(Received for publication, August 5, 1994; and in revised form, December 22, 1994)
From the
The binding of NGF (nerve growth factor) to the rat low affinity
nerve growth factor receptor (p75) has been studied by
site-directed mutagenesis of the receptor. Introduction of non-native N-glycosylation sites within the binding domain indicates that
the second of the characteristic cysteine-rich repeats may be
particularly important to NGF binding. Two mutants of the second
repeat, S42N and S66N, are glycosylated and bind NGF at a drastically
reduced level, while still maintaining a conformation recognized by the
monoclonal antibody against p75, MC192. Alanine substitution at these
sites does not affect NGF binding. Two other mutations that result in
local structural changes in the second repeat also greatly decrease
binding. One of these altered residues, Ser
, appears to
play an essential structural role, since it cannot be replaced by Asn,
Ala, or Thr without loss of both NGF binding and MC192 recognition on a
Western. Glycosylation of selected sites in the other repeats has
little effect on NGF binding or antibody recognition. The introduction
of non-native N-glycosylation sites may provide a generally
useful scanning technique for the study of protein-protein
interactions.
The functional role of the low affinity nerve growth factor
receptor, p75, (
)is still under intensive
study. Although a signal transduction pathway activated by p75
has recently been identified(1) , most of the known
biological responses to NGF do not seem to require p75, whereas trk,
the other known NGF receptor, can transduce a signal without p75
participation(2, 3, 4, 5, 6, 7, 8) .
Moreover, transgenic mice lacking p75 display a rather mild
phenotype(9) . It would therefore seem that the role of p75 is
a fairly subtle one, altering the dose response curve of neurons or
neuronal precursors(10) , perhaps by influencing the kinetics
of trk
activation(11, 12, 13, 14, 15) .
Specifically, certain of the binding data suggest that p75 may
accelerate the on rate of NGF binding to trk by means of ligand
transfer(14) . To understand this mechanism, it will clearly be
necessary to understand binding of NGF to p75 itself.
p75 belongs to a family of cell surface proteins that share a common
four-repeat cystine motif in the extracellular domain. The
three-dimensional structure of the cysteine-rich domain is not known
for p75, but the corresponding domain of the p55
has
been crystallized in complex with its ligand, TNF
, and the
structure determined to 1.8 Å resolution(16) . The four
repeats form a curved wand that binds longitudinally along the groove
between two TNF subunits.
In p75, as in
p55
, the ligand binding site is fully contained within
this cysteine-rich domain(17, 18) . The pattern of
half-cystine residues in the two proteins is almost identical.
Moreover, mutation of at least some pairs of presumably bonded
cysteines in p75 yields active protein, strongly suggesting that the
disulfide connectivity is the same in the two proteins(19) . In
addition, certain residues that have been shown to form critical
hydrogen bonds between
-strands in the p55 structure are conserved
in the p75 structure. It seems highly probable that the backbone
structure of the two proteins will turn out to be very similar,
although the surfaces are expected to vary considerably.
All four repeats of p75 are required for binding, even though the individual repeats seem to fold independently of each other(18) . Neither half of the binding domain can be replaced by the corresponding two repeats of either TNF receptor(20) , and insertions that disrupt the tertiary structure are not tolerated(21) . Deletion of the second repeat prevents expression of the protein(18) , whereas destabilization of the first or the fourth repeat by removal of the first disulfide bridge of the repeat results in a protein that is expressed but unable to bind NGF(19) . It seems that binding is extremely sensitive to conformational changes in any of the four repeats and that the conformational stability of the whole domain requires each repeat to be intact.
It is known from mutagenesis of NGF that multiple contacts between ligand and receptor participate in binding(5, 22) . When our first attempts to disrupt binding by mutagenesis of individual receptor side chains failed, we therefore looked for a way to alter the surface of the receptor over a more extended range than the space sampled by a single side chain, while minimizing the disruption of the backbone conformation.
It occurred to us that we might sterically block specific binding by the introduction of novel N-glycosylation sites. Glycosylation has been shown to prevent aggregation of polypeptides in the unfolded state(23) . In some proteins that utilize the secretion pathway, including the epidermal growth factor and low density lipoprotein receptors(24, 25) , glycosylation is required for transport to the cell surface, a role that might also be attributed to the prevention of unwanted protein-protein interactions. In influenza hemaglutinin, new glycosylation sites have appeared and disappeared spontaneously over much of the surface, possibly as a viral strategy for masking antigenic epitopes(26, 27) . Those regions of hemaglutinin in which the consensus sequence for glycosylation never seems to occur spontaneously are those involved in protein-protein interfaces between monomers, the binding domain for the receptor, or the stem region, where a helical coiled coil undergoes a conformational change(28) . Thus the effect of glycosylation at specific sites can provide information about the surfaces involved in protein interactions.
Here we describe a set of eight mutants in
which extra N-glycosylation sites have been introduced to the
binding domain of p75. In choosing the sites, we looked
for polar residues, likely to be solvent exposed, with a serine or
threonine 2 residues downstream, so that a fairly conservative change
would create a consensus sequence of N-glycosylation. We
cannot assume a priori that a novel N-glycosylation
site will not perturb the backbone conformation. However, we have at
our disposal two monoclonal antibodies against the extracellular domain
of p75, both of which are sensitive to conformational changes in the
protein, and whose epitopes do not overlap with the NGF binding
site(29, 30) . We have used these to assess whether
each individual mutation has resulted in a global conformational
change. Finally, we have made a number of control mutants, mostly with
alanine replacements, to confirm our conclusions.
The mutants fall into three categories: 1) those that bind NGF normally and recognize both monoclonal antibodies even after heat denaturation and Western blotting, 2) those that bind the antibodies even after denaturation, but show greatly reduced binding of NGF, and 3) those that fail to bind well NGF and also fail to recognize one or both of the monoclonal antibodies following denaturation. The mutations that resulted in a drastic loss of NGF binding were all located in the second repeat, and no consensus sequence for N-glycosylation was introduced in this repeat without major loss of NGF binding. We suggest that this repeat plays a crucial role in NGF binding and may form direct contacts with the ligand.
-NGF was purchased from Bioproducts for Science, Inc.
and was iodinated by the lactoperoxidase method(31) .
Sulfosuccinimidobiotin and horseradish peroxidase-conjugated
streptavidin were obtained from Pierce, and endo
F/N-glycosidase F was from Boehringer Mannheim.
Figure 3: Deglycosylation of mutants with novel N-glycosylation sites. Cell surface proteins were biotinylated as in Fig. 3, and the receptor protein was immunoprecipitated with the Parke-Davis polyclonal antibody. A, an aliquot of each sample was reduced and analyzed on a Western blot as in Fig. 3; B, another portion was reduced, boiled briefly with SDS, and then treated with endo F/N-glycosidase F as described under ``Materials and Methods,'' prior to Western analysis as in A.
The p75 mutants considered in this paper are summarized in Table 1. These mutants include the eight mutants with novel N-glycosylation sites and also control mutants with Ala or Thr replacements at certain of these sites. In one case, we made a double mutation, changing Asp to Asn and Ala to Ser. (One other construct, E89N/V35M, has an unintentional second mutation due to a polymerase chain reaction error, but since this mutant binds NGF at an apparently wild-type level, we have included it in the study.) A few other mutants, listed as ``Miscellaneous,'' are included because they shed further light on individual glycosylation mutants. A mutant lacking the naturally occurring N-glycosylation site and one lacking both this site and the O-glycosylated stack region of the receptor are also listed. For the mutants with novel glycosylation sites, the consensus sequence created is given, along with the original triad of amino acids from which it was created.
The approximate
location of each mutation site relative to the cysteine-rich domain is
shown in Fig. 1, a cartoon based roughly on the crystal
structure of p55(16) . This drawing is meant
only to help in orienting the reader and does not imply precise
knowledge of the molecular geometry. The results described below are
summarized in Table 2.
Figure 1:
Cartoon showing approximate positions
of the sites mutated in this study, based roughly on the crystal
structure of p55 by Banner et
al.(15) .
Figure 2: Expression of mutant protein at the cell surface. Transfected COS 7 cells were biotinylated as described under ``Materials and Methods.'' The cells were then lysed, and the p75 protein was immunoprecipitated with the Parke-Davis polyclonal antibody. Fractions were reduced and separated on a Laemmli gel prior to transfer to Immobilon-P(TM) membrane. The blot was stained with peroxidase-conjugated streptavidin and developed by enhanced chemiluminescence.
All other mutants glycosylated at novel sites were transported to the cell surface in the fully glycosylated form (Fig. 3A). When the mutant proteins were immunoprecipitated with the polyclonal antibody, and then treated with endo F/N-glycosidase F, all comigrated on a Laemmli gel with the mutant protein, N32D, which lacks the single naturally occurring N-glycosylation site (Fig. 3B).
Figure 4:
A, cross-linking of
[I]NGF to COS 7 cells transfected with mutants
N32D and N32D/
BS. N32D lacks the naturally occurring N-glycosylation site, and mutant N32D/
BS lacks both N-glycosylation and also the stalk region between the binding
domain and the transmembrane domain. The latter region appears to be
heavily O-glycosylated. B, Scatchard analysis of
I-NGF binding to COS cells transfected with wild-type p75
and with p75-N32D, as described under ``Materials and
Methods.'' The points determined for binding to wild-type cells
were based on 2
10
cells/sample and the binding to
N32D cells on 5
10
cells/sample.
We had previously shown that the stalk region between the cysteine-rich domain and the membrane-spanning domain, which appears to be heavily O-glycosylated, was likewise not essential for NGF binding(18) . Here we show further that when the N-glycosylation site and the stalk region were simultaneously deleted cross-linking still appeared to be normal (Fig. 4A).
Figure 5:
Cross-linking of I-NGF to
COS 7 cells transfected with p75 mutants. A, mutants with
extra potential N-glycosylation sites. B,
miscellaneous mutants C4G/C15G and Y83A/Y85A. In this experiment, the
cross-linked protein was immunoprecipitated with the Parke-Davis
polyclonal antibody prior to electrophoresis and
autoradiography.
To test whether the poor binding of NGF to S42N, S50N, and S66N was directly due to the glycosylation, we constructed control mutants in which the original serine was replaced by Ala instead of by Asn. Fig. 6A shows that mutants S42A and S66A were fully able to bind NGF, but S50A was not. A further variant of this site, S50T, was also unable to bind NGF (Fig. 6B). As a partial control for the double mutant D75N/A77S, we made a single mutant, D75N. This mutant often showed some reduction in cross-linking to NGF, but considerably less reduction than D75N/A77S (data not shown).
Figure 6:
Cross-linking of I-NGF to
mutants of Ser
, Ser
, and Ser
,
showing the effect of Ala substitution at each of these sites (A) or Thr substitution at Ser
(B).
Some, but not all, of the mutants with novel glycosylation sites
recognized the monoclonals on a Western blot, following SDS
denaturation. The reactivity of the different mutants toward MC192
under these conditions is shown in Fig. 7. The results of this
and previous experiments, in which smaller groups of mutants were
tested, are summarized in Table 2. Mutants S42N and S66N, which
were glycosylated and which did not bind NGF well, typically did bind
MC192. In contrast, neither S50N nor the other Ser mutants
reacted well on a Western blot. This is consistent with the lack of NGF
binding to the S50A and S50T mutant proteins; evidently a structural
change, most likely the loss of a critical hydrogen bond, has occurred
due to the loss of the Ser side chain. T10N and T10A in the
corresponding loop of the first repeat were also somewhat sensitive to
the Western blotting procedure (although less so than the Ser
mutants), indicating that Thr
may also contribute a
hydrogen bond that stabilizes some structural feature.
Figure 7: Western analysis of glycosylation mutants. Samples were boiled for 3 min in gel loading buffer without reduction. Proteins were fractionated on a 7.5% Laemmli gel (35) and transferred electrophoretically to Immobilon-P(TM) membrane. The blot was stained with MC192, followed by horseradish peroxidase-conjugated donkey anti-mouse IgG, and the proteins were visualized by enhanced chemiluminescence. D74N/A77S showed less relative stability in this experiment than in others.
All of the
glycosylation mutants except T10N and S50N recognized MC192 on a
Western blot. A similar pattern was seen with 217c staining, except
that the effect of altering Thr or Ser
was
less dramatic (data not shown). This is consistent with other data that
place the 217c epitope toward the C-terminal end of the
repeat(18) . The D75N/A77S mutant behaved anomolously in Fig. 7; in most experiments, this protein reacted strongly with
MC192. It was detected on a blot of a nondenaturing gel (Fig. 8), and was immunoprecipitated by MC192 (Fig. 9).
Figure 8: Detection of mutant proteins on a blot of a nondenaturing gel. Electrophoresis was run with a multiphasic buffer system of Chrambach et al.(42) . The blots were stained with (A) MC192, and (B) 217c, and peroxidase-conjugated goat anti-mouse IgG. Protein was visualized by enhanced chemiluminescence.
Figure 9: Immunoprecipitation of p75 mutant proteins with MC192. The immunoprecipitations were carried out in radioimmune precipitation buffer extracts, as described. The receptor protein was first eluted from the agarose beads and then reduced prior to electrophoresis and Western analysis using the Parke-Davis polyclonal antibody.
To assess binding of the antibodies to undenatured mutant receptors, we have done three kinds of experiment: analysis of blots of nondenaturing gels, immunoprecipitation, and fluorescent surface staining of transfected cells.
All of the mutant proteins, when fractionated on a
nondenaturing gel using a multiphasic buffer system of Chrambach et
al.(41) , and then blotted, stained with both monoclonal
antibodies (Fig. 8). The Thr and Ser
mutants stained less well than some of the others, consistent
with their lesser stability.
Immunoprecipitation with MC192 followed by Western analysis with the polyclonal antibody resulted in a pattern very similar to that obtained when a Western blot was stained with MC192 (Fig. 9). The mutants that failed to precipitate with MC192 were those that showed instability on a Western blot, namely T10N, S50N, S50A, S50T, C4G/C15G, and Y83A/Y85A, although Western analysis of the original extract showed that the mutant proteins were expressed at similar levels (data not shown). Not surprisingly, those proteins that are stable to heat/SDS denaturation were also stable to immunoprecipitation.
Surface staining of transfected cells with
MC192 or 217c, plus rhodamine-conjugated second antibody, likewise
showed some reactivity of all of the constructs to both antibodies,
although staining of the Ser mutants, as well as mutant
C4G/C15G, was less bright with MC192 (Fig. 10). The cell or
cells photographed in each case represented one of the brightest in the
field. Since there was not much difference in surface expression (Fig. 2), and since all of the constructs stained quite evenly
with 217c, the fainter staining of the Ser
and C4G/C15G
mutants must result from decreased affinity for MC192. This result is
consistent with our conclusion that a basic structural change has
occurred as a result of each of these mutations.
Figure 10: Immunostaining of COS 7 cells transfected with p75 mutants. Cells were grown and transfected directly on coverslips, as described, and stained with MC192 or 217c, followed by rhodamine-conjugated donkey anti-mouse IgG. The first and third columns show representative cells stained with MC192; the fifth and seventh columns show representative cells stained with 217c. A phase contrast photograph of approximately the same field is shown immediately to the right of each fluorescent photograph. 1, antisense; 2, wild type; 3, T10N; 4, T10A; 5, S42N; 6, S50N; 7, S50A; 8, S50T; 9, S66N; 10, D75N/A77S; 11, D75N; 12, E89N/V35M; 13, Q114N; 14, E123N; 15, C4G/C15G; 16, Y83A/Y85A.
Figure 11:
Effect of MC192 on I-NGF
binding to p75-S42N and p75-S66N on transfected COS 7 cells. Specific
binding was determined as described under ``Materials and
Methods.'' When MC192 was added (Fig. 12), the final concentration
of purified IgG was 8 µg/ml. &cjs2112;, no MC192;
,
+MC192.
In the p55 several aromatic residues toward
the C-terminal end of the first cystine loop of the third repeat
participate in TNF binding. In p75
, these aromatic
residues are missing, but there is an aromatic cluster at the
N-terminal end of the loop. To see whether these residues might play a
role in ligand binding, we made a double mutant, Y83A/Y85A. This mutant
did not bind NGF appreciably (Fig. 5B). It was also not
recognized by MC192 on a Western blot (Fig. 7), although it
stained well on intact cells (Fig. 10). The mutation therefore
appears to have affected the stability of the receptor, and we cannot
be sure at this stage that the effect on NGF is direct.
The mutants described here strongly implicate the second
cysteine repeat of the cysteine-rich domain in the specific binding of
NGF. This conclusion is based on the markedly position-dependent effect
of adding extra glycosylation sites. Extra glycosylation at two sites
within the second repeat (Ser and Ser
) and
mutations that affect local backbone structure or stability at two
other sites (Ser
and Asp
/Ala
)
greatly impede NGF binding, whereas added glycosylation at two sites in
the third repeat (Glu
and Gln
) and one site
in the fourth repeat (Glu
) do not. In addition, the
naturally occurring N-glycosylation, found on the last
predicted
-strand of the first repeat, does not inhibit binding.
Mutant D75N/A77S was not extensively glycosylated. This mutant contains the amino acid sequence Asn-Asp-Ser. Asp in the middle position of this triad is suspected to prevent glycosylation, either because the local conformation is incompatible with the binding site of the glycosyl transferase (42) or because the charged side chain interferes with the putative hydrogen bonding between the amide proton and the serine oxygen(43) . Moreover, Ser does not function as well as Thr as a proton acceptor in this position, so our result is not surprising.
At another site in
the second repeat (Ser), more extensive structural change
undoubtedly accompanies mutation, since this residue cannot be changed
to Asn (with glycosylation), or to Ala or Thr, without loss of
recognition on a Western blot by MC192. Hydroxyl-containing side chains
that are buried within the hydrophobic core of a protein are nearly
always hydrogen-bonded. The fact that Thr cannot substitute for Ser at
this position makes it likely that this side chain is buried. It might
be involved in the formation of a
-turn or some nonrepetitive
structure within the cysteine loop, or it might form an H-bonded bridge
to the preceding repeat.
The effect of Ser mutations on
MC192 binding is more pronounced than on 217c binding, suggesting that
this part of the second repeat is more critical to the MC192 epitope
than to the 217c epitope. Both antibodies do detect these mutant
proteins after fractionation on a nondenaturing gel, and they both
stain the Ser
mutants on the surface of transfected cells,
although less brightly than the wild-type protein. Evidently these
mutations decrease the stability of the active conformation of
the domain, so the direct effect of glycosylation cannot be assessed.
Thr in the first repeat occupies a position within its
cystine loop similar to that of Ser
in the second repeat,
and its mutants also destabilize the MC192 epitope. They do bind NGF, indicating that any local backbone distortion does not
perturb the binding conformation. Opening of this loop by removal of
the first disulfide bridge did, however, prevent binding of either NGF or MC192, just as did the opening of the corresponding bridge
of the fourth repeat(19) . This reinforces our conclusion that
each repeat serves a role in maintaining the active conformation of the
overall domain. At the same time, it validates Thr
mutants
as position controls for the Ser
mutants.
E89N in the third repeat, and E123N in the fourth repeat, is located
in the first cystine loop, which, as we have said, must play a critical
structural role in the repeat. The glutamic acid side chains may well
extend into the solvent, however, whereas Thr of the first
repeat and Ser
of the second repeat may be hydrogen-bonded
within the loop. Glu
is found toward the C-terminal end of
its cystine loop, whereas Glu
is near the N-terminal end
of its loop.
Gln is found in a putative
-strand
of the third repeat with a local sequence almost identical to that
where the naturally occurring glycosylation site in the first repeat is
found (QNTV in the third repeat compared to NQTV in the first repeat).
The Q114N change therefore might not be expected to disrupt the
backbone structure very much, although glycosylation might break a side
chain to main chain hydrogen bond.
The fact that these mutants bind NGF does not rule out the possibility that the third and fourth repeats make contacts with NGF, nor does it preclude the possibility that these repeats are somewhat misfolded but do not normally contact NGF in the binding reaction. Nonetheless it does indicate that certain surface regions exist where bulky carbohydrate side chains do not interfere with ligand binding.
The consequences of adding extra glycosylation sites have previously been studied in detail in several viral systems in which the secretion of a glycoprotein is dependent on naturally occurring glycan attachment, and it has been shown that some added groups block transport, very likely because they prevent correct folding of the protein, whereas others do not(48, 49, 50, 51) . Our p75 mutants were all expressed in COS 7 cells, and all were transported to the cell surface, except for the glycosylated version of T10N, and thus presumably are not grossly misfolded.
Nonetheless, the simplest interpretation of our results is that the second repeat plays a direct and critical role in the binding of NGF to its p75 receptor. Our failure to identify any critical salt bridge through mutagenesis of acidic residues in this repeat may result from the small contribution of any individual hydrophilic bond to the overall binding energy.
The aromatic residues at the very beginning of the third repeat may contribute to the binding site. Aromatic side chains are
often found in protein-protein interfaces, particularly at the edge (52) . Of the 3 tyrosines in the p75 cluster, the third, by
analogy with conserved aromatic residues in all repeats of
p55, most likely stabilizes the repeat by packing
against the next disulfide bond (16) . The other two, which we
have mutated together, could interact with either NGF or with the
preceding repeat, thus fixing the relative orientation of the two
halves of the domain. The instability of this protein to Western
analysis does indicate a structural role for one or both of these
aromatic side chains.
Finally, the introduction of novel glycosylation sites may have a general application in the study of specific protein-protein interactions, provided each mutant can be adequately characterized. It provides an alternative to homolog scanning (53) in targeting a binding surface, when the tertiary structure of the protein domain in question is as intricate as that of p75 or appropriate homologs are unavailable. It might also be used to identify interactions that are more elusive. In the NGF receptor system, for example, it is still not clear whether p75 forms a physical complex with trk. Some of the glycosylation mutants described here might be useful in testing the effect of p75 on trk activation.