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Nerve growth factor (β-NGF), a neurotrophin required for the development and survival of specific neuronal populations, is translated as a prepro-protein in vivo. While the presequence mediates translocation into the endoplasmic reticulum, the function of the pro-peptide is so far unknown. As the pro-sequences of several proteins are known to promote folding of the mature part, the renaturation behaviour of recombinant human β-NGF pro-protein was compared to that of the mature form. Expression of rh-pro-NGF in Escherichia coli led to the formation of inclusion bodies (IBs). The presence of the covalently attached pro-sequence significantly increased the yield and rate of refolding with concomitant disulfide bond formation when compared to the in vitro refolding of mature NGF (rh-NGF). Physicochemical characterization revealed that rh-pro-NGF is a dimer. The pro-peptide could be removed by limited proteolysis with trypsin yielding biologically active, mature rh-NGF. Furthermore, rh-pro-NGF exhibited biological activity in the same concentration range as rh-NGF.
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Human nerve growth factor (β-NGF) is translated as a prepro-protein consisting of 241 amino-acid residues. The prepeptide comprising 18 amino acids is removed upon translocation into the endoplasmic reticulum. The pro-peptide consisting of 103 residues is subsequently cleaved by cellular pro-protein convertases . Mature β-NGF is biologically active as a homodimer. Within the protomers three disulfide bonds form a cystine knot : Cys58–Cys108 and Cys68–Cys110 form a loop comprising 14 amino acids through which the third disulfide bond (Cys15–Cys80) passes.
β-NGF is a neurotrophic factor that is required for growth and survival of sympathetic and sensory neurons [3–5]. β-NGF also supports growth, differentiation and vitality of cholinergic neurons of the central nervous system . As peripheral nerves are accessible to therapeutic proteins applied systemically, potential therapeutic indications for β-NGF include peripheral neuropathies occurring in patients suffering from diabetes or chemotherapeutic side-effects. Clinical trials with recombinant human β-NGF (rh-β-NGF) for the treatment of peripheral neuropathies caused by diabetes have been promising [7,8]. Further indications for rh-β-NGF are central neuropathies, e.g. Alzheimer's disease concomitant with a degeneration of cholinergic neurons . For further comprehensive clinical trials and the use as a therapeutic, large quantities of homogenous rh-β-NGF are required. A natural source of this growth factor are submaxillar glands of male mice. These preparations, however, are heterogenous mixtures of partially degraded dimers and therefore unsuitable for therapeutic purposes [10–12].
Large amounts of the protein can be obtained by overexpression in procaryotes. However, formation of disulfide bonds cannot occur in the reducing cytosol. Therefore proteins that contain disulfide bonds in their native conformation often form aggregates upon cytosolic synthesis that are deposited as inclusion bodies (IBs) . IBs have to be isolated and subsequently renatured in a suitable redox buffer system. In case of mature rh-β-NGF, renaturation is poor as refolding of the solubilized IB material has to be performed at very low protein concentrations (2 µg·mL−1) and proceeds over more than 100 h (A. Grossmann, unpublished results).
For some proteases such as subtilisin , α-lytic protease  and carboxypeptidase Y , the pro-sequences are known to promote refolding of the mature part. Similarly, folding of bovine pancreatic trypsin inhibitor (BPTI) , transforming growth factor-β1 (TGF-β1) and activin A is stimulated by their pro-peptides . We therefore speculated that this could also be true for β-NGF. Former in vivo investigations on pro-NGF mutants with partial deletions in the pro-sequence showed that the mutations affected maturation and secretion . The authors also demonstrated that two conserved domains in the pro-sequence are required for proper expression and processing. Recently, we could demonstrate that formation of the correct disulfide bonds in the mature growth factor is facilitated by the presence of the covalently attached pro-sequence. Our data indicate that during folding the pro-sequence may mediate association of those thiol groups that form disulfides in the oxidized native protein . Here, we present a detailed comparison of the folding yields and kinetics of both forms together with the physicochemical analysis of pro-NGF and its implications on the structure of the pro-peptide. In addition we show that mature growth factor can be obtained via limited proteolysis of the pro-form. Furthermore, our data indicate that pro-NGF has a comparable biological activity as NGF.
Bacterial strains, culture media and recombinant DNA technology
E. coli strain XL1 blue was used as a host for subcloning and E. coli strain BL21(DE3) for the expression of genes under the T7 promoter control. E. coli cells were transformed with plasmids by electroporation (Gene Pulser II, Bio-Rad). The cells were cultured in Luria–Bertani medium, unless stated otherwise. 100 µg·mL−1 ampicillin and 50 µg·mL−1 kanamycin were used for selection. cDNA for human pro-NGF was PCR-amplified with the vector pMGL-SIG-proNGF (Roche) as DNA template. An NdeI restriction site was generated at the 5′-end by application of the primer ‘FwPro-NGF’ (5′-CGGAATTCCATATGGAAC CACACTCAGAGAGC-3′). Likewise, a BamHI restriction site was introduced at the 3′-end with the reverse primer ‘RevNGF’ (5′-CCGGATCCTTATCATCTCACAGCCTTT CTAGA-3′). The amplification product was inserted into the multiple cloning site of the vector pET11a (Novagen, see also ). The resulting vector, pET11a-Pro-NGF, was cotransformed with plasmid pUBS520 which contains the dnaY gene, encoding the tRNA that recognizes the rare arginine-codons, AGG and AGA, in E. coli. pUBS520 contains the gene for kanamycin resistance and bears the p15A origin of replication which is compatible with ColE1-based pET vectors.
Protein expression and inclusion body isolation
For gene expression, BL21(DE3) transformants were grown at 37 °C in 2 × YT medium (17 g tryptone, 10 g yeast extract and 5 g NaCl per L supplemented with the appropriate antibiotics) to a D600 of 0.5–0.8. Gene expression was induced by addition of 3 mm isopropyl thio-β-d-galactoside (IPTG) and subsequent cultivation for four hours. Cells were harvested by centrifugation. Inclusion bodies were isolated and solubilized as described . Protein concentration was determined according to Bradford . For the concentration determination of purified rh-pro-NGF, the absorption at 280 nm was measured in a Beckman DU 640 spectrophotometer. A molar extinction coefficient of 25 320 L·mol−1·cm−1 was used for calculation of the protein monomer concentration .
Analysis of rh-NGF and rh-pro-NGF refolding by RP-HPLC
To generate denatured, reduced rh-NGF, the native protein (Roche Diagnostics, Penzberg, Germany) was dialyzed overnight against 100 mm Tris/HCl pH 8.0, 6 m guanidinium hydrochloride (GdnHCl), 10 mm EDTA. Solid dithiothreitol was added to a final concentration of 100 mm. After incubation for 2 h at room temperature, the sample was acidified and dialyzed three times against 6 m GdnHCl pH 4.0, to remove excess dithiothreitol. Protein concentration was determined according to Bradford  or by measuring the absorption at 280 nm in a Beckman DU 640 spectrophotometer using a molar extinction coefficient of 19 630 L·mol−1·cm−1 for rh-NGF and 25 320 L·mol−1·cm−1 for rh-pro-NGF (monomers) . The thiol content was measured as described . Samples of 925 µL were withdrawn at certain time points during renaturation. Refolding was stopped by the addition of 75 µL 32% HCl. Protein samples were loaded onto a Poros 10 R1 column (4.6 mm × 100 mm) equilibrated with 6% solvent B [solvent A: 0.13% (v/v) trifluoroacetic acid in water; solvent B: 0.1% (v/v) trifluoroacetic acid in 80% (v/v) aqueous acetonitrile]. The protein was eluted at 20 °C at a flow rate of 0.8 mL·min−1 by a nonlinear gradient (0–4 min, 6% solvent B; 4–9 min, 6–30% B; 9–24 min: 30–69% B; 24–25 min: 69–100% B; 25–30 min: 100% B). Peaks were detected at 220 nm with the Beckman Gold HPLC system (125NM solvent module, 168 detector, autosampler 507). Data were collected using ‘Gold V 8.10’ software. Peaks were fitted with the program peakfit version 2.01. For quantification of the yields, calibration curves with the purified refolded protein were determined in the range between 1 and 5.5 µg·mL−1. As rh-pro-NGF IB material was almost homogenous (≥ 95% according to SDS/PAGE), calculation of the yield of refolded protein was based on the total amount of IB protein used for a refolding experiment.
Analytical gelfiltration and ultracentrifugation studies
A Superdex 75 column (bed volume: 130 mL, Amersham Pharmacia Biotech) was used for analytical gel filtration. Chromatography was performed at 7 °C with 50 mm Na-phosphate pH 7.0, 100 mm NaCl, 1 mm EDTA. A 500-µL sample of 0.67 mg of rh-pro-NGF was injected. The elution profile was monitored by absorption at 280 nm. The column was calibrated with the following marker proteins: RNase A from bovine pancreas (molecular mass 13 700); chymotrypsinogen A from bovine pancreas (molecular mass 25 000); hen ovalbumin (molecular mass 43 000) and bovine serum albumin (molecular mass 67 000). The void volume was determined with Blue Dextran 2000.
Analytical ultracentrifugation was carried out with a Beckman Optima XL-A ultracentrifuge and an An-60 Ti rotor at 20 °C. The rh-pro-NGF concentrations were adjusted to 0.1, 0.25 and 0.6 mg·mL−1 in 50 mm Na-phosphate pH 7.0, 1 mm EDTA. Sedimentation runs were performed at 40 000 r.p.m., equilibrium measurements at 20 000 r.p.m. Data were collected and analyzed with the Beckman xla data aquisition and analysis program provided by the manufacturer. The partial specific volume of rh-pro-NGF was calculated from the average partial specific volume of amino acids (0.73 mL·g−1) .
CD measurements were carried out with an Aviv Model 62ADS circular dichroism spectrometer at 20 °C in 50 mm Na-phosphate pH 7.0. Far-UV CD (190–250 nm) spectra were recorded at protein concentrations of 0.5 mg·mL−1 in a 0.02-cm cell, averaged over 20 accumulations (acquisition time: 2 s). Near-UV (250–350 nm) spectra were determined at 0.76 mg·mL−1 for rh-pro-NGF and 0.46 mg·mL−1 for rh-NGF. Measurements were performed in a 1-cm cuvette (4 accumulations; acquisition time: 15 s). Spectra were buffer-corrected. Mean ellipticity values were calculated according to Schmid . Deconvolution of the spectra was achieved by using the program cdnn, version 2.1 .
Bioassay for rh-NGF and rh-pro-NGF
The biological activity of rh-pro-NGF was measured with the dorsal root ganglion (DRG) assay . Dorsal root ganglions were prepared from 7- to 8-day-old chick embryos and incubated with rh-pro-NGF or rh-NGF at final concentrations of 0.02–20 ng·mL−1. After 48 h incubation, the number of surviving cells was determined. Purified rh-NGF was taken as a standard.
For purification of renatured rh-pro-NGF, the renaturation solution was dialyzed against 50 mm Na-phosphate pH 7.0, 1 mm EDTA and applied onto a Poros 20 HS column (4.6 × 100 mm). Refolded species eluted in a single peak at 980 mm NaCl after application of a linear gradient from 0 to 2 m NaCl. For chemical crosslinking, 20 µL of 0.3 mg·mL−1 rh-pro-NGF in 50 mm Na-phosphate pH 7.0, 1 mm EDTA were mixed with 20 µL 0.02% (v/v) glutaraldehyde in the same buffer. The mixture was incubated at room temperature (22 °C) for 1 h. The crosslinking reaction was stopped by addition of 5 µL 2 m sodium borohydride in 0.1 m NaOH. After incubation for another 20 min, the sample was subjected to SDS/PAGE.
High yields of native rh-pro-NGF can be obtained by in vitro refolding
Recombinant overexpression of rh-pro-NGF in E. coli led to the formation of inclusion bodies (IBs) (Fig. 1). Accumulation of rh-pro-NGF in IBs was expected as native NGF contains three intramolecular disulfide bonds that cannot be formed inside the host cells leading therefore to the deposition of the protein in aggregates. Peptide sequencing of the IB material indicated that the N-terminal methionine was retained. In a typical experiment, ≈ 300 mg IB protein (> 95% purity according to SDS/PAGE) was obtained from 1 L cell culture (D600 = 3.0).
Prior to refolding of rh-pro-NGF, solubilized IB protein was completely reduced by dithiothreitol. To allow refolding of the reduced material to the correctly disulfide-bridged native protein, renaturation was tested in the presence of a redox system containing reduced and oxidized glutathione (GSH/GSSG, respectively). Furthermore, l-arginine, a low molecular mass additive which is known to improve refolding of proteins from IB material , was included in the refolding buffer.
In order to establish an optimum renaturation protocol, refolding was assayed by variation of the refolding conditions. First, renaturation was recorded at different pH values (Fig. 2A). Refolding was monitored by RP-HPLC using native rh-pro-NGF as a standard. Up to pH 7, no refolded protein was detected. Above this pH, an increase in yield was observed. A beneficial effect of alkaline buffer conditions was expected as the thiolate anion (pKa 8.6)  is the reactive species in the disulfide exchange mechanism. Optimum yields could be achieved after 3 h at 10 °C between pH 9.25 and 9.5. Beyond pH 9.5, a decrease in yield was observed, probably due to the increasing instability of the protein at high pH values.
Next, the concentration of l-arginine was varied. For these experiments, renaturation was carried out at pH 9.5. Whereas in the absence of l-arginine no refolding occurred, a linear increase in yield up to 0.75 m l-arginine was observed (Fig. 2B). No further improvement was obtained with higher concentrations of l-arginine.
By modifying the redox potential, refolding could be further optimized. Variation of the GSSG-concentration in the range from 0.5 to 20 mm GSSG with keeping the GSH-concentration at 5 mm had no significant effect on the yield of refolded protein (about 35% after 3 h with 1 m l-arginine at pH 9.5, data not shown). On the other hand, in the absence of GSSG, no renaturation of rh-pro-NGF occurred. Keeping the GSSG concentration constant at 1 mm and varying the GSH concentration, a maximum yield of refolding at 5 mm GSSG was obtained (36%, data not shown). Omission of GSH still gave a final yield of 11% native protein. The moderate refolding in the absence of a reduced redox compound can be explained by the formation of GSH due to redox shuffling between GSSG and the thiol groups of the protein.
Finally, the protein concentration was varied from 50 to 500 µg·mL−1 during refolding. The yield of refolded protein decreased from 35% at 50 µg·mL−1 to 9% at 500 µg·mL−1 protein (data not shown). The effect of residual guanidine hydrochloride in the renaturation buffer on the refolding yield was also examined. No loss in yield occurred up to 500 mm guanidine hydrochloride. Taken together, optimal refolding of rh-pro-NGF was obtained by the following conditions: 100 mm Tris/HCl pH 9.5, 0.75 m l-arginine, 5 mm GSH, 0.5 mm GSSG, 5 mm EDTA. The yield of renaturation under these conditions was ≈ 35% after 3 h of folding. Mass spectrometry data of the refolded protein indicated a molecular mass of 24 868.1 Da which was in accordance with the calculated molecular mass of 24 869.2 Da for the fully oxidized monomer (amino acids −103 to 118 and the retained start methionine).
Rh-pro-NGF is dimeric with its pro-peptide containing a limited amount of secondary structure
Mature NGF is a homodimer with an interface area of 1166 Å2 per monomer . To investigate whether rh-pro-NGF is monomeric or oligomeric, the association state was analyzed by analytical ultracentrifugation. Sedimentation experiments at 40 000 r.p.m. gave an s-value of 3.2 S. The equilibration run performed at 20 000 r.p.m. yielded a molecular mass of 49 190 Da for rh-pro-NGF (Fig. 3A). As the calculated mass of the rh-pro-NGF dimer is 49 738 Da, these data clearly indicate the dimeric state of the pro-protein. At the protein concentrations used for analytical ultracentrifugation, all samples were homogenous and no monomeric species were detected. The native molecular mass of rh-pro-NGF was also assessed by crosslinking with glutaraldehyde and subsequent SDS/PAGE. A cross-linked product corresponding in size to the dimer was observed (apparent molecular mass: 50 kDa; Fig. 3B). To confirm the dimeric state with a third experiment, analytical gel filtration of rh-pro-NGF was performed. rh-pro-NGF eluted earlier than mature rh-β-NGF with an apparent molecular mass of 46 kDa, confirming our results that suggest the dimeric form (Fig. 3C).
Next, circular dichroism spectra were determined to detect structural differences between rh-NGF and rh-pro-NGF. The far-UV CD spectrum of rh-NGF showed a slight signal decrease from 235 to 210 nm which is in accordance with a β sheet structure and a broad minimum at around 206 nm (Fig. 4A) (see also [32,33]). The far-UV CD spectrum of rh-pro-NGF was similar to that of rh-NGF in the 235–210 nm region. In contrast, the spectrum of rh-pro-NGF exhibited a deeper minimum at 202 nm possibly indicating secondary structure of the pro-peptide. Calculation of the secondary structure elements yielded 24% antiparallel β sheet, 4% parallel β strand and 25% β turn using the data starting from 195 nm . As our results suggest the dimeric form of rh-pro-NGF, we assume that the mature part of the pro-form does not significantly differ in structure from rh-NGF. To test this hypothesis, the difference spectrum of rh-pro-NGF and rh-NGF in the far UV region was calculated. Deconvolution of this spectrum predicts a content of 20% antiparallel β strand, 4% parallel β sheet and 27% β turn, and indicates that the pro-peptide contains a limited amount of secondary structure (β sheet). The near-UV CD spectrum of rh-pro-NGF showed some fine-structure in the 280–300 nm range that is absent in the NGF spectrum (Fig. 4B) which could indicate that the single tryptophan residue of the pro-peptide is in an asymmetric environment. The three tryptophane residues of the mature moiety are known to be fixed in the subunit interface .
Biologically active rh-NGF is obtained after limited proteolysis of rh-pro-NGF
A major goal of our studies was to obtain biologically active, mature rh-NGF. Posttranslational proteolysis is assumed to be an essential step during biogenesis of NGF. As many other precursor proteins, the pro-peptide contains at the C-terminus two basic amino acids, which are recognized in vivo by furin-like proteases. In order to obtain mature NGF from the refolded rh-pro-NGF in vitro, limited proteolysis with trypsin was carried out. A cleavage product corresponding in size to mature rh-NGF was detected by SDS/PAGE analysis (Fig. 5). Mass determination and sequencing of the N-terminus confirmed that the cleavage product was identical to mature rh-NGF. The pro-peptide was completely degraded by trypsin. rh-NGF produced by limited proteolysis exhibited a similar biological activity as the NGF standard in the dorsal root ganglion assay (Fig. 6). It is important to note that the pro-form of NGF also revealed activity in this bioassay. Taking into account that pro-NGF is about twice as large as mature NGF, both proteins have comparable biological activities.
The pro-peptide stimulates folding of NGF
To learn more about the biological role of the pro-peptide, refolding kinetics of mature and rh-pro-NGF were monitored by RP-HPLC. For renaturation, the fully reduced proteins were diluted into refolding buffer at equimolar concentrations (26 µg·mL−1 for rh-NGF and 50 µg·mL−1 for rh-pro-NGF). At the indicated time points, refolding was stopped by acidification or by direct injection onto the RP-HPLC-column. In case of rh-pro-NGF, a maximum folding yield of 35% at 10 °C was observed after 3 h (Fig. 7). Incubation at higher temperatures led to a reduction in yield probably because aggregation reactions are accelerated at increased temperatures and compete effectively with folding. In contrast to rh-pro-NGF, refolding of mature rh-NGF proceeded very slowly: after 25 h renaturation at 4 °C and 10 °C, yields of 1.1% and 0.4%, respectively, were obtained.
In vivo, NGF attains its native structure in the pro-form in the endoplasmic reticulum . Therefore, we rationalized that the NGF pro-sequence could stimulate refolding of the mature part and exert an analogous function as the pro-peptides of subtilisin BPN′ and other proteases. Cytosolic synthesis of rh-pro-NGF in E. coli led to deposition of the protein in IBs. Renaturation of IBs to the folded protein was optimized. The renaturation buffer included a redox buffer system containing 0.75 m l-arginine. Comparison of refolding yields showed that the yield of native protein is dramatically enhanced by the presence of the pro-peptide (35%). In contrast, mature protein could be refolded with a yield of 0.4% under identical conditions. Furthermore, folding kinetics of the pro-protein were significantly accelerated compared to mature NGF. Our results demonstrate that the pro-peptide plays an important role during oxidative folding of β-NGF.
Analysis of the quarternary structure of rh-pro-NGF was assessed by analytical ultracentrifugation, analytical gel filtration and chemical crosslinking. These techniques revealed that the pro-form is a dimer. Hence proteolytic cleavage of the pro-peptide is likely to occur in vivo after dimerization. CD analysis of rh-pro-NGF revealed a predominance of β sheet structures. Given that the secondary structure of the mature part does not significantly differ between pro- and mature form, the difference spectrum of rh-pro-NGF and rh-NGF indicates a limited amount of secondary structure for the pro-peptide. However, as the pro-peptide was readily degraded by trypsin the packing of the pro-peptide into secondary structural elements is probably not very tight. As the near-UV CD-spectrum of rh-pro-NGF revealed some fine structure we propose that the single tryptophane of the pro-sequence is in an asymmetric environment, i.e. associated with the mature part.
Former investigations showed that the NGF-precursor obtained from extracts of transfected mouse fibroblasts had a 10-fold reduced biological activity when compared to the mature protein . These data were based exclusively on the number of neurons with neurites as long as the cell body. It was also reported by a separate investigation that NGF precursor from rat displays biological activity as evidenced by flattening and neurite outgrowth of PC-12 cells . In accordance with these data, we could demonstrate biological activity for rh-pro-NGF in the DRG assay. Here the number of surviving neurons, regardless of the neurite lengths, was counted. This could explain why we observe comparable biological activities of rh-pro-NGF and mature rh-NGF and not the 10-fold lower activity of the pro-form reported by Edwards et al. . It should be noted that we cannot exclude that rh-pro-NGF is processed to the mature form during cultivation of the neurons. However, if that were the case processing would be 100% as the EC50 values for rh-pro-NGF and rh-NGF show that both proteins are active in the same molar concentrations (inset of Fig. 6). Thus, we assume that pro-NGF besides mature NGF is a physiologically relevant form. The mechanism by which pro-NGF functions as a neurotrophic factor is presently unknown. We would propose that pro-NGF binds to TrkA, the receptor for mature NGF. Certainly, comprehensive analyses are required to unravel this issue.
Upon limited proteolysis, refolded rh-pro-NGF was converted to the mature protein with full biological activity. The hydrolysis product was the correctly processed mature protein as evidenced by N-terminal sequencing and mass analysis. Moreover, in vitro processed rh-NGF eluted at the same ionic strength from a cation exchange column as the rh-NGF standard (Roche Diagnostics) (data not shown).
Presently, it is unclear whether proteolytic cleavage of the pro-peptide occurs before or after dimerization in vivo. Our data on the association state of rh-pro-NGF revealed that this protein exists in the dimeric state at protein concentrations as low as 5 µg·mL−1 as determined by chemical crosslinking. However, as rh-pro-NGF displayed biological activity in the same concentration range as mature NGF and as it is probably only the dimeric form of NGF which is physiologically relevant, we assume that dissociation of pro-NGF occurs at a concentration lower than 300 pg·mL−1. It should be noted, however, that these considerations are based on the assumption that the pro-peptide is not proteolytically removed in the cell culture assay. Further experiments are required to determine the dissociation constant of rh-pro-NGF.
Taken together, the data presented here clearly indicate that the pro-peptide of NGF aids to oxidative folding in vitro. In a recent work we could show that the pro-peptide accelerates structure formation via facilitating formation of the correct disulfide bonds. However, folding in trans, i.e. by the separated pro-peptide was not successful . Furthermore, though a comprehensive analysis of this issue is required, our data suggest that not only mature NGF, but also pro-NGF is biologically active in vivo. The physiological relevance of this finding has to be explored by careful cell biological investigations.
We thank Peter Rücknagel for N-terminal sequencing, Angelika Schierhorn for mass analysis and Olaf Mundigl and Monika Heidrich for performing the DRG assay. The authors are indebted to Gennaro Marino for critically reading the manuscript. This work was supported by the Federal German Ministry for Education and Research (BMBF), grant no. 0311149.
*Present address: Department of Dermatology, University of Münster, Von-Esmarch-Str. 58, D-48149 Münster, Germany.