Histidine residue modification inhibits binding of murine β nerve growth factor to its receptor

The reaction of the β subunit of murine nerve growth factor (NGF) with diethylpyrocarbonate (DEP) results in the quantitative modification of histidine residues and the loss of binding to rabbit superior cervical ganglia microsomes. No conformational changes accompanied the conversion as judged by fluorescence spectra. Hydroxylamine converted the carbethoxy derivatives back to the unmodified imidazoles and simultaneously restored the capacity of NGF to bind to its receptor. Modification of des (1–9) NGF, from which His-4 and His-8 have been quantitatively removed, results in the same loss in binding activity, suggesting that His-75 and/or His-84 may play an important role in hormone-receptor interactions.     

Identification of essential histidine residues in a recombinant alpha-amylase of thermophilic and alkaliphilic Bacillus sp. strain TS-23

To understand the structure-function relationships of a truncated Bacillus sp. strain TS-23 -amylase, each of His-137, His-191, His-239, His-269, His-305, His-323, His-361, His-436, and His-475 was replaced with leucine. The molecular masses of the purified wild-type and mutant enzymes were approximately 54 kDa. The specific activity of His323Leu and His436Leu was decreased by more than 52%, while His239Leu, His305Leu, and His475Leu showed activity similar to that of the wild-type enzyme. As compared with the wild-type enzyme, His323Leu and His436Leu exhibited a 62% decrease in the value of k_cat/K_m. Alterations in His-191, His-239, His-305, and His-475 did not cause a significant change in the K_m or k_cat values. At 70°C, a decreased half-life was observed in His436Leu. These results indicate that His-137, His-269, and His-361 of Bacillus sp. strain TS-23 -amylase are important for proper catalytic activity and that His-436 may contribute to the thermostability of the enzyme.Communicated by K. Horikoshi     

Residue Histidine 669 Is Essential for the Catalytic Activity of Bacillus anthracis Lethal Factor

The lethal factor (LF) of Bacillus anthracis is a Zn²⁺-dependent metalloprotease which plays an important role in anthrax virulence. This study was aimed at identifying the histidine residues that are essential to the catalytic activities of LF. The site-directed mutagenesis was employed to replace the 10 histidine residues in domains II, III, and IV of LF with alanine residues, respectively. The cytotoxicity of these mutants was tested, and the results revealed that the alanine substitution for His-669 completely abolished toxicity to the lethal toxin (LT)-sensitive RAW264.7 cells. The reason for the toxicity loss was further explored. The zinc content of this LF mutant was the same as that of the wild type. Also this LF mutant retained its protective antigan (PA)-binding activity. Finally, the catalytic cleavage activity of this mutant was demonstrated to be drastically reduced. Thus, we conclude that residue His-669 is crucial to the proteolytic activity of LF.Anthrax is a zoonotic disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis (24). Because infections are highly fatal, the organisms are easily produced, and the spores spread easily, B. anthracis has been used as a bioweapon in biological war and biological terrorism (38). If inhaled, the spores are phagocytosed by alveolar macrophages, where they germinate to produce vegetative bacteria (10, 24). The vegetative bacteria further release anthrax toxins, which inhibit the innate and adaptive immune responses of the hosts. This enables the capsulated bacteria to escape the lymph node defense barrier to reach the blood system, causing bacteremia and toxemia, which can rapidly kill the hosts (24, 26). The great threat posed by anthrax to the public is not only due to the highly lethal rate of inhaled anthrax, but also is due to the social panic caused by the lethality. Therefore, efficient ways to defend against anthrax infection and spreading are greatly needed. This mostly depends on a full understanding of the mechanisms of anthrax infection and toxicities.Anthrax toxins are the dominant virulence factors of Bacillus anthracis (6, 33, 37). They consist of three proteins: protective antigen (PA; 83 kDa), lethal factor (LF; 90 kDa), and edema factor (EF; 89 kDa). The 83-kDa PA (PA₈₃) directly binds to cellular membrane receptors and was cleaved to an active fragment of 63-kDa PA (PA₆₃) by cellular proteases of the furin family or by serum proteases. The receptor-bound portion of PA₆₃ self-assembles into either ring-shaped heptamers, which bind to three molecules of LF and/or EF, resulting in (PA₆₃)₇(LF/EF)₃ (21), or octamers which bind up to four molecules of these moieties, resulting in (PA₆₃)₈(LF/EF)₄ complexes (16, 17). The catalytic partners (EF and/or LF) are subsequently transported across the membrane to the cell cytosol (24, 27). EF is a Ca²⁺- and calmodulin-dependent adenylate cyclase that, together with PA, forms edema toxin. EF causes a rapid increase in intracellular cyclic AMP (cAMP) levels in host cells and alters the elaborate balance of intracellular signaling pathways (20, 23). LF is a Zn²⁺-dependent protease that, together with PA, forms lethal toxin (LT). It is a dominant virulence factor and the major cause of death for the B. anthracis-infected animals (1, 29, 30). LF specifically cleaves the N-terminal domain of mitogen-activated protein kinase kinases (MAPKKs) (11, 35). Because the N-terminal domain of MAPKKs is essential for the interaction between MAPKKs and MAPKs, the cleavage of this domain impairs the activation of MAPKs (8, 11, 15) and leads to the inhibition of three major cellular signaling pathways—the ERK (extracellular signal-regulated kinase), p38, and JNK (c-Jun N-terminal kinase) pathways (29, 31)—and thus induces the lysis of the host cells in an unknown mechanism.The crystal structure of LF with the N-terminal domain of MEK2 has been reported (28). LF has 776 amino acids and comprises four different domains. Domain I (residues 1 to 254) is a PA-binding domain which delivers the remaining domains of the LF to the cell cytoplasm (3). The interface among domains II, III, and IV creates long, deep, 40-Å-long catalytic grooves into which the N terminus of MEK fits and forms an active site complex (28). Domain IV is central to catalytic activities of LF, containing two zinc-binding motifs (residues 686 to 690 and residues E735 to E739) and bound to a single Zn ion (18). However, which residues of LF are critical for efficient catalytic activities and execute the substrate cleavage remains unclear.Histidine is the only naturally occurring amino acid to contain an imidazole residue as a side chain. The catalytic activity of histidine mostly depends on the special features of the imidazole residue. The logarithm of the proton dissociation constant of imidazolyl in the histidine residue is about 6.5; thus, under the physiological condition, it tends to form hydrogen bonds and shares donor and acceptor properties that can take part in either nucleophilic or base catalysis. The speed of the imidazole residue to give or accept protons is very fast, with a half-life of less than 10 s. So in the process of natural selection, histidine was chosen as the catalytic structure, indicating that it plays an important role in the catalysis process of enzymes (9, 12, 14). There are 21 histidines in LF, with 9 of them in LF domain I and 12 of them in domains II, III, and IV. The histidine residues important to LF activities in domain I have been identified (2, 22). The other 12 histidine residues in the remaining three domains include His-277, His-280, and His-424 in domain II; His-309 in domain III; and His-588, His-645, His-654, His-669, His-686, His-690, His-745, and His-749 in domain IV (28). His-686 and His-690 in domain IV were demonstrated to form a zinc binding site constituting a thermolysin-like zinc metalloprotease motif, HEXXH (18). The activities of the remaining 10 histidine residues in domains II, III, and IV have not been explored yet. In this study, we replaced these 10 histidine residues separately with alanine residues by site-directed mutagenesis. By the cytotoxicity assay of all these mutants, the H669A mutant was found to lose cell toxicity completely. Further assay revealed that residue His-669 was involved in neither zinc stabilization nor PA binding but participated in the substrate proteolytic activity of LF.     

Induction of the high-affinity nerve growth factor receptor on embryonic chicken sensory nerve cells by elevated potassium

Culture medium with elevated K⁺ has been shown to enhance the survival of neurons isolated from several different regions of the nervous system. Nerve growth factor binds to binding sites on sensory and sympathetic neurons through two sites, one of high-affinity (K _d13×10^–11 M) and the other of low-affinity (K _d22×10^–9 M). Equilibrium binding data generated on dissociated cells derived from E9 chicken embryo dorsal root ganglia, has shown that there is a two-fold increase in the number of high affinity (type I) receptors, with no effect on the affinity, when cells are incubated for 2 hours in buffer containing 59 mM K⁺. There does not appear to be a significant change in the affinity or the number of low-affinity binding sites. This two-fold increase in type I receptors is dependent on temperature, Ca²⁺, and active protein synthesis. There does not appear to be an intracellular pool of the type I receptor sufficient to account for this increase. The induction is not observed on sensory nerve cells cultured in 59 mM K⁺ for 24 hours, either in the presence or absence of nerve growth factor. Additionally, the induction in the number of type I receptors requires that both nerve growth factor and K⁺ be present simultaneously. Taken in total, this data suggests that there may be a critical period in which the sensory neurons require nerve growth factor exposure to respond. Evidence is presented which indicates that nerve growth factor responsive cells are able to elicit neurites after an acute exposure to nerve growth factor of as little as 4 hours. Finally, there is an approximate two-fold decrease in the concentration of nerve growth factor needed to elicit maximal fiber outgrowth, consistent with the two-fold increase in the number of type I receptors.Abbreviations NGF nerve growth factor - 7S NGF the high molecular weight form of NGF - NGF the -subunit of 7S NGF - ¹²⁵I-NGF ¹²⁵I-labeled NGF - mNGF–_rAb polyclonal rabbit IgG raised against mouse NGF - DRG dorsal root ganglia - K_d the equilibrium dissociation constant - N the maximal number of binding sites for the ligand NGF - NGFR the biologically relevant receptor through which the neurite outgrowth and neuron survival are mediated - GBS Gey's balanced salts - HKGBS high K⁺ GBS - PBG phosphate buffered GBS - HKPBG high K⁺ PBG - CFHKPBG Ca⁺² free high K⁺ PBG - PBG-cyt c PBG containing 2 mg/ml cytochrome c - HKPBG-cyt c HKPBG containing 2 mg/ml cytochrome c - AbU antibody unit - BU biological unit PBS, phosphate buffered saline - HKPBS high K⁺ PBS Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Improvement of Sciatic Nerve Regeneration Using Laminin-Binding Human NGF-β

Background

Sciatic nerve injuries often cause partial or total loss of motor, sensory and autonomic functions due to the axon discontinuity, degeneration, and eventual death which finally result in substantial functional loss and decreased quality of life. Nerve growth factor (NGF) plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical applications. We reported here by fusing with the N-terminal domain of agrin (NtA), NGF-β could target to nerve cells and improve nerve regeneration.

Methods

Laminin-binding assay and sustained release assay of NGF-β fused with NtA (LBD-NGF) from laminin in vitro were carried out. The bioactivity of LBD-NGF on laminin in vitro was also measured. Using the rat sciatic nerve crush injury model, the nerve repair and functional restoration by utilizing LBD-NGF were tested.

Findings

LBD-NGF could specifically bind to laminin and maintain NGF activity both in vitro and in vivo. In the rat sciatic nerve crush injury model, we found that LBD-NGF could be retained and concentrated at the nerve injury sites to promote nerve repair and enhance functional restoration following nerve damages.

Conclusion

Fused with NtA, NGF-β could bind to laminin specifically. Since laminin is the major component of nerve extracellular matrix, laminin binding NGF could target to nerve cells and improve the repair of peripheral nerve injuries.     

Tyrosine kinase activity of nerve growth factor and estrogen in embryonic septal neurons cultured from the rat

Alzheimer's disease (AD) is a neurodegenerative disease characterized by dementia, senile plaques, fibrillary tangles, and a reduction of cholinergic neurons in the septal nucleus of the brain. Nerve growth factor (NGF) and estrogen were studied to observe effects on tyrosine kinase activity in septal neurons. The time course of tyrosine kinase activation and number of cells in which tyrosine kinase was activated were measured. Tissue from embryonic day 16 rats was microdissected and the septal neurons obtained were treated with estrogen (10 M) or NGF (100 ng/mL) at intervals of 1, 2, 3, 4, 5, or 10 min. Immunostaining for phosphotyrosine revealed that cells treated with NGF showed an increase in phosphotyrosine activity within 2-4 min followed by a decline to control levels of enzyme activity. Treatment with estrogen led to an increase in phosphotyrosine immunostaining within 2-3 min followed by a decline to control levels. This time course suggests a mechanism for estrogen activity other than the traditional method involving binding to nuclear receptors followed by protein synthesis.     

Effect of coexisting cells on the NGF-inducing neurite growth from PC12h-R

Possible roles of coexisting cells in inducing neurite growth from a nerve cell were studied. Nerve growth factor (NGF)-inducing neurite growth from PC12h-R (a cell line derived from cultured nerve cells) was investigated at various cell densities. At the cell density 10²10⁴ cells/ml neurites appeared even without NGF. In contrast, no neurite appeared without NGF in single cell culture. The neurite growth observed in plural cell culture without NGF was only partially inhibited by antibody to NGF receptor (Ab-NGFR). However, the effect of the used medium alone was mostly inhibited by Ab-NGFR. These results suggest that the neurite inducing potency of coexisting cells is via different sites than the NGF receptor.Abbreviations Ab-IgG-FITC anti-mouse-IgG labeled with fluorescein isothiocyanate - Ab-NF monoclonal antibody to neurofilament 160 kD - Ab-NGFR monoclonal antibody to NGF receptor - BDNF brain-derived neurotrophic factor - D-medium medium for differentiation culture - DMEM Dulbecco's modified Eagle's medium - M-medium medium for multiplication culture - NGF nerve growth factor - NGFR NGF receptor - NT-3 neurotrophin-3 - PC12 pheochromocytoma cell line - PC12h-R subclone of PC12 - Sup-D supernatant of D-medium     

Improved procedure for the purification of an iodinated protein: Beta nerve growth factor

An improved procedure for the isolation of iodinated Nerve Growth Factor (¹²⁵I-NGF) has been devised. Use of Centricon microconcentrators (Amicon) has allowed the facile and efficient recovery of ultrapure¹²⁵I-NGF in high yields. Centricon microconcentrators are supplied with two molecular weight cutoffs of 10 K and 30 K daltons. NGF is a basic protein with a molecular weight of 26 K daltons. It is therefore possible to filter the¹²⁵I-NGF through the 30K filter (30 K Filtrate) leaving behing any aggregates or reactants greater than 30 K while the¹²⁵I-NGF can be retained and concentrated on the 10 K filter (10 K Retentate). Any free¹²⁵I is easily removed, passing through the 10 K filter and then being discarded. In this way¹²⁵I-NGF can be easily purified.     

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     

Nerve growth factor antibodies recognize neurotrophin-3

The immunological properties of the neurotrophins NGF, BDNF, and NT-3 were compared using polyclonal and monoclonal antibodies against the subunit of mouse NGF. Affinity-purified anti-NGF IgG consistently recognized NGF and NT-3 on Western blots, and inhibited the trophic activity of NGF and NT-3 but not BDNF. In contrast, anti-NGF monoclonal antibodies did not block the trophic activity of either NT-3 or BDNF. These results are consistent with the greater structural overlap between NGF and NT-3 than between NGF and BDNF.