(Received for publication, October 13, 1994; and in revised form, December 20, 1994)
From the
The role of the NH-terminal region of nerve growth
factor (NGF) was studied with an NGF
9/13 deletion mutant,
overexpressed in a baculovirus system, and mouse NGF truncated at Met-9
by cleavage with CNBr (des-(1-9)-NGF). Structural studies have
been performed on the purified proteins, in addition to biological
activity assessment, in order to determine effects of such
modifications on global conformation and stability. The activity of
NGF
9/13 was reduced below detectable levels, and the activity of
the des-(1-9)-NGF form was decreased by at least a 50-fold in a
PC12 bioassay. Competitive binding of NGF
9/13 to low affinity
receptors on PC12 cells was not impaired; however, the mutant was not
capable of competing for the cold chase-stable, high affinity binding
of NGF to the cells. The binding of NGF
9/13 to Sf21 cells
ectopically expressing the TrkA NGF receptor was also abolished. Thus,
deletion of residues 9-13 significantly altered the binding
affinity for the high affinity receptors on PC12 cells and for the TrkA
receptor, but not for the low affinity receptor. Neither the secondary
structure, determined by circular dichroism, nor the conformational
stability determined by equilibrium denaturation of NGF
9/13 was
altered as compared with wild type NGF. Slight conformational and
stability perturbations of des-(1-9)-NGF were revealed by the
same analysis; however, these changes were found to reflect the
influence of the formic acid treatment, not the truncation of 9
residues. Our results support the conclusion that the
NH
-terminal domain encompassing residues 1-9 and
9-13 is essential for maintaining the binding capability of NGF
for high affinity TrkA receptors. Moreover, conformational and
stability data show that the functional results of these modifications
of the NH
-terminal region are directly due to receptor
binding and not to secondary effects of improper folding or other
indirect structural changes.
Nerve growth factor (NGF) ()is a member of the
targetderived neurotrophin family that is essential for the development
and maintenance of sympathetic neurons, neural crest-derived sensory
neurons in the peripheral nervous system, and basal forebrain
cholinergic neurons in the central nervous system (Levi-Montalcini,
1987; Thoenen et al., 1987). The other structurally related
neurotrophins include brain-derived neurotrophic factor (Barde et
al., 1982; Leibrock et al., 1989), neurotrophin-3 (Hohn et al., 1990; Maisonpierre et al., 1990; Ernfors et al., 1990; Rosenthal et al., 1990; Jones and
Reichardt, 1990; Kaisho et al., 1990), and neurotrophin-4/5
(Berkemeier et al., 1991; Hallbook et al., 1991). The
mature neurotrophins are highly conserved across species and share over
50% amino acid identity (Rodriguez-Tebar et al., 1990; Squinto et al., 1991; Snyder, 1991; Hynes et al., 1994). The
specific biological function of each neurotrophin is generated by the
selective interaction between the different neurotrophins and their
receptors on the surfaces of distinct but overlapping neuronal
populations.
Two distinct NGF receptors have been identified,
p75 (Johnson et al., 1986; Radeke et
al., 1987; Large et al., 1989) and p140
(Kaplan et al., 1991a, 1991b; Klein et
al., 1991). The low affinity neurotrophin receptor (LANR) is a
member of a superfamily that includes tumor necrosis factor receptors I
and II, Fas, OX40, CD30, and CD40 (Smith et al., 1990). The
LANR glycoprotein binds the other neurotrophins with similar affinity,
but the rates of association and dissociation are markedly distinct
among the different neurotrophins (Rodriguez-Tebar et al.,
1990, 1992). The protein-tyrosine kinase receptor,
p140
, is a member of the Trk family that
includes TrkB and TrkC (reviewed by Barbacid(1993)). Specific binding
of NGF to p140
activates the receptor and
results in p140
dimerization and
autophosphorylation, which are thought to be crucial for ligand
signaling (Jing et al., 1992; Ullrich and Schlessinger, 1990).
NGF interacts with these two receptors, resulting in two distinct
classes of receptors with different affinity (Sutter et al.,
1979; Landreth and Shooter, 1980; Schechter and Bothwell, 1981;
Woodruff and Neet, 1986). Although it is generally accepted that high
affinity binding is required for the initiation of biological responses
(Sutter et al., 1979), whether both receptors are involved in
the formation of a high affinity receptor or whether p140
is the sole component contributing to high affinity binding
remains controversial (Klein et al., 1991; Hempstead et
al., 1991; Chao, 1992; Barbacid, 1993; Mahadeo et al.,
1994).
Mature NGF is a noncovalently associated homodimer of
118-amino acid monomeric subunits (Angeletti and Bradshaw, 1971;
Angeletti et al., 1973). The three-dimensional structure of
NGF (McDonald et al., 1991) consists of predominantly
-structure, consistent with Raman (Williams et al., 1982)
and circular dichroism (Timm and Neet, 1992) spectroscopy. However,
some regions, including both amino and carboxyl termini, were not
defined, suggesting that these sequences are likely to be flexible and
solvent-accessible. Surface loop regions containing most of the
variable residues are, in general, thought to be important for receptor
binding of growth factors (Daopin et al., 1992; Oefner et
al., 1992; Schlunegger and Gruter, 1992).
Recent studies with
recombinant NGF have suggested that both amino and carboxyl termini
contribute significantly to the biological activity, receptor binding,
or regulation of their specificity (Luo and Neet, 1991; Ibanez et
al., 1993; Drinkwater et al., 1993; Kahle et
al., 1992; Burton et al., 1992; Schmelzer et
al., 1992), but potential structural consequences of the mutations
have not been addressed. Monoclonal anti-NGF antibodies specifically
inhibit the biological activity of NGF by blocking the accessible
region encompassing the amino and carboxyl termini and the loop on the
surface containing residues 60-80 to the p140 receptor, suggesting that these regions are essential for
receptor binding and biological activity (Nanduri et al.,
1994). A homolog scanning site-directed mutagenesis technique was
employed to create mutants that have altered specificity of binding and
activation of the p140
receptor (Suter et
al., 1992; Ibanez et al., 1991, 1993). The variable
amino-terminal residues were found to be more important for TrkA
receptor binding than for receptor activation or biological activity
and to be important for specificity against TrkB (Ibanez et
al., 1993). Furthermore, this region may elicit synergistic
effects by the interaction with the residues in other variable domains.
Proteolytic removal of the first 9 residues from wild type recombinant
human NGF (
-subunit) caused a 50-100-fold reduced
bioactivity, although the 109-residue species still had the ability to
form dimers with intact recombinant human NGF or mNGF (Burton et
al., 1992). This lower bioactivity was caused by reduced binding
and activation of p140
, with a smaller effect
on p75
(Kahle et al., 1992). Since none of
these studies of NH
-terminally modified NGF had considered
possible global changes in the molecular structure, we felt it was
important to measure conformational properties of interesting NGF
variants.
In this report, the functional role of the NH terminus of NGF and the conformation and stability of the altered
proteins were examined after purification. The regions encompass
residues 9-13 of mouse NGF by deletion mutagenesis and residues
1-9 by CNBr cleavage. The deleted residues (MGEFS) are positioned
between the proteolytically labile 8 amino-terminal residues and the
first structurally well defined
-sheet (residues 15-22)
(McDonald et al., 1991). Comparisons of circular dichroism
spectra and stability from the equilibrium denaturation analysis have
also been made. These spectroscopic studies help support the
interpretation that the NH
-terminal portion of NGF has a
direct role in receptor binding and bioactivity of NGF.
Fully processed
NGF9/13 was produced as a secreted protein at a level of
0.25-0.6 mg/liter medium at 3 days post-infection in XL401 medium
(JRH Biosciences) with 2.5% fetal bovine serum. The time course of
expression and processing of the preproregion of the mutant resembled
those of recombinant wild type NGF (Luo and Neet, 1992). The
recombinant protein was purified by three steps according to Luo and
Neet(1992). A pepPRO FPLC column (Pharmacia Biotech Inc.) was used with
an acetonitrile linear gradient for purification to homogeneity, and
fractions with a distinct NGF
9/13 peak were pooled and lyophilized
to get a concentrated protein solution. The final recovery during the
purification was
20% of the expressed NGF
9/13 in the
supernatant.
Figure 1:
SDS-polyacrylamide gel electrophoresis
of NGF9/13 and des-(1-9)-NGF. Purified preparations were run
on a 16% reducing SDS-polyacrylamide gel stained with Coomassie
Brilliant Blue R-250. Lanes a and h, prestained
molecular mass standards from Bio-Rad; lane b, 6 µg of NGF
(control); lanes c and d, 6 and 2 µg of
NGF
9/13 (purified as described under ``Materials and
Methods''), respectively; lane e, 10 µg of NGF
purified by reversed-phase HPLC; lane f, 10 µg of the
second peak from reversed-phase HPLC containing proteolyzed forms; lane g, 10 µg of des-(1-9)-NGF purified by
reversed-phase HPLC as described under ``Materials and
Methods.''
Figure 2:
Dose-response curve of modified NGF in the
PC12 bioassay. The biological activity of NGF9/13 and
des-(1-9)-NGF was compared with NGF activity. PC12 bioassays were
carried out in defined medium by using a dilution series of 10 nM to 1 pM NGF (
) (means ± S.D. of three
independent experiments), NGF
9-13 (
),
des-(1-9)-NGF (
), or formic acid (FA)-treated
control NGF (
). Neurite-bearing PC12 cells were counted after
48 h, and cells possessing a neurite more than one cell body in length
were scored as positive.
Figure 3:
NGF and NGF9/13 competition for
I-NGF binding to PC12 cells. Binding of
I-NGF to PC12 cells was determined in the presence of
increasing amounts of unlabeled NGF (
) or NGF
9/13 (
).
The concentration of
I-NGF was fixed at 100 pM,
while that of a competing species was varied from 1 pM to 1
nM. The incubation was initiated by adding PC12 cells (final
concentration of 1
10
cells/ml) and was continued
for 40 min at 37 °C (A) or for 90 min at 0.5 °C (B).
The differences in biological activity could be determined
by differences in specificity for high affinity receptors on PC12
cells. Therefore, cold chase-stable binding, which is usually
correlated with high affinity binding, was examined to determine if the
affinity of NGF9/13 for this receptor class was affected. All of
the fast kinetic component and essentially none of the slow component
are dissociated during the first 30 min of the cold chase paradigm with
an excess of NGF (Sutter et al., 1979; Landreth and Shooter,
1980; Schechter and Bothwell, 1981; Woodruff and Neet, 1986). Two
concentrations of ligand were incubated with PC12 cells in the presence
of 100 pM
I-NGF for 1 h at 37 °C, followed
by the cold chase at 0.5 °C for 30 min with an excess of NGF. Under
these conditions, 0.1 nM NGF was enough to inhibit >60% of
the cold chase-stable binding, and 1.0 nM NGF completely
inhibited the stable binding (Fig. 4). However, NGF
9/13 did
not significantly inhibit the cold chase-stable binding even at 10
nM. This experiment indicated that deletion of these 5 amino
acid residues in the NH
-terminal region significantly
affected the high affinity binding, as defined by cold chase-stable
binding in PC12 cells.
Figure 4:
Cold chase-stable binding to PC12 cells.
Binding of I-NGF to PC12 cells proceeded in the presence
of unlabeled NGF or NGF
9/13 for 40 min at 37 °C and then was
cold-chased for 30 min at 0.5 °C with the addition of 1 µM unlabeled NGF. The concentrations of iodinated NGF and PC12 cells
were 100 pM and 10
cells/ml, respectively. The first and secondcolumns contained 0.1 and
1.0 nM NGF, respectively; the third through fifthcolumns contained 0.1, 1.0, and 10 nM NGF
9/13, respectively.
Figure 5:
Competitive binding assay to the TrkA
receptor in insect cells. Competition for I-NGF binding
to TrkA ectopically expressed in Sf21 insect cells for 3 days was
assayed in the presence of increasing concentrations of unlabeled NGF
(
) or NGF
9/13 (
). 100 pM iodinated NGF was
incubated with Sf21-TrkA cells (final concentration of 5
10
cells/ml) for 2 h at 0.5 °C in the presence of
unlabeled competitor.
Figure 6:
Immunoprecipitation of I-NGF
cross-linked to Sf21-TrkA cells. Sf21-TrkA cells (2
10
cells/ml) were harvested at 3 days post-infection, incubated with
I-NGF (1 nM) for 2 h at 0.5 °C, and then
cross-linked with disuccinimidyl suberate for 30 min at room
temperature. Cell lysates were immunoprecipitated with either anti-NGF
antibodies (lanes 1-5) or anti-Trk antibodies (lanes
6-8). 5 µg of each antibody were used for
immunoprecipitation. Lanes1 and 6 had no
competitors; lanes2, 3, and 7 contained 1.0, 10, and 10 nM unlabeled NGF, respectively;
and lanes4, 5, and 8 contained 1,
10, and 10 nM unlabeled NGF
9/13,
respectively.
Figure 7:
CD spectra of native wild type and
modified NGFs. The CD spectra were measured at a concentration of 0.2
mg/ml in 10 mM sodium phosphate buffer, pH 7.0, in a
cylindrical 2.0-mm light path quartz cell at room temperature. Each
spectrum was averaged over six scans and converted to mean residue
ellipticity (degrees cm/dmol
10
). A, NGF (thinline,
+) and NGF
9/13 (thickline,
); B, NGF (thinline), formic acid (FA)-treated NGF control (thickline), and
des-(1-9)-NGF (
).
Figure 8:
Guanidine hydrochloride equilibrium
denaturation. The relative fluorescence intensity following excitation
at 280 nm was plotted versus the guanidine HCl concentration.
Samples (5 µg/ml) were incubated for 24 h at room temperature with
guanidine HCl at pH 7.0. The solidlines were
obtained by fitting the 338 nm (NGF9/13) or 320 nm
(des-(1-9)-NGF) emission data to the two-state dimer model (Timm
and Neet, 1992) to obtain the conformational stability,
G
,
as reported in Table 1. A, NGF (
) and NGF
9/13
(
). Inset, emission measurements made at 320 nm. B, formic acid (FA)-treated NGF (
) and unfolded
(
) and refolded (
)
des-(1-9)-NGF.
Similar results were obtained with des-(1-9)-NGF (Fig. 8B and Table 1). In this case, the
stability of CNBr-treated NGF must again be compared with the stability
of control NGF treated with formic acid. Treatment of NGF at low pH
with formic acid destabilizes the structure by 6 kcal/mol; whether
these differences are due to noncovalent denaturation effects or to
covalent modifications induced under the acidic conditions is unknown
at present. Thus, the formic acid treatment does not affect activity (Fig. 2), but does affect both conformation (Fig. 7B) and stability (Fig. 8B).
Perhaps the dilute conditions in the presence of cellular receptor
promote the reversal of any acid-induced changes. However,
des-(1-9)-NGF has a stability not significantly different from
that of the formic acid-treated control as judged by the midpoints of
the denaturation curves (Fig. 8B) or from the
calculated
G
values, 15.8 versus 13.3 kcal/mol (Table 1).
Therefore, no change in stability can be attributed to removal of 9
residues by CNBr. We conclude that cleavage at Met-9 does not affect
conformation or stability beyond that caused by the formic acid
treatment since the CD spectra and
G
values
are similar between des-(1-9)-NGF and the formic acid control.
Nevertheless, the removal of the 9 NH
-terminal residues
does reduce activity by at least 50-fold relative to the formic acid
control (Fig. 1), implicating a direct role for the NH
terminus in biological activity.
Studies on the role of the NH-terminal region
traditionally have depended on desocta-(1-8)-NGF purified from
the male mouse submaxillary gland (Moore et al., 1974; Mobley et al., 1976) or CNBr-cleaved des-(1-9)-NGF (Dunbar et al., 1984; Springer and Vernon, 1987); these products were
thought to have virtually equal bioactivity compared with full-length
NGF. However, recent results obtained from recombinant human NGF
expressed in Chinese hamster ovary cells have shown that
proteolytically generated recombinant human NGF-(10-118) retained
only 1-2% of the bioactivity in neuronal survival and
differentiation assays (Burton et al., 1992; Kahle et
al., 1992), and this reduced activity was correlated with the
down-regulated immediate early responses in PC12 cells, such as
tyrosine phosphorylation of key enzymes in the signaling pathway and
induction of the transcription factor c-Fos (Kahle et al.,
1992). Since the 1-8 deletion mutant was not correctly processed
(Drinkwater et al., 1993), however, the route to studying
deletions from Ser-1 is still dependent on enzymatic or chemical
modification of intact NGF. In this study, we have studied the detailed
structure, stability, and activity of an internal deletion mutant in
the NH
terminus, NGF
9/13, and an NGF chemically
cleaved at residue 9, des-(1-9)-NGF.
In contrast, a chimera, in which residues 1-9 or
3-9 of NGF were substituted with residues 1-7 of
brain-derived neurotrophic factor, retained full activity with only
5-fold decreased TrkA binding (Ibanez et al., 1993),
while in an earlier study on NGF/brain-derived neurotrophic factor
chimeric proteins, it was revealed that substitution of residues
3-9 of NGF affected bioactivity 2-4-fold, but did not
affect binding to PC12 cells (Suter et al., 1992). Thus,
residues 3-9 and 9-13 could synergistically affect the
bioactivity as well as TrkA receptor binding.
One of the important
structural features in the deleted region of NGF9/13 is Phe-12,
which was shown to interact with Trp-76 of the second subunit (McDonald et al., 1991) and has been suggested to play a role in dimer
stabilization (Drinkwater et al., 1993). Furthermore, previous
chemical modification studies suggested that Trp-76 was deeply buried,
solvent-inaccessible, and not involved in bioactivity or receptor
binding (Frazier et al., 1973; Cohen et al., 1980).
Deletion of residues 9-13 shortens the length of the flexible,
solvent-accessible NH
-terminal docking region and replaces
Phe-12 with what was once Phe-7, i.e. a Phe moiety is still
three positions from the first critical Cys residue. Thus, retention of
a Phe residue in a position to optimally interact with Trp-76 might
explain why there was no effect of the deletion on dimer-dimer
interactions (Table 1). These changes could modify the local
geometry formed between the NH
terminus and the
``south'' side of the molecule, which was previously proposed
to be an important TrkA-binding site in NGF (Nanduri et al.,
1994). Prevention of both the initial docking and subsequent
rearrangement of the ligand-TrkA conformations could be critical for
the synergistic reinforcement of the binding and lead to disruption of
initiation of signal transduction (Ibanez et al., 1993;
Nanduri et al., 1994). However, these structural changes in
the mutant would not severely affect the binding specificity for LANR
because the amino acid residues involved in the binding of NGF to LANR
have been identified in the ``northern'' loop regions (Ibanez et al., 1992).
From these and other studies of the NH terminus, we may conclude that residues 9-13 of mouse NGF
(or the corresponding residues shown in parentheses for the human
sequence), Met(Arg)-Gly-Glu-Phe-Ser, and residues 1-8,
Ser-Ser-Thr(Ser)-His-Phe-Val(Ile)-Phe-His, directly play a role in TrkA
receptor binding, without being critical for conformation or stability.
On the other hand, Ser-1 may be important for processing.
Note Added in Proof-Shih et al.(1994) have provided further evidence with deletion mutants that residues 1-9 play an important role in the TrkA binding and activity of NGF.