Robert Rosenfeld

Expression of brain-derived neurotrophic factor protein in the adult rat central nervous system

We have generated and characterized a multi-functional polyclonal anti-brain-derived neurotrophic factor antibody. Western blot analysis, dorsal root ganglion neurite outgrowth and dorsal root ganglion neuron survival assays showed that this antibody specifically recognized brain-derived neurotrophic factor and not the other neurotrophins. Furthermore, it was capable of blocking the functional effects of brain-derived neurotrophic factor. Using this antibody, we examined the expression of brain-derived neurotrophic factor in adult rat brains by immunohistochemistry. We found distinct brain-derived neurotrophic factor immunoreactivity in several structures of the brain. These included the neocortex, piriform cortex, amygdaloid complex, hippocampal formation, claustrum, some thalamic and hypothalamic nuclei, the substantia nigra and some brainstem structures. In contrast to brain-derived neurotrophic factor messenger RNA expression, brain-derived neurotrophic factor immunoreactivity was also found in the lateral septum, bed nucleus of the stria teminalis, medial preoptic nucleus, olivery pretectal nucleus, lateral paragigantocellular nucleus and the dorsal horn of the spinal cord. In normal adult rat brains, there was little or no staining in the CA1 region or the granule cell layer of the dentate gyrus of the hippocampus. However, kainate treatments greatly increased brain-derived neurotrophic factor immunoreactivity in the pyramidal cells of the CA1 region, as well as in the dentate gyrus, CA2 and CA3 hippocampal regions. We present evidence for both the subcellular localization and anterograde transport of endogenous brain-derived neurotrophic factor in the central nervous system. The detection of brain-derived neurotrophic factor protein in several discrete regions of the adult brain, and brain-derived neurotrophic factors dramatic up-regulation following kainate treatment, strongly supports a role of brain-derived neurotrophic factor in the maintenance of adult neurons and synapses. Since several populations of neurons lost during neurodegenerative diseases synthesize brain-derived neurotrophic factor protein, modulation of brain-derived neurotrophic factor levels may be clinically beneficial. The antibody described in this paper will be helpful in determining more precisely the functional activities of brain-derived neurotrophic factor in the adult.

Structure and Stability Changes of Human IgG1 Fc as a Consequence of Methionine Oxidation

The Fc region has two highly conserved methionine residues, Met 33 (C(H)3 domain) and Met 209 (C(H)3 domain), which are important for the Fcs structure and biological function. To understand the effect of methionine oxidation on the structure and stability of the human IgG1 Fc expressed in Escherichia coli, we have characterized the fully oxidized Fc using biophysical (DSC, CD, and NMR) and bioanalytical (SEC and RP-HPLC-MS) methods. Methionine oxidation resulted in a detectable secondary and tertiary structural alteration measured by circular dichroism. This is further supported by the NMR data. The HSQC spectral changes indicate the structures of both C(H)2 and C(H)3 domains are affected by methionine oxidation. The melting temperature (Tm) of the C(H)2 domain of the human IgG1 Fc was significantly reduced upon methionine oxidation, while the melting temperature of the C(H)3 domain was only affected slightly. The change in the C(H)2 domain T m depended on the extent of oxidation of both Met 33 and Met 209. This was confirmed by DSC analysis of methionine-oxidized samples of two site specific methionine mutants. When incubated at 45 degrees C, the oxidized Fc exhibited an increased aggregation rate. In addition, the oxidized Fc displayed an increased deamidation (at pH 7.4) rate at the Asn 67 and Asn 96 sites, both located on the C(H)2 domain, while the deamidation rates of the other residues were not affected. The methionine oxidation resulted in changes in the structure and stability of the Fc, which are primarily localized to the C(H)2 domain. These changes can impact the Fcs physical and covalent stability and potentially its biological functions; therefore, it is critical to monitor and control methionine oxidation during manufacturing and storage of protein therapeutics.

A novel chordin-like BMP inhibitor, CHL2, expressed preferentially in chondrocytes of developing cartilage and osteoarthritic joint cartilage

We have identified a novel chordin-like protein, CHL2, which is structurally most homologous to CHL/neuralin/ventroptin. When injected into Xenopus embryos, CHL2 RNA induced a secondary axis. Recombinant CHL2 protein interacted directly with BMPs in a competitive manner to prevent binding to the type I BMP receptor ectodomain, and inhibited BMP-dependent induction of alkaline phosphatase in C2C12 cells. Thus, CHL2 behaves as a secreted BMP-binding inhibitor. In situ hybridization revealed that CHL2 expression is restricted to chondrocytes of various developing joint cartilage surfaces and connective tissues in reproductive organs. Adult mesenchymal progenitor cells expressed CHL2, and its levels decreased during chondrogenic differentiation. Addition of CHL2 protein to a chondrogenic culture system reduced cartilage matrix deposition. Consistently, CHL2 transcripts were weakly detected in normal adult joint cartilage. However, CHL2 expression was upregulated in middle zone chondrocytes in osteoarthritic joint cartilage (where hypertrophic markers are induced). CHL2 depressed chondrocyte mineralization when added during the hypertrophic differentiation of cultured hyaline cartilage particles. Thus, CHL2 may play negative roles in the (re)generation and maturation of articular chondrocytes in the hyaline cartilage of both developing and degenerated joints.

Yeast-derived recombinant human insulin-like growth factor I: production, purification, and structural characterization.

Recombinant human insulin-like growth factor I (IGF-I) is efficiently expressed and secreted fromSaccharomyces cerevisiae using a yeast α-factor leader to direct secretion. However, approximately 10–20% of the IGF-I was in a monomeric form, the remaining materials being disulfide-linked aggregates. When the purified material was subjected to reverse-phase high-performance liquid chromatography (rp-HPLC), it gave two doublet peaks, I and II. Upon reduction, doublet peaks I and II converged to one doublet peak. This suggests that peaks I and II result from different disulfide structures, and the doublet feature of each peak results from other causes. Different disulfide structures between peaks I and II were also suggested from the near UV circular dichroism of these proteins. Only the peak II was biologically active, indicating that peak II has the correct disulfide structure. Concanavalin A affinity chromatography of the purified peak II doublet showed binding of the subpeak with an earlier rp-HPLC retention time, indicating that it was glycosylated. Sequence analysis of tryptic peptides suggested that Thr29 was the site of glycosylation. Site-directed mutagenesis was used to convert Thr29 to Asn29. This substitution reduced, but did not eliminate IGF-I glycosylation, suggesting additional glycosylation sites. The site of carbohydrate addition was consistent with the model that O-glycosylations occur on hydroxyl amino acids near proline residues in β-turns.

The candidate neuroprotective agent artemin induces autonomic neural dysplasia without preventing peripheral nerve dysfunction.

Artemin (ART) signals through the GFRα—3/RET receptor complex to support sympathetic neuron development. Here we show that ART also influences autonomic elements in adrenal medulla and enteric and pelvic ganglia. Transgenic mice over-expressing Art throughout development exhibited systemic autonomic neural lesions including fusion of adrenal medullae with adjacent paraganglia, adrenal medullary dysplasia, and marked enlargement of sympathetic (superior cervical and sympathetic chain ganglia) and parasympathetic (enteric, pelvic) ganglia. Changes began by gestational day 12.5 and formed progressively larger masses during adulthood. Art supplementation in wild type adult mice by administering recombinant protein or an Art-bearing retroviral vector resulted in hyperplasia or neuronal metaplasia at the adrenal corticomedullary junction. Expression data revealed that Gfrα—3 is expressed during development in the adrenal medulla, sensory and autonomic ganglia and their projections, while Art is found in contiguous mesenchymal domains (especially skeleton) and in certain nerves. Intrathecal Art therapy did not reduce hypalgesia in rats following nerve ligation. These data (1) confirm that ART acts as a differentiation factor for autonomic (chiefly sympathoadrenal but also parasympathetic) neurons, (2) suggest a role for ART overexpression in the genesis of pheochromocytomas and paragangliomas, and (3) indicate that ART is not a suitable therapy for peripheral neuropathy.

Conformational changes of brain-derived neurotrophic factor during reversed-phase high-performance liquid chromatography

Recombinant human brain-derived neurotrophic factor (r-HuBDNF) is eluted as two peaks under reversed-phase liquid chromatographic conditions with gradient elution. Sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed identical molecular weights in the two peaks, while rechromatography of the separated peaks showed interconvertibility. The two peaks are identified as the monomeric forms of the parent molecules. The molecular weight of the components in the peaks was determined by on-line 90 degree light scattering using a fluorescence detector as a scatterograph. The early eluted peak is a folded form of the r-HuBDNF monomer, while the later eluted peak is an unfolded form of the BDNF monomer. The conformational states were established using a fluorescence detector both at a fixed wavelength and in the scanning mode.

Mutation of Arg55/56 to Leu55/Ala56 in insulin-like growth factor-I results in two forms different in disulfide structure and native conformation but similar under reverse-phase conditions

Folding of recombinant human insulin-like growth factor-I (IGF-I) results in two distinct species as resolved by reversed-phase high-performance liquid chromatography (RP-HPLC). The earlier eluting peak (PI) has a nonnative disulfide structure, while the later eluting peak (PII) assumes the native disulfide structure. This folding problem causes a lower yield and requires expensive RP-HPLC separation. In contrast, IGF-II folds mainly into a single form with all three disulfide bonds correctly formed. Sequence comparison of the two molecules revealed that IGF-I has arginine at residues 55 and 56, while IGF-II has alanine and leucine, respectively, at these positions. Two analogs of IGF-I, IGF-I (Ala55/Leu56) and IGF-I (Leu56), behave similarly to IGF-II upon refolding and RP-HPLC; that is, a single peak eluted from the RP-HPLC column. However, when the peaks isolated by RP-HPLC were subjected to hydrophobic interaction chromatography, circular dichroism, and peptide mapping, they were found to be a mixture of PI and PII. It was then concluded that factors other than just these two residues contribute to correct folding of IGF-II and that the PI and PII of the above two IGF-I mutants assume different conformation at neutralpH but similar conformation under the RP-HPLC condition.

Human neurotrophin-3: a one-step peptide mapping method and complete disulfide characterization of the recombinant protein.

Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an intial denaturation of NT-3 in 6 M guanidinium chloride (pH 6) for 2 hr at 37°C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl2 at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys14 to Cys79, Cys57 to Cys108, and Cys67 to Cys110. This disulfide structure is homologous to the NGF family of neurotrophic factors.