Since the introduction of the substitution F189N resulted in low levels of receptor expression (Kreienkamp et al., 1994), larger amounts of cellular material were required, resulting in the detection of background protein bands. PNGase-F treatment alters the diffuse appearance and slower mobility of the mutant alpha-subunits to equal the relatively sharp appearance and faster mobility of the wild-type alpha-subunit (Fig. 3, lanes D-F), indicating that additional glycosylation contributes to both the observed molecular weight shifts and the diffuse protein banding patterns of the mutant alpha-subunits. The wild-type alpha-subunit shifted in mobility following PNGase-F treatment. It contains a conjugated oligosaccharide at Asn that is also present in the mutants.


Figure 3: PNGase-F treatments of wild-type and mutant alpha-subunits with the substitutions F189N, P194L, and D111N. Detergent extracts of whole cells were blotted and mAb 210 was used for detection. Five times the amount of cellular material was used for the mutant compared to wild-type alpha-subunit lanes due to lower expression of mutants. Lanes A and F, untreated and PNGase-F-treated cellular extracts, respectively, of wild-type alpha-subunits; lanes B and E, untreated and PNGase-F-treated detergent extracts, respectively, of cells transfected with alpha-subunit DNA containing the substitutions F189N and P194L; lanes C and D, untreated and PNGase-F-treated detergent extracts, respectively, with the substitutions F189N, P194L, and D111N in the alpha-subunit. The region between the arrows and the 43K markers is where the alpha-subunits reside.



Evidence for glycosylation at Asn for alpha-subunits expressing the substitutions W187N,F189T is displayed on the Western blots of Fig. 4. Lanes A-H are Western blots of detergent extracts of whole cells, which should detect both cell surface and intracellular alpha-subunits. Lanes I and J are blots of receptors immunoprecipitated from the cell surface. The insertion of the Asn glycosylation site reduces the mobility of the mutant alpha-subunit on SDS-polyacrylamide gel electrophoresis, presumably due to the conjugation of an additional oligosaccharide chain (Fig. 4, lanes C and D). The protein banding pattern of this mutant alpha-subunit is not as diffuse as those expressing the Asn glycosylation site, indicating that oligosaccharide processing is more uniform at Asn. Since receptors with the mutation W187N,F189T show expression levels higher than wild-type, less cellular material was required to detect the alpha-subunit and therefore background protein bands are almost undetectable in these Western blots.


Figure 4: Western blots of wild-type and mutant alpha-subunits containing the substitutions W187N, F189T, and P194L. The substitutions W187N,F189T introduce a glycosylation signal present in mongoose. The length of exposure to film of these blots was much shorter than in Fig. 2and Fig. 3, which contributes to an absence of background protein bands. Lanes A-H are detergent extracts of whole cells, with each gel lane derived from approximately one-third of a 10-cm plate of confluent cells. Lanes I and J are blots of alpha-subunits immunoprecipitated from the cell surface and are derived from approximately 200 fmol of alpha-subunit protein. Lanes C-J show transfections of mutant and wild-type cDNAs encoding the alpha-subunit, along with beta-, -, and -subunit cDNAs. Lane A, extracts of cells not transfected with receptor subunit cDNAs; lane B, extracts of cells not transfected with receptor subunit cDNAs but treated with PNGase-F; lane C, cells transfected with wild-type alpha-subunit cDNA: lane D, mutant alpha-subunit containing the substitutions W187N,F189T which adds a new glycosylation site and P194L; lanes E and F, wild-type and the mutant alpha-subunit, respectively, treated with PNGase-F; lanes G and H, wild-type and the mutant alpha-subunit, respectively, treated with Endo-H; lanes I and J, wild-type and the mutant alpha-subunit, respectively, immunoprecipitated from the cell surface.



Cell extracts were treated with PNGase-F (Fig. 4, lanes E and F) and Endo-H (Fig. 4, lanes G and H) to characterize the additional oligosaccharide at Asn. Similar gel mobility changes are observed for the mutant alpha-subunits treated with PNGase-F or Endo-H (lanes F and H), indicating that oligosaccharides present at the Asn site are equivalently susceptible to cleavage by both of these enzymes and are therefore most likely high mannose structures. The higher mobility of the mutant subunit when compared to the wild-type after PNGase-F treatment is possibly due to substitution of aromatic side chains W, F, or the proline ring in wild-type with the 3 aliphatic amino acid residues Asn, Thr, and Leu in the mutant. Western blots of mutant receptors immunoprecipitated from the cell surface also display a decrease in migration, indicating that the majority of alpha-subunits with the substitution W187N,F189T possess oligosaccharides conjugated to Asn (Fig. 4, lanes I and J). In summary, all glycosylation signals on the wild-type and mutant alpha-subunits result in the expression of oligosaccharides in HEK-293 cells. The F189N mutation appears to impart far more heterogeneity of glycosylation than does W187N,F189T or the Asn glycosylation site present in the wild-type and mutant alpha-subunits.

Multiple Mutations Influencing alpha-Bungarotoxin Affinity

To serve as a frame of reference for comparison, the previously investigated templates (Kreienkamp et al., 1994) containing the wild-type sequence and the substitutions F189N and W187N,F189T were expressed and assayed for alpha-toxin association rates and dissociation rates ( Fig. 5and Fig. 6), and K(D) values were calculated (Table 1). The introduction of the mutation D111N into the wild-type template, which introduces the second glycosylation signal present in the cobra sequence, reduces the toxin association rate by approximately 3-fold (Fig. 5A, Table 1), and displays a negligible effect on the dissociation rate (Fig. 6A, Table 1).


Figure 5: Association kinetics for I-alpha-bungarotoxin to nAchRs expressed in HEK-293 cells. A, receptors expressing wild-type and mutant alpha-subunits with the substitution D111N. The inset shows the initial association of toxin with the receptor. B, receptors expressing mutant alpha-subunits with glycosylation sites at Asn, Asn, and Asn, and the substitution P194L. All measurements were made with 18 nMI-alpha-bungarotoxin in stoichiometric excess over the receptor sites. The lines shown represent computer estimates of the first order approach to equilibrium. Data are presented from representative experiments. , wild-type (solid line); bullet, D111N (dashed line); (box), W187N,F189T,P194L; +, F189N,P194L,D111N.




Figure 6: Dissociation kinetics of I-alpha-bungarotoxin from nAchRs expressed in HEK-293 cells. A, receptors expressing wild-type and mutant alpha-subunits with the substitution D111N. I-alpha-Bungarotoxin at 20 nM was used to form the initial complex. B, receptors expressing mutant alpha-subunits with glycosylation sites at Asn, Asn, and Asn and the substitution P194L. I-alpha-Bungarotoxin at 40 nM was used to form the initial complex. Data from representative experiments are presented. , wild-type; bullet, D111N; box, W187N,F189T,P194L; , F189N,P194L; +, F189N,P194L,D111N.





Introducing the mutation P194L into a cDNA template containing the substitution F189N, which encodes a glycosylation site present in the cobra sequence, reduces the association rate approximately 8-fold (Fig. 5B, Table 1) and increases the dissociation rate in the fast phase approximately 8-fold over alpha-subunits possessing the F189N substitution alone (Fig. 6B, Table 1). The dissociation constant K(D) for the fast phase of this double mutant is approximately 65-fold larger than for the template expressing F189N alone (Table 1), indicating that the emplacement of the mutation P194L near the oligosaccharide at Asn significantly lowers toxin affinity. Coexpression of the substitution P194L with the glycosylation signal at Asn increases the rate of alpha-toxin dissociation to approximately that observed for alpha-subunits encoding the glycosylation signal at Asn (Fig. 6B, Table 1). Furthermore, coexpression of the mutation P194L and F189N results in a dissociation profile that is distinctly multiphasic (Fig. 6B). These results contrast with the expression of P194L in an otherwise wild-type template, which caused only a 2-fold decrease in affinity (Kreienkamp et al., 1994).

Adding the substitution D111N to the template expressing the F189N and P194L mutations results in small changes in the association and dissociation rates and also a minor increase in the K(D) value (Fig. 5B and Fig. 6B, respectively; Table 1), demonstrating that emplacement of the second oligosaccharide unique to the cobra sequence has only a slight impact on alpha-bungarotoxin affinity. Coexpressing the substitution P194L with W187N,F189T (encodes the Asn glycosylation site as present in the mongoose cDNA sequence), does not significantly decrease toxin affinity further (Fig. 5B and Fig. 6B, Table 1).

To illustrate the free energy contributions to alpha-toxin binding of the mutants (mt) in relation to the wild-type (wt) alpha-subunit, the change in free energy was calculated using following equation:

Fig. 7shows that the additions of glycosylation at Asn and Asn are the predominant factors that reduce ligand affinity and that a further lowering of alpha-toxin affinity occurs when the P194L substitution is coexpressed with the Asn glycosylation site. These findings establish that substitutions unique to both the cobra and mongoose between residues 183-200 in the alpha-subunit contribute to reducing alpha-toxin affinity.


Figure 7: Free energy differences (DeltaDeltaG) associated with the substitutions found for the snake (F189N, D111N, and P194L) and mongoose (F187N and P194L) acetylcholine receptors. DeltaDeltaG was calculated from the formula DeltaDeltaG = RT ln(K/K) where K and K are the respective dissociation constants for the mutant and wild-type receptors. The K value for the substitution P194L was from Kreienkamp et al.(1994). sp, slow phase; fp, fast phase.




DISCUSSION

Transfection of mutant and wild-type alpha-subunits from mouse along with corresponding beta-, -, and -subunits leads to expression of intact nicotinic acetylcholine receptors on the surface of cells originally lacking these receptors (Sine and Claudio, 1991). Using site-directed mutagenesis to insert glycosylation signals in the alpha-subunit results in the expression of additional oligosaccharides in our mammalian expression system. By systematically substituting residues found in the alpha-toxin-resistant snake and mongoose sequences, we have been able to analyze the influence of individual amino acid side chains and conjugated oligosaccharides on the kinetics of alpha-toxin association and dissociation. By examining combinations of mutations and correlating the kinetic data with estimates of the extent of glycosylation, we have extended our initial observation that the introduction of glycosylation signals is the predominant factor in reducing alpha-toxin affinity (Kreienkamp et al., 1994). We find that other mutations found in the snake or mongoose show little influence on alpha-toxin affinity in the absence of glycosylation. Hence, the influence of glycosylation is 2-fold: first, to directly influence the kinetics of alpha-toxin binding, and second, to induce changes in the conformation of the assembled receptor such that additional amino acid substitutions further reduce alpha-toxin affinity.

The role of these substitutions had been investigated previously by expressing bacterial peptides which contain portions of the Torpedo alpha-subunit sequence. These studies showed single amino acid substitutions Tyr Asn and Pro Leu without glycosylation virtually abolished alpha-bungarotoxin binding (Chaturvedi et al., 1992; Ohana, et al. 1991). However, results from these studies are contradictory to those in Kreienkamp et al.(1994), which demonstrated that substitutions to Asn without the emplacement of an entire glycosylation signal (Asn-X-Ser/Thr), and the mutation P194L when inserted alone into a wild-type template, have negligible effects on alpha-toxin affinity in the intact receptor.

alpha-Toxin dissociation constants for peptides containing the Torpedo alpha-subunit sequence between amino acid residues 160-220 vary from 7.8 nM (Chaturvedi et al., 1992) to 63 nM (Ohana et al., 1991) whereas picomolar dissociation constants are found for the intact receptor. Competition of agonists and antagonists with alpha-toxin binding also requires far higher competing ligand concentrations with the expressed peptides.

Divergent results with peptides expressed in bacteria from those obtained with intact receptors expressed in mammalian cells are possibly due to peptide fragments or fusion proteins not being subjected to the same conformational constraints of being positioned within an intact alpha-subunit and the entire pentameric receptor, and the inability of bacteria to glycosylate peptides. Hence, post-translational modifications in primary structures such as glycosylation or disulfide bond formation are important to the spatial distribution of residues important for the alpha-toxin-receptor interaction. The assembly of subunits into a pentameric molecule is also likely to further induce small conformational changes. In addition, substantial evidence has accumulated from several sources to show that the ligand and alpha-toxin-binding sites are located at the alpha-- and alpha--subunit interfaces (Karlin, 1993). All of these factors raise concerns as to whether the peptides reveal a binding fidelity representative of the intact receptor.

Previous studies have shown the presence of an oligosaccharide at the conserved Asn site on wild-type alpha-subunits in BC3H-1 cells and in injected oocytes by observing mobility shifts on gels (Covarrubias et al., 1989; Buller and White, 1990). We employed similar antibody precipitation techniques to identify oligosaccharides at the 111, 187, and 189 glycosylation sites on the alpha-subunits exported to the cell surface. Mutants with one and two added glycosylation sites could be distinguished from each other and from the wild-type alpha-subunit which contains a glycosylation site at Asn. PNGase-F digestion verified the gel shifts are due to the addition of oligosaccharide chains at the 111, 187, and 189 glycosylation sites. For alpha-subunits containing the Asn glycosylation site, Endo-H and PNGase-F cleavage resulted in an equivalent increase in mobility, indicating that all conjugated oligosaccharides are most likely high mannose structures and complex oligosaccharides are absent. Since a single sharp band appeared on gels for the alpha-subunit expressing the Asn glycosylation site, the processed oligosaccharides conjugated to this site are of approximately the same molecular size.

Diffuse and multiple gel banding patterns for alpha-subunits containing the Asn glycosylation site indicate that oligosaccharide processing is heterogeneous. Endo-H treatment of alpha-subunits with the Asn glycosylation site resulted in only partial cleavage of oligosaccharides under conditions where complete cleavage was observed for the Asn glycosylation site (data not shown). This indicates that all of the oligosaccharides may not be susceptible to Endo-H cleavage and that complex oligosaccharides could be present at the Asn glycosylation site. An alternative explanation of variable glycosylation, which would also result in the presence of larger oligosaccharides, is that partial cleavage by the processing enzymes involved in the trimming reactions of N-linked oligosaccharides failed to truncate all oligosaccharides expressed at Asn.

Oligosaccharides when introduced at Asn and Asn, but not at Asn, in the alpha-subunit appear as the predominant factors lowering toxin affinity (Fig. 7). Other amino acid changes present in the cobra or mongoose sequence when inserted alone show a relatively minor role in influencing alpha-toxin affinity compared to the emplacement of oligosaccharides at positions Asn or Asn. It is noteworthy that residues 116 and 117 in the -subunit contribute to d-tubocurarine binding specificity (Sine, 1993). These residues are in comparable positions in the alpha-subunit. Since binding sites exist at the alpha- and alpha- interfaces, residues 116 and 117 in the alpha-subunit should reside on the face distal to the binding site. Accordingly, a glycosylation site at 111 might be expected to be positioned some distance away from the alpha-toxin-binding site.

It also appears that other amino acid changes can act synergistically with nearby oligosaccharides to further reduce alpha-toxin affinity, as observed for the addition of the substitution P194L in the alpha-subunit that expressed an oligosaccharide at Asn. Leucine at residue 194, five residues from the oligosaccharide at Asn, could allow for more rotational freedom of the polypeptide backbone that was constrained by the presence of the 194 residue and enable the oligosaccharide to alter the conformation of the receptor's alpha-toxin-binding domain. Consistent with this notion, the presence of the oligosaccharide is the driving force that distorts the polypeptide backbone, and therefore the substitution P194L alone should not significantly lower toxin affinity, as was observed in Kreienkamp et al.(1994). The substitution P194L in the presence of an oligosaccharide at Asn may be too distant from the oligosaccharide, or the oligosaccharide is too small, to act synergistically in a manner that further lowers alpha-toxin affinity.

Mutations contributing to alpha-toxin resistance have a larger impact on the dissociation kinetics, with the rate of dissociation increasing up to 800-fold compared to a 22-fold lowering of the association rate over wild-type for the alpha-subunit coexpressing the mutations F189N and P194L. This suggests that the influence of glycosylation on alpha-toxin binding is far more complex than simply restricting diffusional access of relatively large alpha-toxin molecules. The association kinetics for the alpha-subunits with oligosaccharides at Asn and Asn suggest alpha-bungarotoxin molecules diffuse into the receptor's binding domain without a large degree of steric hindrance caused by a conjugated oligosaccharide, but the dissociation kinetics indicate that these substitutions lower ligand affinity primarily by precluding the formation of a stable binding conformation for alpha-toxin on the receptor. A free energy profile for alpha-toxin binding would be reflected in the greatest diminution of free energy of activation between the alpha-toxin-receptor complex and the transition state for alpha-toxin dissociation.

Oligosaccharides on receptors typically facilitate protein transport from intracellular organelles to the membrane, as does the conserved oligosaccharide at Asn on the alpha-subunit (Merlie et al., 1982; Prives and Olden 1980), or influence the polypeptide backbone conformation to maintain a high affinity ligand-binding domain, as in the receptors to thyrotropin (Russo et al., 1994) and luteinizing hormone (Petaja-Repove et al., 1993). In the cobra and mongoose, oligosaccharides at positions 187 and 189 on the alpha-subunit appear to function in an opposite manner, to distort the polypeptide backbone in regions that influence the binding of relatively large neurotoxin molecules, but do not significantly alter the stability of the complex for smaller ligands such as lophotoxin, carbamylcholine, and conotoxin M1 (Kreienkamp et al., 1994). To be compatible with function, the insertion of an oligosaccharide maintains a viable ligand-binding domain in the receptor for molecules other than alpha-toxins. Additional structural factors such as interactions at the subunit interfaces with the adjoining - and -subunits that may also possess unique amino acid substitutions encoded in the cobra and mongoose may act to further reduce alpha-toxin affinity in the resistant receptors.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant GM24437 (to P. T.) and a Deutsche Forschungsgemeinschaft Fellowship (to H-J. K.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Visiting Fellow, Department of Psychiatry, Yokohama City University, Yokohama, Japan.

(^1)
The abbreviations used are: nAChR, nicotinic acetylcholine receptor; PNGase-F, peptide-N-glycosidase-F; Endo-H, endo-beta-N-acetylglucosaminidase H; PBS, phosphate-buffered saline; mAb, monoclonal antibody.


ACKNOWLEDGEMENTS

We thank Drs. Darwin Berg, William Conroy, and John Lindstrom for generously providing monoclonal antibodies.


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