(Received for publication, November 7, 1994)
From the
Sequences of the -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
-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
-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
-toxin-receptor complex, suggesting
that the decrease for
-toxin affinity is not simply a consequence
of restricted diffusional access, rather glycosylation affects the
conformation and stability of the bound complex.
The nicotinic acetylcholine receptor (nAChR) ()at the
neuromuscular junction is a ligand-gated cation channel displaying a
pentameric configuration of four subunits (
,
,
, and
) in the stoichiometric ratio of
(cf.Unwin, 1993). Ligand-binding domains are
positioned at the
-
and
-
interfaces (cf. Karlin, 1993). The nAChRs in mouse and humans have a high affinity
for snake neurotoxins, such as
-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
-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 -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
-bungarotoxin, and cDNA sequences of their
-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 -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
-subunit known to bind
-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 -subunits along
with the
-,
-, and
-subunits (Kreienkamp et
al., 1994). This study demonstrates that the mutation F189N in the
-subunit, which introduced one of the glycosylation signals
present in the cobra, lowers
-bungarotoxin affinity 140-fold.
Likewise, expression of the mongoose glycosylation signal W187N,F189T
reduces
-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.
Figure 1:
Amino acid sequences of the nicotinic
acetylcholine receptor -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
-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.
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
-subunit, which limits the expression of the
-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.
Figure 2:
Western blots of wild-type and mutant
-subunits containing the substitutions F189N, P194L, and D111N.
Except for lanes B and F, cells were transfected with
DNA coding for the
-,
-,
-, and
-subunits;
-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
-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
-subunit immunoprecipitated from the cell
surface; lane B, wild-type
-subunit with the exclusion of
the
-subunit in the transfection, which prevents receptor export
to the cell surface; lane C, mutant
-subunit with the
substitutions F189N which introduces a glycosylation site and P194L; lane D, mutant
-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
-subunit DNA; lane F excludes the
-subunit in the
transfection; lane G, the mutant
-subunit with the
substitutions F189N and P194L; lane H, mutant
-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 -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
-subunit, as determined by their presence at
similar migration distances on gels displaying
-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
-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
-subunits to equal
the relatively sharp appearance and faster mobility of the wild-type
-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
-subunits. The wild-type
-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 -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
-subunit lanes due to lower
expression of mutants. Lanes A and F, untreated and
PNGase-F-treated cellular extracts, respectively, of wild-type
-subunits; lanes B and E, untreated and
PNGase-F-treated detergent extracts, respectively, of cells transfected
with
-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
-subunit. The region between the arrows and the 43K markers is where the
-subunits
reside.
Evidence
for glycosylation at Asn for
-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
-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
-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
-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
-subunit and therefore background protein bands are almost
undetectable in these Western blots.
Figure 4:
Western blots of wild-type and mutant
-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
-subunits immunoprecipitated
from the cell surface and are derived from approximately 200 fmol of
-subunit protein. Lanes C-J show transfections of mutant
and wild-type cDNAs encoding the
-subunit, along with
-,
-, 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
-subunit cDNA: lane D, mutant
-subunit containing
the substitutions W187N,F189T which adds a new glycosylation site and
P194L; lanes E and F, wild-type and the mutant
-subunit, respectively, treated with PNGase-F; lanes G and H, wild-type and the mutant
-subunit,
respectively, treated with Endo-H; lanes I and J,
wild-type and the mutant
-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
-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
-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
-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
-subunits.
Figure 5:
Association kinetics for I-
-bungarotoxin to nAchRs expressed in HEK-293
cells. A, receptors expressing wild-type and mutant
-subunits with the substitution D111N. The inset shows
the initial association of toxin with the receptor. B,
receptors expressing mutant
-subunits with glycosylation sites at
Asn
, Asn
, and Asn
, and the
substitution P194L. All measurements were made with 18 nM
I-
-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);
, D111N (dashed line);
(
), W187N,F189T,P194L; +,
F189N,P194L,D111N.
Figure 6:
Dissociation kinetics of I-
-bungarotoxin from nAchRs expressed in HEK-293
cells. A, receptors expressing wild-type and mutant
-subunits with the substitution D111N.
I-
-Bungarotoxin at 20 nM was used to form
the initial complex. B, receptors expressing mutant
-subunits with glycosylation sites at Asn
,
Asn
, and Asn
and the substitution P194L.
I-
-Bungarotoxin at 40 nM was used to form
the initial complex. Data from representative experiments are
presented.
, wild-type;
, D111N;
,
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 -subunits possessing the F189N substitution alone (Fig. 6B, Table 1). The dissociation constant K
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
-toxin dissociation to approximately that observed for
-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 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
-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 -toxin binding
of the mutants (mt) in relation to the wild-type (wt)
-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
-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
-subunit contribute to reducing
-toxin
affinity.
Figure 7:
Free energy differences
(G) associated with the substitutions found for the
snake (F189N, D111N, and P194L) and mongoose (F187N and P194L)
acetylcholine receptors.
G was calculated from the
formula
G = 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.
Transfection of mutant and wild-type -subunits from
mouse along with corresponding
-,
-, 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
-subunit results in the expression of additional
oligosaccharides in our mammalian expression system. By systematically
substituting residues found in the
-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
-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
-toxin affinity (Kreienkamp et
al., 1994). We find that other mutations found in the snake or
mongoose show little influence on
-toxin affinity in the absence
of glycosylation. Hence, the influence of glycosylation is 2-fold:
first, to directly influence the kinetics of
-toxin binding, and
second, to induce changes in the conformation of the assembled receptor
such that additional amino acid substitutions further reduce
-toxin affinity.
The role of these substitutions had been
investigated previously by expressing bacterial peptides which contain
portions of the Torpedo -subunit sequence. These studies
showed single amino acid substitutions Tyr
Asn and
Pro
Leu without glycosylation virtually abolished
-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
-toxin affinity in the intact receptor.
-Toxin dissociation constants for peptides containing the Torpedo
-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
-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 -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
-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
-toxin-binding sites are located at the
-
- and
-
-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
-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
-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
-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
-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
-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
-subunits containing the Asn
glycosylation site
indicate that oligosaccharide processing is heterogeneous. Endo-H
treatment of
-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
-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
-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
-subunit. Since
binding sites exist at the
-
and
-
interfaces,
residues 116 and 117 in the
-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
-toxin-binding site.
It also appears that other amino acid
changes can act synergistically with nearby oligosaccharides to further
reduce -toxin affinity, as observed for the addition of the
substitution P194L in the
-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
-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
-toxin affinity.
Mutations contributing to -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
-subunit coexpressing the
mutations F189N and P194L. This suggests that the influence of
glycosylation on
-toxin binding is far more complex than simply
restricting diffusional access of relatively large
-toxin
molecules. The association kinetics for the
-subunits with
oligosaccharides at Asn
and Asn
suggest
-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
-toxin on the
receptor. A free energy profile for
-toxin binding would be
reflected in the greatest diminution of free energy of activation
between the
-toxin-receptor complex and the transition state for
-toxin dissociation.
Oligosaccharides on receptors typically
facilitate protein transport from intracellular organelles to the
membrane, as does the conserved oligosaccharide at Asn on
the
-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
-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
-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
-toxin affinity in the
resistant receptors.