Biological and Immunological Properties of Recombinant Human, Rat, and Chicken Nerve Growth Factors: A Comparative Study

Biological and immunological properties of recombinant human, rat, and chicken nerve growth factors (NGFs) were studied and compared. Recombinant NGF proteins were produced in a transient expression system using COS cells and levels of secreted NGF protein were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of conditioned media from in vivo [³⁵S]cysteine-labeled cell cultures. Antigenic differences among the three NGFs were studied by immunoblotting and immunoprecipitation of secreted cell products using a rabbit polyclonal antiserum against purified mouse NGF, and by a two-site enzyme im-munoassay (EIA) with a monoclonal antibody against mouse NGF. Although all three NGFs were recognized equally well in the immunoblotting, only one-third of the chicken NGF protein could be detected by immunoprecipitation or by the EIA as compared to the rat and human NGFs. Thus, changes in the three-dimensional structure of the NGF molecule are most likely responsible for the antigenic differences between avian and mammalian NGFs. The three NGF proteins were also compared in their ability to displace ¹²⁵I-mouse NGF from low-affinity NGF receptors on rat pheochromocytoma PC12 cells. Similar displacement curves and values were obtained for each NGF protein, indicating that structural differences among these molecules do not affect low-affinity binding to NGF receptors. Biological activities were studied by the ability of the conditioned media to promote neurite outgrowth from explants of 9 chick sympathetic ganglia and from PC12 ceils. Although the rat system showed a slight preference for the homologous molecule, the morphological changes, dose-response curves, and maximal stimulation values obtained with the different NGFs were practically indistinguishable in the chicken bioassay. The observed differences and similarities among the three NGF proteins are discussed in the context of their evolutionary relationship and their potential therapeutic applications.     

Highly sensitive enzyme immunoassay for beta-nerve growth factor (NGF): a tool for measurement of NGF level in rat serum

A sensitive two-site enzyme immunoassay (EIA) system was established for mouse beta nerve growth factor (NGF) isolated from mouse submaxillary gland. Our EIA system is based on the sandwiching of antigen between anti-mouse beta NGF antibody IgG coated on a polystyrene plate and biotinylated anti-mouse beta NGF antibody IgG. The bound antibody complex was quantified with streptavidin linked-beta-D-galactosidase (beta-D-galactosidase, EC 3.2.1.23). With this system NGF concentrations as low as 0.02 pg/well (corresponding to 8 x 10(-19) mol) could be measured reproducibly. The sensitivity of this EIA system permitted the quantification of endogenous immunoreactive beta NGF in rat serum. The mean level in serum of male rats (153.2 pg/ml) was found to be almost the same as that of female rats (127.6 pg/ml).     

Human fibroblast cells synthesize and secrete nerve growth factor in culture.

Using our enzyme immunoassay system developed for recombinant hNGF, we examined the synthesis and secretion of human NGF (hNGF) by human fibroblast (WS-1) cells. The amount of the factor secreted by WS-1 cells increased linearly and a significant amount of NGF was detected in the conditioned medium of WS-1 cultures. WS-1 NGF showed properties identical to those of recombinant human NGF in immunoreactivity and molecular weight. An increase in cell density or the withdrawal of serum from the culture medium caused a drastic decrease in the rate of NGF secretion. These results suggest that WS-1 cells are able to synthesize and secrete hNGF in culture and that the synthesis/secretion is regulated in a growth phase-dependent manner.     

性别及日龄对转人神经生长因子基因小鼠唾液中蛋白分泌的影响

神经生长因子(Nerve growth factor,NGF)是一种能促进神经元发育、分化、再生的蛋白。为高效生产药效更佳的人源NGF (hNGF)药物,最近,笔者实验室构建出唾液腺特异表达hNGF的转基因小鼠,并从该转基因小鼠唾液中纯化获得具有高生物学活性的h NGF蛋白。为了选择性别和日龄最适宜的转hNGF基因小鼠用于收集纯化hNGF蛋白,文中比较了28日龄(性成熟前)雄性、雌性,63日龄(性成熟后)雄性、雌性转hNGF基因小鼠,共4组转hNGF基因小鼠分泌的唾液量、唾液总蛋白量、唾液鼠源NGF (mNGF)蛋白量和唾液h NGF蛋白量等指标。结果显示,63日龄的转hNGF基因小鼠分泌的唾液量、唾液总蛋白量、唾液mNGF蛋白量和唾液hNGF蛋白量显著高于28日龄同一性别的转hNGF基因小鼠,且63日龄的雄性转hNGF基因小鼠分泌的唾液hNGF蛋白量显著高于同一日龄的雌性转hNGF基因小鼠;在4组小鼠中,63日龄的雄性转hNGF基因小鼠分泌的唾液hNGF含量最高,比28日龄雌性转hNGF基因小鼠高出约46倍,最适宜用于收集唾液并从中纯化hNGF。     

Functional Characterization of Human ProNGF and NGF Mutants: Identification of NGF P61SR100E as a “Painless” Lead Investigational Candidate for Therapeutic Applications

Background

Nerve Growth Factor (NGF) holds a great therapeutic promise for Alzheimer''s disease, diabetic neuropathies, ophthalmic diseases, dermatological ulcers. However, the necessity for systemic delivery has hampered the clinical applications of NGF due to its potent pro-nociceptive action. A “painless” human NGF (hNGF R100E) mutant has been engineered. It has equal neurotrophic potency to hNGF but a lower nociceptive activity. We previously described and characterized the neurotrophic and nociceptive properties also of the hNGF P61S and P61SR100E mutants, selectively detectable against wild type hNGF. However, the reduced pain-sensitizing potency of the “painless” hNGF mutants has not been quantified.

Objectives and Results

Aiming at the therapeutic application of the “painless” hNGF mutants, we report on the comparative functional characterization of the precursor and mature forms of the mutants hNGF R100E and hNGF P61SR100E as therapeutic candidates, also in comparison to wild type hNGF and to hNGF P61S. The mutants were assessed by a number of biochemical, biophysical methods and assayed by cellular assays. Moreover, a highly sensitive ELISA for the detection of the P61S-tagged mutants in biological samples has been developed. Finally, we explored the pro-nociceptive effects elicited by hNGF mutants in vivo, demonstrating an expanded therapeutic window with a ten-fold increase in potency.

Conclusions

This structure-activity relationship study has led to validate the concept of developing painless NGF as a therapeutic, targeting the NGF receptor system and supporting the choice of hNGF P61S R100E as the best candidate to advance in clinical development. Moreover, this study contributes to the identification of the molecular determinants modulating the properties of the hNGF “painless” mutants.     

The Distribution of Nerve Growth Factor in the Male Sex Organs of Mammals

Abstract: The Nerve Growth Factor (NGF) content of male sex organs of the mouse, rat, guinea pig, hamster, rabbit, human, and bull has been investigated using both a biological assay and a two-site radioimmunoassay. The prostate glands of the rabbit and bull have been found to contain moderate levels of NGF, these being lower than the concentrations found in the guinea pig prostate and mouse submaxillary glands. The sex organs investigated of the mouse, rat, hamster, and human contained no detectable NGF activity. Genital organs, other than the prostate glands, of the guinea pig and rabbit were also devoid of NGF. The NGFs from the rabbit and bull are immunologically related to those found in the submaxillary glands of the mouse and the prostate glands of the guinea pig, but immunodiffusion and radioimmunoassay experiments show that there are also clear differences between the NGFs. The use of a two-site radioimmunoassay, based on purified antibodies against mouse submaxillary gland NGF, for the determination of NGF levels in species other than the mouse, is described. It is essential during such applications to compensate for the fact that the NGFs from different species are sufficiently distinct that only part of the antibody population (raised against mouse NGF) is capable of recognizing NGF from species other than the mouse. The results of radioimmunoassay and biological assay determinations are in reasonable agreement, if corrections for this feature are made.     

Probing the structure-function relationship of nerve growth factor

We compared the receptor binding, antigenicity, biological activation, and cell-mediated proteolytic degradation properties of mouse nerve growth factor (mNGF) and human NGF (hNGF). The affinity of hNGF toward human NGF-receptor is greater than that of mNGF, but the affinity of mNGF toward rat NGF-receptor is greater than that of hNGF. Thus, the specificity of the interaction between NGF and its receptor resides both on the NGF and on its receptor. Using a group of anti-NGF monoclonal antibodies that competitively inhibit the binding of NGF to receptor, sites differing between mNGF and hNGF were detected. Together, these results indicate that the sites on hNGF and mNGF, responsible for binding to NGF-receptor, are similar but not identical. In comparing the relative abilities of mNGF and hNGF to stimulate a biological response in PC12 cells, we observed that mNGF was better at stimulating neurite outgrowth than was hNGF, consistent with the differences observed for receptor binding affinity. However, the ED₅₀ for biological activation is approximately 100-fold lower than theK _d for receptor occupancy, and, thus, the dose-response curve is not consistent with a simple activation proportional to receptor occupancy. The data are consistent with a model requiring a low-level threshold occupancy of NGF-receptor (K _d=10^–9 M) in order to stimulate full biological activity. Finally, we observed the degradation of NGF by PC12 cells. We found that the NGF molecule is significantly degraded via a receptor-mediated uptake mechanism. Together, the data provide insight into regions of the NGF molecule involved in contacts with the receptor leading to formation of the NGF: NGF-receptor complex. Additionally, they establish the link between occupancy of receptor and biological activation and the requirement for receptor-mediated uptake in order to degrade NGF proteolytically in cultured PC12 cells.     

A sensitive two-site enzyme immunoassay for human epidermal growth factor (urogastrone)

A sensitive enzyme immunoassay (EIA) was developed for human epidermal growth factor (hEGF) or urogastrone, which was isolated from human urine. Our EIA system is based on the sandwiching of an antigen between anti-hEGF IgG coated on a polystyrene tube and anti-hEGF antibody Fab'-linked beta-D-galactosidase (beta-D-galactosidase, EC 3.2.1.23). This method has the advantages that the procedures are simple and rapid and that the antibody Fab'-beta-D-galactosidase complex is more stable than radioisotope-labeled IgG. Purified hEGF is detectable at as low as 100 pg/ml, which is very sensitive compared to the radioimmuno-assays or radioreceptor assays already reported. Using this new EIA system, hEGF levels in human urine were examined. The values for normal males and females were 48.4 and 83.5 ng/mg creatinine, respectively, which shows that females excrete 1.7 times more hEGF than males.     

Human nerve growth factor: Lack of immunocrossreactivity with mouse nerve growth factor

Human β-nerve growth factor (hNGF) was purified from term human placenta. The biological potency of hNGF in the chick dorsal root ganglion assay did not differ significantly from that of mouse NGF (mNGF). Molecular weight determinations of mNGF and hMGF were also similar. No immunological crossreactivity was noted between hNGF, at a concentration of 100 μg/ml, and mNGF in a radioimmunoassay for mNGF using 6 different antisera to mNGF. hNGF shares several properties with mNGF but is immunological distinct. The results of studies in man using antisera to mNGF should be interpreted with caution.     

Overproduction of biologically-active human nerve growth factor in Escherichia coli.

A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the beta-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the beta-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.     

Biologically active human and mouse nerve growth factors secreted by the yeast Saccharomyces cerevisiae

Nerve growth factor (NGF) is a trophic agent that is essential for the development and survival of sympathetic and sensory nerves. A chemically-synthesized DNA fragment encoding human NGF (hNGF) and a cDNA encoding mouse NGF (mNGF) were engineered for expression in the yeast, Saccharomyces cerevisiae. Expression and secretion of hNGF and mNGF was attempted under the direction of the yeast PGK promoter and with various leader sequences. Among the leader sequences tested, that of the yeast -factor successfully directed secretion of both hNGF and mNGF that were correctly processed. The content of the recombinant NGF (reNGF) in the culture supernatant was estimated to be 1 g/ml. The yeast-produced reNGF was able to bind to NGF receptors in rat pheochromocytoma (PC12) cells as efficiently as the standard mNGF, and partially purified reNGF could induce neurite outgrowth of PC12 cells. Thus, we have demonstrated that biologically active human and mouse reNGF can be produced in yeast cells. Correspondence to: M. Nishizawa     

Over-Expression of hNGF in Adult Human Olfactory Bulb Neural Stem Cells Promotes Cell Growth and Oligodendrocytic Differentiation

The adult human olfactory bulb neural stem/progenitor cells (OBNC/PC) are promising candidate for cell-based therapy for traumatic and neurodegenerative insults. Exogenous application of NGF was suggested as a promising therapeutic strategy for traumatic and neurodegenerative diseases, however effective delivery of NGF into the CNS parenchyma is still challenging due mainly to its limited ability to cross the blood–brain barrier, and intolerable side effects if administered into the brain ventricular system. An effective method to ensure delivery of NGF into the parenchyma of CNS is the genetic modification of NSC to overexpress NGF gene. Overexpression of NGF in adult human OBNSC is expected to alter their proliferation and differentiation nature, and thus might enhance their therapeutic potential. In this study, we genetically modified adult human OBNS/PC to overexpress human NGF (hNGF) and green fluorescent protein (GFP) genes to provide insight about the effects of hNGF and GFP genes overexpression in adult human OBNS/PC on their in vitro multipotentiality using DNA microarray, immunophenotyping, and Western blot (WB) protocols. Our analysis revealed that OBNS/PC-GFP and OBNS/PC-GFP-hNGF differentiation is a multifaceted process involving changes in major biological processes as reflected in alteration of the gene expression levels of crucial markers such as cell cycle and survival markers, stemness markers, and differentiation markers. The differentiation of both cell classes was also associated with modulations of key signaling pathways such MAPK signaling pathway, ErbB signaling pathway, and neuroactive ligand-receptor interaction pathway for OBNS/PC-GFP, and axon guidance, calcium channel, voltage-dependent, gamma subunit 7 for OBNS/PC-GFP-hNGF as revealed by GO and KEGG. Differentiated OBNS/PC-GFP-hNGF displayed extensively branched cytoplasmic processes, a significant faster growth rate and up modulated the expression of oligodendroglia precursor cells markers (PDGFRα, NG2 and CNPase) respect to OBNS/PC-GFP counterparts. These findings suggest an enhanced proliferation and oligodendrocytic differentiation potential for OBNS/PC-GFP-hNGF as compared to OBNS/PC-GFP.     

Molecular cloning of bovine and chick nerve growth factor (NGF): delineation of conserved and unconserved domains and their relationship to the biological activity and antigenicity of NGF.

Previous experiments with purified mouse and bovine nerve growth factor (NGF) have shown that the biological activities of these two NGFs are identical, whereas the immunological cross-reactivity of antibodies produced against the two NGF molecules is very limited. This observation, together with the fact that antibodies to mouse NGF do not affect the development of sympathetic and sensory neurons in chick embryos, suggests that the domain of the NGF molecules responsible for the biological action has been highly conserved during evolution, whereas other domains determining the immunological properties were under less rigorous evolutionary constraint. The nucleotide sequences of bovine and chick NGF were determined from a cDNA clone prepared from mRNA of bovine seminal vesicles and from cloned chick genomic DNA, and the amino acid sequences deduced therefrom were compared with the available sequences of mouse and human NGF. All six cysteine residues were conserved in agreement with the previous finding that the biological activity of NGF is conformation-dependent requiring intact disulfide bridges. Amino acid changes are mainly confined to hydrophilic regions expected to be potential antigenic determinants, thus providing an explanation for the poor immunological cross-reactivities between the different NGFs. One single hydrophilic region is conserved in all NGFs and this region could be involved in the biological activity. The carboxy termini of bovine and chick NGF differ from that of mouse NGF, the changes in the amino acid sequences suggest that chick and bovine NGF are probably not processed by the gamma-subunit and that no 7S complex can be formed as in the mouse submandibular gland.     

Intranasal "painless" human Nerve Growth Factors slows amyloid neurodegeneration and prevents memory deficits in App X PS1 mice

Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease (AD) treatment but the clinical application is hindered by its potent pro-nociceptive activity. Thus, to reduce systemic exposure that would induce pain, in recent clinical studies NGF was administered through an invasive intracerebral gene-therapy approach. Our group demonstrated the feasibility of a non-invasive intranasal delivery of NGF in a mouse model of neurodegeneration. NGF therapeutic window could be further increased if its nociceptive effects could be avoided altogether. In this study we exploit forms of NGF, mutated at residue R100, inspired by the human genetic disease HSAN V (Hereditary Sensory Autonomic Neuropathy Type V), which would allow increasing the dose of NGF without triggering pain. We show that "painless" hNGF displays full neurotrophic and anti-amyloidogenic activities in neuronal cultures, and a reduced nociceptive activity in vivo. When administered intranasally to APPxPS1 mice ( n?=?8), hNGFP61S/R100E prevents the progress of neurodegeneration and of behavioral deficits. These results demonstrate the in vivo neuroprotective and anti-amyloidogenic properties of hNGFR100 mutants and provide a rational basis for the development of "painless" hNGF variants as a new generation of therapeutics for neurodegenerative diseases.     

Human Ovarian Reserve from Conception to the Menopause

The human ovary contains a fixed number of non-growing follicles (NGFs) established before birth that decline with increasing age culminating in the menopause at 50–51 years. The objective of this study is to model the age-related population of NGFs in the human ovary from conception to menopause. Data were taken from eight separate quantitative histological studies (n = 325) in which NGF populations at known ages from seven weeks post conception to 51 years (median 32 years) were calculated. The data set was fitted to 20 peak function models, with the results ranked by obtained correlation coefficient. The highest ranked model was chosen. Our model matches the log-adjusted NGF population from conception to menopause to a five-parameter asymmetric double Gaussian cumulative (ADC) curve ( = 0.81). When restricted to ages up to 25 years, the ADC curve has  = 0.95. We estimate that for 95% of women by the age of 30 years only 12% of their maximum pre-birth NGF population is present and by the age of 40 years only 3% remains. Furthermore, we found that the rate of NGF recruitment towards maturation for most women increases from birth until approximately age 14 years then decreases towards the menopause. To our knowledge, this is the first model of ovarian reserve from conception to menopause. This model allows us to estimate the number of NGFs present in the ovary at any given age, suggests that 81% of the variance in NGF populations is due to age alone, and shows for the first time, to our knowledge, that the rate of NGF recruitment increases from birth to age 14 years then declines with age until menopause. An increased understanding of the dynamics of human ovarian reserve will provide a more scientific basis for fertility counselling for both healthy women and those who have survived gonadotoxic cancer treatments.     

Bitis arietans nerve growth factor is a disulphide-linked homodimer.

1. Nerve growth factor from Bitis arietans venom was isolated in high yield and purified to homogeneity using a rapid two-step procedure involving gel exclusion chromatography and reversed-phase HPLC. 2. On polyacrylamide gel electrophoresis in SDS, the NGF migrates as a 25 kDa homodimer and is thus atypical of other Viperid NGFs. 3. Evidence suggests that, unlike mammalian beta NGFs, the subunits of the Bitis arietans homodimer are covalently linked by a disulphide bond(s). 4. Partial sequence analysis shows that only 6 out of the first 21 amino acids are identical with those of cobra NGF including cys-14 and val-21 which are known to be important for NGF activity.     

Overproduction of Biologically-active Human Nerve Growth Factor in Escherichia coli

A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the β-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the β-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.     

促进CHO细胞生长及其产物hNGF表达的培养条件的初步研究

以稳定表达人神经生长因子（hNGF）的重组工程CHO细胞株为对象,采用无血清流加悬浮培养（Fed batch culture）方式,考察使用基础培养基(无特殊添加物),分别添加丁酸钠、DMSO、KH2PO4的培养基及不同培养温度（32℃和37℃）对细胞生长和重组蛋白表达的影响。每日取样检测细胞密度、细胞活率、葡萄糖浓度、重组蛋白浓度。结果表明细胞培养温度由37℃下降至32℃,细胞生长周期明显延长,重组蛋白产量增加。5mmol/L丁酸钠和2% DMSO的加入虽然提高了重组蛋白的表达量,但严重抑制细胞生长。最大的蛋白比生成速率（qNGF）出现在37℃培养且添加2% DMSO的培养条件下,而最高蛋白表达量则出现于32℃培养添加3.65mmol/L KH2PO4的培养条件下。研究表明,将培养温度设为32℃,在基础培养基中添加3.65mmol/L KH2PO4或1% DMSO是提高hNGF表达水平的有效方法。     

A common mechanism for recombinant human NGF, BDNF, NT-3, and murine NGF slow unfolding

The recombinant human nerve growth factor (hNGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin 4/5 (NT4/5), and murine NGF (mNGF) dimers all undergo rapid unfolding and dissociation to monomer in GdnHCl. Fluorescence spectroscopy, reversed-phase high-performance liquid chromatography, and size-exclusion chromatography were used to show that this monomer M1 converts slowly to a more fully unfolded monomer, M2, by a first order process with half-lives of 22, 2.5, 1.6, and 0.73 h for hNGF, mNGF, NT-3, and BDNF, respectively, at 25 degrees C. Linear Arrhenius plots for the conversion of M1 to M2 yielded activation energies of 27, 22, 24, and 24 kcal/mol for hNGF, mNGF, NT-3, and BDNF, respectively. The refolding of these neurotrophins from 5 M GdnHCl was also first order with NT-3 the slowest to refold and BDNF the fastest. Threading of the N-terminus out through the cystine-knot loop present in each of these proteins is proposed as the slow step in unfolding. The number of amino acids in the cystine-knot loop (14 for hNGF, mNGF, NT-3, and BDNF; 21 for NT4/5), and the number and position of the proline residues in this loop (2 for hNGF; 1 for mNGF, NT-3, BDNF, and NT4/5) correlate with the relative rates of unfolding. The smaller the loop and the greater the number of prolines, the more hindered and slower the unfolding.     

A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor

Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than ¹²⁵I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.