Inhibition of biological activity of mouse beta-nerve growth factor by monoclonal antibody.

A continuous hybrid cell line was derived that secretes monoclonal antibody capable of inhibiting the biological activity of mouse beta-nerve growth factor (beta-NGF). Results obtained with monovalent fragments indicate that the monoclonal antibody inhibits activity by interfering with the direct interaction between beta-NGF and the cell membrane receptor rather than by precipitating the dimeric form of beta-NGF. This monoclonal antibody binds to an antigenic determinant common to mouse beta-NGF, snake venom (Naja naja) beta-NGF, and human beta-NGF. These antibodies should provide specific molecular probes for a variety of studies of nerve growth factor including its tissue distribution and mechanism of action.

A common pentapeptide conformation occurs in viral acid proteases and other proteins.

We found a pentapeptide conformation, termed a type I twist, which has a strikingly high propensity (56%) for aspartic acid in the first position. Type I twists include the active site loops from cellular and viral aspartic proteases, with the catalytic Asp in the first position. Fifteen other type I twists, from non-homologous proteins, were found among high-resolution structures in the Protein Data Bank using a comparison method based on main-chain torsion angles. We propose that the Asp affects electrostatic interactions and thus plays a major structural role in the formation of this recurring motif, in addition to its catalytic role in the aspartic proteases.

Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation.

The conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2-state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, delta Gu (H2O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing delta Gu (H2O) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed.

Equilibrium denaturation studies of mouse beta-nerve growth factor.

Equilibrium denaturation of dimeric mouse beta-nerve growth factor (beta-NGF) has been studied by monitoring changes in the protein's spectroscopic characteristics. Denaturation of beta-NGF in guanidine hydrochloride and urea resulted in an altered intrinsic fluorescence emission spectrum, fluorescence depolarization, and diminished negative circular dichroism. Native-like spectroscopic properties and specific biological activity are restored when denaturant is diluted from unfolded samples, demonstrating that this process is fully reversible. However, refolding of denatured beta-NGF is dependent on the three disulfide bonds present in the native protein and does not readily occur when the disulfide bonds are reduced. Graphical analysis and nonlinear least-squares fitting of beta-NGF denaturation data demonstrate that denaturation is dependent on the concentration of beta-NGF and is consistent with a two-state model involving native dimer and denatured monomer (N2 = 2D). The conformational stability of mouse beta-NGF calculated according to this model is 19.3 +/- 1.1 kcal/mol in 100 mM sodium phosphate at pH 7. Increasing the hydrogen ion concentration resulted in a 25% decrease in beta-NGF stability at pH 4 relative to pH 7.

Characterization of the recombinant extracellular domain of the neurotrophin receptor TrkA and its interaction with nerve growth factor (NGF).

Nerve growth factor (NGF) is the prototype of a family of neurotrophins that support important neuronal programs such as differentiation and survival of a subset of sympathetic, sensory, and brain neurons. NGF binds to two classes of cell surface receptors: p75LANR and p140TrkA. NGF binding to p140TrkA initiates the neuronal signaling pathway through activation of the tyrosine kinase activity, which subsequently results in a rapid signal transduction through a phosphorylation cascade. To examine this crucial signaling step in more detail, the TrkA extracellular domain polypeptide (TrkA-RED) was overexpressed in Sf21 insect cells and purified to homogeneity. The recombinant TrkA-RED is a 70 kDa acidic glycoprotein with a pI of 5.1, and mimics the intact TrkA receptor for NGF binding with a dissociation constant, Kd, of 2.9 nM. Thus, the recombinant TrkA-RED is functionally competent and can be used to elucidate the interaction of NGF and TrkA receptor. Circular dichroism difference spectra indicated that, upon association of NGF with TrkA-RED, a minor conformational change occurred to form a complex with decreased ordered secondary structure. Interaction between NGF and TrkA-RED was also demonstrated by size exclusion chromatography, light scattering, and chemical crosslinking with evidence for formation of a higher molecular weight complex consistent with a (TrkA-RED)2-(NGF dimer) complex. Association and dissociation rates of 5.6 x 10(5) M(-1) s(-1) and 1.6 x 10(-3) s(-1), respectively, were determined by biosensor technology. Thus, initiation of signaling may stem from NGF-induced receptor dimerization concomitant with a small conformational change.

Spectroscopic and chemical studies of the interaction between nerve growth factor (NGF) and the extracellular domain of the low affinity NGF receptor.

Nerve growth factor (NGF) interacts with a cell surface receptor on responsive neurons to initiate a series of cellular events leading to neuronal survival and/or differentiation. The first step in this process is the binding of NGF to a low affinity and/or a high affinity receptor. In the present report, we have studied the conformation and stability of recombinant receptor extracellular domain (RED) from the human low affinity receptor and the structural basis of its interaction with NGF. Circular dichroism (CD) studies indicate that the RED is primarily random coil in nature with little regular secondary structure. Thermal stability studies have shown that this irregular conformation is a specific structure that can undergo a reversible two-state thermal denaturation with a concomitant fluorescent and CD change. During heating at 100 degrees C for 15 min, the structure of RED is sufficiently unfolded for a reducing agent, dithiothreitol, to inactivate the receptor toward NGF binding and cross-linking. The complex formation between the RED and NGF has been examined by differential CD measurements, and we have shown that a small, reproducible change in conformation occurs in RED or NGF upon interaction. These results are interpreted in terms of the initiation of NGF cell surface binding and possible modes of signal transduction.

Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low-affinity neurotrophin receptor.

Interactions between the purified recombinant receptor extracellular domain (RED) of the human low-affinity neurotrophin receptor (LANR) and recombinant human brain-derived neurotrophic factor, neurotrophin-3 (NT-3) and neuotrophin-4/5 have been studied by chemical crosslinking and circular dichroism. Conformational changes subsequent to binding have been shown by these procedures. First, relative affinities of the neurotrophins for RED were determined by binding competition assays in which radioiodinated nerve growth factor (NGF) from mouse submaxillary gland was crosslinked to RED in the presence of varying amounts of unlabeled neurotrophin competitors. RED bound each of the 3 recombinant human neurotrophins with affinities that were indistinguishable from authentic mouse NGF. These results are the first measurement of binding of the neurotrophin family to their common receptor using purified components. In order to study the effect of binding on the conformation of the proteins, CD measurements were made before and after mixing neurotrophins and RED, as had previously been done with NGF and RED (Timm DE, Vissavajjhala P, Ross AH, Neet KE, 1992, Protein Sci 1:1023-1031). Similar changes in CD spectra occurred upon combination of each of the neurotrophins and RED, with negative changes near 220-225 nm and positive changes near 190-200 nm; however, significant differences existed among the various neurotrophin-RED difference spectra. The NT-3/RED complex showed the largest spectral change and NGF the smallest. Thus, specific conformational changes in secondary structure of neurotrophin, RED, or both accompany the binding of each neurotrophin to the extracellular domain of the LANR.

Differences in the amino acid distributions of 3(10)-helices and alpha-helices.

Local determinants of 3(10)-helix stabilization have been ascertained from the analysis of the crystal structure data base. We have clustered all 5-length substructures from 51 nonhomologous proteins into classes based on the conformational similarity of their backbone dihedral angles. Several clusters, derived from 3(10)-helices and multiple-turn conformations, had strong amino acid sequence patterns not evident among alpha-helices. Aspartate occurred over twice as frequently in the N-cap position of 3(10)-helices as in the N-cap position of alpha-helices. Unlike alpha-helices, 3(10)-helices had few C-termini ending in a left-handed alpha conformation; most 3(10) C-caps adopted an extended conformation. Differences in the distribution of hydrophobic residues among 3(10)- and alpha-helices were also apparent, producing amphipathic 3(10)-helices. Local interactions that stabilize 3(10)-helices can be inferred both from the strong amino acid preferences found for these short helices, as well as from the existence of substructures in which tertiary interactions replace consensus local interactions. Because the folding and unfolding of alpha-helices have been postulated to proceed through reverse-turn and 3(10)-helix intermediates, sequence differences between 3(10)- and alpha-helices can also lend insight into factors influencing alpha-helix initiation and propagation.

Mutational studies of conserved residues in the dimer interface of nerve growth factor.

An understanding of the structure-function relationship of nerve growth factor (NGF) requires precise knowledge of all the residues and regions that participate in NGF receptor binding, receptor activation, and biological activity. Seven recombinant human NGF mutants having alanine substituted for residues located either in the NGF dimer interface or beta-strand region were studied to determine the role of each amino acid residue in NGF biological activity. F86A, T91A, R100A, and R103A remained nearly full active with 61, 120, 91, and 73% of wild-type activity, respectively, in the PC12 cell bioassay. Hydrophobic core and dimer interface residues Y52, F53, and F54 were studied in more detail. Y52A and F54A were expressed in very low levels, suggesting that these two residues may be important for protein stability. Y52A retained full biological activity (91%). F53A had a 20- and 70-fold reduction in biological activity and TrkA phosphorylation, respectively, with only a 5- to 10-fold effect on TrkA binding and no effect on low-affinity receptor binding. F54A had significantly decreased TrkA phosphorylation and biological activity (40-fold). The results suggest that F53 and F54 may play a structural role in TrkA receptor activation subsequent to binding.

Expression of the cDNA for mouse beta-nerve growth factor protein in Escherichia coli.

The cDNA coding for the mature beta-nerve growth factor (beta-NGF) has been cloned into a plasmid expression vector, pAS1, and expressed in Escherichia coli. The cDNA fragment in pAS1 is under the control of strong phage transcriptional and translational initiation elements that provide for regulated expression of cloned genes in E. coli. The protein, produced in bacteria at a level of about 0.0005-0.1% of cell protein, was purified by ammonium sulfate precipitation and ion exchange chromatography. The recombinant NGF was biologically active in the PC12 neurite outgrowth assay, and formed a band at Mr of about 11,000 to 12,000, when electrophoresed on sodium dodecyl sulfate-polyacrylamide gel and Western-blotted.

Nerve growth factor and the concept of neural-epithelial interactions. Immunohistochemical observations in two cases of vasitis nodosa and six cases of prostatic adenocarcinoma.

The so-called perineural invasion is a well-recognized, but poorly understood, phenomenon occurring in vasitis nodosa, a benign epithelial proliferation of the vas deferens; such unusually close epithelial-neural interactions also occur, among others, in benign and malignant conditions of the prostate. In the present study, immunoreactive nerve growth factor was found in the epithelium of these organs. This nerve growth factor is thought to possibly play a role in the process of the so-called perineural invasion.

Nerve growth factor in human amniotic and cerebrospinal fluid.

Nerve growth factor (NGF) is a polypeptide hormone involved in development of the sympathetic and central nervous systems. The detection and measurement of NGF in clinical samples would be useful in evaluating its role in various disease states. In this report, NGF activity and protein levels have been investigated in human amniotic fluid and cerebrospinal fluid samples. In amniotic fluid, NGF activity was found at levels ranging from less than 10 pM to nanomolar. The activity in all samples was blocked by polyclonal and monoclonal antibodies to mouse NGF. The finding of NGF in clinically obtainable samples raises the possibility of correlating NGF levels with a variety of disorders in which changes in NGF levels or activity have been implicated.