©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alteration of NH-terminal Residues of Nerve Growth Factor Affects Activity and Trk Binding without Affecting Stability or Conformation (*)

(Received for publication, October 13, 1994; and in revised form, December 20, 1994)

Sang B. Woo David E. Timm (§) Kenneth E. Neet (¶)

From the Department of Biological Chemistry, Finch University of Health Sciences/Chicago Medical School, North Chicago, Illinois 60064

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The role of the NH(2)-terminal region of nerve growth factor (NGF) was studied with an NGFDelta9/13 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 NGFDelta9/13 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 NGFDelta9/13 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 NGFDelta9/13 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 NGFDelta9/13 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(2)-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(2)-terminal region are directly due to receptor binding and not to secondary effects of improper folding or other indirect structural changes.


INTRODUCTION

Nerve growth factor (NGF) (^1)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 beta-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 (beta-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(2)-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(2) 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 beta-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(2)-terminal portion of NGF has a direct role in receptor binding and bioactivity of NGF.


MATERIALS AND METHODS

NGF

Mouse NGF was prepared from 7 S NGF isolated from the mouse submaxillary glands of adult male Swiss Webster mice as described previously (Stach et al., 1977; Woodruff and Neet, 1986). NGF was further purified by Mono S FPLC according to Luo and Neet (1992).

Antibodies

Polyclonal anti-TrkA antibody was from Oncogene Science Inc.; polyclonal anti-rabbit NGF antibody was from CRC, Inc. Monoclonal antibody to NGF(N60) has been previously described (Neet et al., 1987; Nanduri et al., 1994).

PC12 Bioassay

Cell culture and defined medium bioassay were carried out as described by Reinhold and Neet(1989). Neurite-bearing cells were counted 48 h after the addition of NGF, and the experiment was repeated three times.

Preparation of NGFDelta9/13

Deletion of the DNA bases corresponding to residues 9-13 was carried out by polymerase chain reaction-based gene splicing by overlap extension (Horton et al., 1989) in the pTZ19U vector, which encodes full-length NGF-(1-120) and the preproregion. The oligomer pair used was 5`-CAGTCTTCCACGTGTGTGACAG-3` and 5`-CTGTCACACACGTGGAAGACTG-3`. The full-length cDNA for NGFDelta9/13 in pBluescript II SK (Stratagene) was cut at BamHI/PstI sites and subcloned into pBlueBac III (Invitrogen). Deletion of residues 9-13 (MGEFS) was verified by dideoxy-DNA sequencing of 200 base pairs using the Sequenase sequencing kit (United States Biochemical Corp.). Plasmid pBBD913 was amplified in Escherichia coli NM522 and purified by CsCl gradient ultracentrifugation. The plasmid was introduced into log phase Sf21 insect cells by cotransfection with linearized wild type Autographica california nuclear polyhedrosis virus DNA following the protocol supplied by Invitrogen. Recombinant baculoviruses were identified visually by blue color and further purified by plaque purification.

Fully processed NGFDelta9/13 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 NGFDelta9/13 peak were pooled and lyophilized to get a concentrated protein solution. The final recovery during the purification was 20% of the expressed NGFDelta9/13 in the supernatant.

Preparation of Des-(1-9)-NGF

Des-(1-9)-NGF was prepared (Dunbar et al., 1984) by dissolving 1 mg of NGF and 50 mg of CNBr in 1-2 ml of 70% formic acid, incubating at room temperature overnight, and lyophilizing. Cleavage was >90% as determined from SDS and isoelectric focusing gels. The dried material was dissolved in 0.1% trifluoroacetic acid and purified by reversed-phase HPLC (RP-8 column) with a linear acetonitrile gradient. Des-(1-9)-NGF eluted at 34% acetonitrile, whereas native NGF eluted at 33% (2 min apart). The formic acid control used for comparison was treated identically, except for omission of CNBr.

Receptor Binding Assays

Iodination of NGF (specific activity of 50-75 cpm/pg mNGF) and suspension binding assays with PC12 cells in the one-step sucrose cushion were performed as described previously (Woodruff and Neet, 1986; Luo and Neet, 1992). The cold chase experiment was performed to differentiate the slow (stable) binding from the fast (labile) binding. For p140 binding, Sf21 cells at log phase were transfected with TrkA baculovirus for 1 h (kindly provided by D. R. Kaplan) and then ectopically overexpressed for 3 days (Stephens et al., 1994). TrkA was characterized in solubilized cells by Western blotting with anti-TrkA antibody. Cells were harvested and washed twice with cell suspension buffer; 0.5 million cells/ml were incubated with 100 pMI-NGF in the presence of competing ligand at various concentrations for 2 h at 0.5 °C and sedimented. Radioactivity was determined in a Packard Auto-Gamma 5780 counter.

Cross-linking Studies

Sf21 cells containing ectopically overexpressed p140 were prepared as described for binding assays. One million cells were incubated with 1 nMI-NGF for 90 min at 0.5 °C either with or without unlabeled NGF or NGFDelta9/13. The chemical cross-linker disuccinimidyl suberate (Pierce) dissolved in dimethyl sulfoxide was added to a final concentration of 150 µM as described previously (Hosang and Shooter, 1985; Meakin and Shooter, 1991). Cross-linked cells were lysed in radioimmune precipitation buffer with brief sonication, centrifuged at 15,000 rpm for 30 min, and immunoprecipitated using polyclonal anti-p140 antibody. The resulting immunoprecipitates were electrophoresed on SDS-7% acrylamide gel and analyzed by autoradiography (Nanduri et al., 1994).

Fluorescence and Circular Dichroism

Steady-state fluorescence of NGF was measured in an SLM-AMINCO C-8000 spectrofluorometer in a 1.0-cm quartz cuvette with excitation at 280 nm and emission at 340 nm. Nonlinear least-square analysis of guanidine HCl equilibrium denaturation data to obtain DeltaG(D)^O was performed as described previously (Timm and Neet, 1992). However, to conserve on mutant and modified NGF samples, experiments were done at 5 µg/ml, resulting in somewhat lower quality data. CD spectra were measured by a computer-controlled Jasco 710 spectropolarimeter. NGF, modified protein samples, or appropriate controls were measured in 2-mm path length quartz cuvettes at 200 µg/ml protein in 10 mM sodium phosphate, pH 7.0. Spectra were typically recorded as an average of six scans from 250 to 185 nm.


RESULTS

Expression and Purification of NGFDelta9/13

The deletion mutant was generated by polymerase chain reaction overlap extension. Higher expression was obtained with pBlueBac III as a transfer vector than with pVL1393. Supplementation of the serum-free medium with 2.5% serum increased yields by preventing lysis of Sf21 insect cells infected with NGFDelta9/13 baculovirus. NGFDelta9/13 was purified from the culture supernatant by CM52 cation-exchange, N60 monoclonal antibody immunoaffinity, and Mono S FPLC cation-exchange columns (Luo and Neet, 1992). Note that the N60 monoclonal antibody used in the immunoaffinity column reacts only with NGF species in a native conformation (Luo and Neet, 1992; Nanduri et al., 1994), suggesting that purified NGFDelta9/13 is correctly folded for this epitope. The purified protein was homogeneous as judged by the protein staining of SDS-polyacrylamide gels (Fig. 1) and the Western blot pattern obtained with polyclonal anti-rabbit NGF antibody (data not shown).


Figure 1: SDS-polyacrylamide gel electrophoresis of NGFDelta9/13 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 NGFDelta9/13 (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.''



NGFDelta9/13 Is Biologically Inactive

The biological activity of NGFDelta9/13 was assayed using the defined medium PC12 bioassay and compared to that of mNGF. NGFDelta9/13 did not elicit neurite outgrowth at the highest concentrations of this preparation that could be tested in the assay (Fig. 2). Less than 3% of the cells responded at 1 nM. Comparison of the EC for NGF suggested that NGFDelta9/13 shows at least 100-fold lower biological activity than mNGF. This lack of activity was also noted in insect cell supernatants expressing the mutant and therefore is not due to a loss of activity during purification. Lack of biological activity of the NGFDelta9/13 mutant might be caused by the decreased binding to the high affinity receptors or by an inability to activate TrkA in PC12 cells.


Figure 2: Dose-response curve of modified NGF in the PC12 bioassay. The biological activity of NGFDelta9/13 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 (circle) (means ± S.D. of three independent experiments), NGFDelta9-13 (bullet), des-(1-9)-NGF (), or formic acid (FA)-treated control NGF (box). 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.



NGFDelta9/13 Binding to Both Classes of Receptors on PC12 Cells

To determine the binding affinity for PC12 cells, equilibrium displacement binding studies were carried out at two different temperatures, 37 and 0.5 °C; the lower temperature suppresses internalization of the ligand-receptor complex. PC12 cells were incubated with 100 pMI-NGF in the presence of various concentrations of nonradioactive competitive ligands at 37 °C for 1 h and at 0.5 °C for 2 h, and total binding was measured (Fig. 3). Both NGFDelta9/13 and NGF inhibited specific binding of I-NGF at concentrations higher than 1.0 nM at both temperatures, suggesting that the binding of mutant and wild type NGFs to the low affinity receptors on PC12 cells is very similar.


Figure 3: NGF and NGFDelta9/13 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 (bullet) or NGFDelta9/13 (circle). 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 times 10^6 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 NGFDelta9/13 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 pMI-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, NGFDelta9/13 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(2)-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 NGFDelta9/13 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 10^6 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 NGFDelta9/13, respectively.



NGFDelta9/13 Binding to TrkA

The effect of the Delta9/13 mutation on NGF binding to TrkA as the sole component was assessed in the suspension binding assay using Sf21 insect cells ectopically expressing TrkA (Fig. 5). Mouse NGF inhibited the TrkA binding with an EC of 2 nM, whereas NGFDelta9/13 did not compete with I-NGF for the TrkA binding at 10 nM. The results from this experiment and the PC12 cell experiments suggest that NGFDelta9/13 has significantly lower affinity for p140, but retains normal binding affinity for p75.


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 (bullet) or NGFDelta9/13 (circle). 100 pM iodinated NGF was incubated with Sf21-TrkA cells (final concentration of 5 times 10^5 cells/ml) for 2 h at 0.5 °C in the presence of unlabeled competitor.



NGFDelta9/13 Cross-linking to TrkA

The binding of the Delta9/13 mutant to TrkA was also assessed by cross-linking of NGFDelta9/13 to TrkA in the Sf21-TrkA cells using disuccinimidyl suberate as a chemical cross-linker (Fig. 6). Nonradioactive NGF (10M) inhibited 100 pMI-NGF, and no cross-linking of NGF to p140 occurred, whereas the same concentration of NGFDelta9/13 did not inhibit the TrkA binding. The same results were obtained either with the sonicated samples after cross-linking or with the immunoprecipitated samples with polyclonal anti-Trk antibody. This result is consistent with previous results from the binding assay with p140.


Figure 6: Immunoprecipitation of I-NGF cross-linked to Sf21-TrkA cells. Sf21-TrkA cells (2 times 10^6 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 NGFDelta9/13, respectively.



The Biological Activity of Des-(1-9)-NGF with PC12 Cells Is Markedly Reduced

The activity of des-(1-9)-NGF was tested in the standard PC12 bioassay (Fig. 2) and found to be 50-fold less than that of native NGF. This decrease in activity was not due to the low pH, formic acid treatment of the CNBr cleavage since the formic acid control (without CNBr) retained full activity (Fig. 2). The homogeneity of purified des-(1-9)-NGF was assessed by SDS gel electrophoresis (Fig. 1, laneg) and was found by Coomassie Blue staining to be a single band that migrated slightly faster, consistent with a slightly smaller size. Purity was also checked by nonequilibrium isoelectric focusing (Mobley et al., 1976), which showed a shift of the major and two minor bands of NGF to a slightly more acidic migrating position (data not shown), consistent with removal of positive charges in residues 1-9. The possibility cannot be discounted that the remaining 2% of activity may be due to incomplete cleavage and residual native NGF even after purification of des-(1-9)-NGF; if this were the case, the removal of the terminal residues may have completely eliminated activity. In either event, the activity of des-(1-9)-NGF is dramatically reduced compared with control NGF.

Determination of the Conformation of NGFDelta9/13 and Des-(1-9)-NGF by Circular Dichroism

CD spectra were measured to determine if the deletion of NH(2)-terminal residues significantly altered the secondary structure of NGF. The CD spectra of NGF and the two deletion NGFs (Fig. 7) reflect the predominant beta-structure and random coil previously reported (Timm and Neet, 1992). Comparison of the CD spectra suggests that the conformation of NGFDelta9/13 is not greatly altered from the native NGF conformation (Fig. 7A). The spectrum of des-(1-9)-NGF is somewhat different from that of authentic NGF (Fig. 7B), but closely resembles the control NGF that had been treated with formic acid. Apparently, the treatment with formic acid is sufficiently harsh to modify the secondary structure of NGF slightly without affecting the bioactivity (Fig. 2). Thus, the secondary structure conformation of NGF, as judged by CD, is not greatly altered by removal or deletion of portions of the NH(2) terminus.


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^2/dmol times 10). A, NGF (thinline, +) and NGFDelta9/13 (thickline, bullet); B, NGF (thinline), formic acid (FA)-treated NGF control (thickline), and des-(1-9)-NGF (circle).



Determination of the Stability of NGFDelta9/13 and Des-(1-9)-NGF by Equilibrium Solvent Denaturation

Denaturation in guanidine hydrochloride has been shown to be an effective measure of the stability of neurotrophins (Timm and Neet, 1992; Timm et al., 1994) and other dimeric proteins (Neet and Timm, 1995). Moreover, small differences can be determined and associated with sequence differences (Timm et al., 1994). Accordingly, equilibrium denaturation was utilized to determine if the deletion of NH(2)-terminal residues significantly altered the structure (Fig. 8). A better fit to the two-state denaturation model was obtained with the emission data at 338 nm than with the data at 320 nm; nevertheless, the data at 320 nm also show good correspondence between wild type and mutant NGFs (Fig. 8A, inset). Analysis of the shift in max from 338 to 350 nm also agreed with the fluorescence intensity results for wild type and mutant NGFs (data not shown). Qualitative examination of the denaturation curves shows that the EC for NGFDelta9/13 appeared to be shifted only slightly (Fig. 8A), suggesting that the stability of NGFDelta9/13 is similar to that of native NGF. This conclusion was confirmed by calculation of the stability with the fitting program; the DeltaG(D)^O for NGFDelta9/13 was the same as that for native NGF (Table 1). The line after the transition region for denaturation of NGFDelta9/13 (Fig. 8A) has a greater slope than that for native NGF (Timm and Neet, 1992), indicating that the influence of guanidine on the fluorescence of the denatured form of NGFDelta9/13 is more significant than with NGF; the interpretation and significance of such a difference are unknown. Nevertheless, the stability calculated from the transition region is still reliable since the algorithm used to analyze the data takes into account the pre- and post-transition slopes.


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 (NGFDelta9/13) or 320 nm (des-(1-9)-NGF) emission data to the two-state dimer model (Timm and Neet, 1992) to obtain the conformational stability, DeltaG^O, as reported in Table 1. A, NGF (bullet) and NGFDelta9/13 (circle). Inset, emission measurements made at 320 nm. B, formic acid (FA)-treated NGF (bullet) and unfolded () and refolded (up triangle) 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 DeltaG(D)^O 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 DeltaG(D)^O values are similar between des-(1-9)-NGF and the formic acid control. Nevertheless, the removal of the 9 NH(2)-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(2) terminus in biological activity.


DISCUSSION

Studies on the role of the NH(2)-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(2) terminus, NGFDelta9/13, and an NGF chemically cleaved at residue 9, des-(1-9)-NGF.

Expression and Characterization of NGFDelta9/13

Recombinant NGFDelta9/13 was secreted into the medium as the fully processed mature NGF species by transfected insect cells, but with a 5-fold reduced level of expression compared with wild type mNGF (Luo and Neet, 1992). The molecular mass of 13 kDa for the monomer and the characteristic purification by three different columns suggest that deletion of residues 9-13 did not interfere with either processing or secretion of the mutant molecule; the recombinant mutant protein is structurally intact and similar in overall conformation.

Spectroscopic Studies

Changes in global conformation or stability of dimeric NGF that might result from deletion of residues in the NH(2) terminus could be detected by appropriate biophysical measurements. The CD spectrum of NGFDelta9/13 was nearly the same as those of NGF and wild type mNGF (Fig. 7). Solvent denaturation curves showed that the transition occurs in the range of guanidine concentrations for the transition of mNGF with a DeltaG(D)^O of 19.3 kcal/mol (Fig. 8). Similar conclusions were made for des-(1-9)-NGF when comparisons were made with the appropriate formic acid-treated control since the acidic treatment itself led to changes in CD spectra and thermodynamic stability ( Fig. 7and Fig. 8). These results suggest that neither deletion of 5 residues at positions 9-13 in the NH(2)-terminal domain nor removal of residues 1-9 significantly changed the overall folding and stability of the modified NGF. Since solvent denaturation includes measurement of dissociation of the native dimer N(2) to unfolded monomer (D) (Timm and Neet, 1992), the dimerization constant of these modifications must not have been perturbed either. Therefore, the dramatic decrease in the biological activity of NGFDelta9/13 and des-(1-9)-NGF can be attributed to direct effects on the interaction of the protein with the receptor.

Receptor Binding Specificities

We investigated whether the loss of bioactivity of NGFDelta9/13 is due to lack of receptor binding or to a subsequent cellular step. NGF-initiated signal transduction has been shown to be mediated by interaction with Trk receptors (Hempstead et al., 1991; Klein et al., 1991; Jing et al., 1992). Our results demonstrated that NGFDelta9/13 had almost the same affinity as NGF for LANR in PC12 cells (Fig. 3), whereas the mutant had little or no affinity for the TrkA receptor expressed in insect cells (Fig. 5). In cold chase experiments performed with PC12 cells, which express both TrkA and LANR, NGFDelta9/13 was unable to compete with wild type NGF at 10-fold higher concentrations (Fig. 4). Furthermore, the binding of NGFDelta9/13 to TrkA in PC12 cells was abolished as shown by chemical cross-linking with disuccinimidyl suberate (Fig. 6). Since recombinant human des-(1-9)-NGF does not bind TrkA (Kahle et al., 1992), mouse des-(1-9)-NGF probably does not interact with TrkA either, although we have not directly tested this conclusion.

Functional Sites in the NH(2) Terminus of NGF

Some residues in the NH(2) terminus were found to be required for proper processing of the preprosequence as well as in bioactivity (Drinkwater et al., 1993). Deletion of residues 1-8 resulted in secretion of the unprocessed 32-34-kDa protein, but with retention of some bioactivity in the unprocessed precursor. Deletion of residues 3-13 or 3-14 resulted in secretion of unprocessed inactive proteins. Deletion of residues 9-14 decreased the bioactivity and receptor binding by at least 100-fold with consequently impaired signal transduction. These results suggest that the region responsible for biological activity or receptor binding is extended toward the vicinity of the first disulfide bond. Our results support these previous observations, but also show that deletion of the 5 residues from positions 9 to 13 does not significantly affect the disulfide bonding between Cys-15 and Cys-80. The region from residues 9 to 13 did not affect the processing of the NGF precursor. The mRNA level for NGFDelta9/13 expression in Sf21 insect cells was slightly down-regulated, but not severely suppressed (data not shown). NGFDelta9/13 did not support neurite outgrowth from PC12 cells, consistent with previous results with similar mutants (Drinkwater et al., 1993). In addition, our physical biochemical studies substantiate the conclusion that these effects are due to residues that are directly involved in the NGF-receptor interactions and are not a consequence of global conformational changes.

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 NGFDelta9/13 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(2)-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(2) 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(2) 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.


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grant NS24380 from the National Institutes of Health. 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.

§
Supported by Cell and Molecular Biology Training Grant GM08056 from the National Institutes of Health to Case Western Reserve University (Cleveland, OH). Present address: Structural Molecular Biology Unit, Birkbeck College, London WC1E 7HX, Great Britain.

To whom correspondence should be addressed: Dept. of Biological Chemistry, Finch University of Health Sciences/Chicago Medical School, 3333 Green Bay Rd., North Chicago, IL 60064. Tel.: 708-578-3220; Fax: 708-578-3240.

(^1)
The abbreviations used are: NGF, nerve growth factor (beta-subunit); LANR, low affinity neurotrophin receptor; mNGF, mouse nerve growth factor (beta-subunit); FPLC, fast protein liquid chromatography; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Dr. D. R. Kaplan for providing the baculovirus vector containing the TrkA receptor.

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.


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