Metal-thiolate clusters in neuronal growth inhibitory factor (GIF)

Human neuronal growth inhibitory factor (GIF) is a metallothionein-like protein specific to the central nervous system, which has been linked to Alzheimer's disease. In this article a short overview of the biological and structural properties of native Cu4,Zn3-GIF are described. Moreover, metal-thiolate clusters formed in the synthetic beta-domain (residues 1-32) and the alpha-domain (residues 32-68) both with native CuI and ZnII, and as a spectroscopic probe also with Cd(II) are discussed. The cluster formation was followed by electronic absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and 113Cd NMR spectroscopy and, in the special case of Cu(I) complexes, by luminescence spectroscopy at 77 K. These structural features are compared with those of recombinant Zn7- and 113Cd7-GIF. The structural studies suggest the existence of distinct MeII4S11 and MeII3S9 clusters located in the mutually interacting alpha- and beta-domains, respectively, of Cd7-GIF. In addition, evidence for a highly dynamic and flexible structure of this protein is presented.     

Incorporation of a glycine within the conserved TCPCP motif of human neuronal growth inhibitory factor significantly reduces its bioactivity

It has been reported that the ⁶CPCP⁹ motif near the N-terminus is pivotal to the inhibitory activity of human neuronal growth inhibitory factor (hGIF). In order to better understand the biological significance of this region on the structure, property and function of hGIF, we introduced a highly flexible residue, Gly, either in front of the ⁶CPCP⁹ motif (the IG6 mutant, TGCPCP) or in the middle of it (the IG8 mutant, TCPGCP) and investigated their structural and metal binding properties in detail. The results showed that the overall structure and the stability of the metal-thiolate clusters of the two mutants were comparable to that of hGIF. However, the bioassay results showed that the bioactivity of the IG6 mutant decreased significantly, while the bioactivity of the IG8 mutant was almost abolished. Molecular dynamics simulation results showed that the backbone of the IG6 mutant exhibited high similarity to that of hGIF, and the two prolines could still induce structural constraints on the ⁶CPCP⁹ tetrapeptide and form a similar conformation with that of hGIF, however, the conformation of the first five amino acid residues in the N-terminus was quite different. In hGIF, the five residues are twisted and form a restricted conformation, while in the IG6 mutant this peptide extends more naturally and smoothly, which is similar to that of MT2. As to the IG8 mutant, the Gly insertion broke the ⁶CPCP⁹ motif, thus probably abolishing the interactions with other molecules and eliminating its inhibitory activity. Based on these results, we suggested that although the structure adopted by the ⁶CPCP⁹ motif is the determinant factor of the inhibitory bioactivity of hGIF, other residues within the N-terminal fragment (residue 1-13) may also influence the peptide conformation and contribute to the protein’s bioactivity.     

Important roles of the conserved linker-KKS in human neuronal growth inhibitory factor

Metallothinein-3 (MT3), also named neuronal growth inhibitory factor (GIF), is attractive by its distinct neuronal growth inhibitory activity, which is not shared by other MT isoforms. The polypeptide chain of GIF is folded into two individual domains, which are connected by a highly conserved linker, KKS. In order to figure out the significance of the conserved segment, we constructed several mutants of human GIF (hGIF), including the K31/32A mutant, the K31/32E mutant and the KKS-SP mutant by site-directed mutagenesis. pH titration and DTNB reaction exhibited that all the three mutations made the β-domain lower in stability and looser. More significantly, change of KKS to SP also altered the general backbone conformation and metal–thiolate cluster geometry. Notably, bioassay results showed that the bioactivity of the K31/32A mutant and the K31/32E mutant decreased obviously, while the KKS-SP mutant lost inhibitory activity completely. Based on these results, we proposed that the KKS linker was a crucial factor in modulating the stability and the solvent accessibility of the Cd₃S₉ cluster in the β-domain through domain–domain interactions, thus was indispensable to the biological activity of hGIF.     

Crystal structure of human epidermal growth factor and its dimerization.

Epidermal growth factor (EGF) is a typical growth-stimulating peptide and functions by binding to specific cell-surface receptors and inducing dimerization of the receptors. Little is known about the molecular mechanism of EGF-induced dimerization of EGF receptors. The crystal structure of human EGF has been determined at pH 8.1. There are two human EGF molecules A and B in the asymmetric unit of the crystals, which form a potential dimer. Importantly, a number of residues known to be indispensable for EGF binding to its receptor are involved in the interface between the two EGF molecules, suggesting a crucial role of EGF dimerization in the EGF-induced dimerization of receptors. In addition, the crystal structure of EGF shares the main features of the NMR structure of mouse EGF determined at pH 2.0, but structural comparisons between different models have revealed new detailed features and properties of the EGF structure.     

Study on structure-property-reactivity-function relationship of human neuronal growth inhibitory factor (hGIF)

Human metallothionein-3 (hMT3), also named as human neuronal growth inhibitory factor (hGIF), can inhibit the outgrowth of embryonic cortical neurons in the presence of brain extracts. In order to systematically study the structure-property-reactivity-function relationship of hGIF, our laboratory designed a series of mutants and studied their structure, property, reactivity and functions by a series of chemical and biological tools including UV spectroscopy, CD spectroscopy, NMR, chemical reaction and primary neuronal culture assays. In summary, we concluded that the bioactivity of hGIF was regulated by multiple factors, including the ⁶CPCP⁹ motif, an additional threonine insert at sequence position 5, domain-domain interactions, the structure and stability of the metal-thiolate cluster and the linker. Our studies provide more and more evidences which revealed that the bioactivity of hGIF is mainly related to the essential metal release and its characteristic conformation.     

Evidence for a surface receptor for human migration inhibitory factor on a B-lymphoid cell line.

Reception by PGLC-33H target cells for the migration inhibitory factor (MIF) produced by this established line has been investigated by pulse time and temperature dependence, MIF absorption, and abrogation by trypsinization. PGLC-33H supernatants containing MIF were concentrated 5× with Carbowax and dialyzed against serum free RPMI-1640 before use. Prior to standard capillary migration assay a minimum 30 min pulse of MIF at 37 °C is required for significant migration inhibition (MI > 20%). No significant MI is observed when cells are pulsed at 4 °C for up to 2 hr. Preincubation with PGLC-33H for 1 hr at 37 °C reduces activity of supernatants from 38 to 13% MI; at 4 °C to 27% MI. Trypsinization of target cells for 30 min at 25 °C abrogates response to MIF (43 to ?14% MI). Trypsinized cells did not reduce activity of supernatants. MIF activity is abolished (32 to 3% MI) in samples preincubated with supernatants of the trypsinized cells inactivated with serum. These data suggest that cells from the human B-lymphoid cell line PGLC-33H have a surface receptor for human MIF.     

Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three-dimensional structure. Since all chimeras retained full biological activity and receptor-binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF-R were unaltered by inter-species domain swapping. Consequently, the site on the hLIF molecule that confers species-specific binding to the hLIF-R and higher affinity binding to the mLIF-R, must constitute an additional interaction site to that used by both mLIF and hLIF to bind to the mLIF-R. These studies define a maximum of 15 amino acid differences between hLIF and mLIF that are responsible for the different properties of these proteins.     

Molecular cloning of human growth inhibitory factor cDNA and its down-regulation in Alzheimer''s disease.

In previous studies, we discovered a growth inhibitory factor (GIF) that was abundant in normal human brain, but greatly reduced in Alzheimer's disease (AD) brain. Molecular cloning of a full-length cDNA for human GIF revealed that the GIF had striking homology to metallothioneins. Furthermore, it was determined that the GIF gene was on chromosome 16, as are the metallothionein genes. GIF, in contrast to metallothioneins, was found to be expressed exclusively in the nervous system. The GIF protein produced by Escherichia coli harboring the GIF cDNA in a prokaryotic expression vector inhibited the growth of neonatal rat cortical neurons. These results indicate that GIF is a new member of the metallothionein family with distinct tissue-specific expression and functions. Northern blot analysis revealed that expression of the GIF mRNA is drastically decreased in AD brains. The result raises the possibility that down-regulation of the GIF gene in AD brain plays an important role in the pathogenesis of AD.     

The structure of human acidic fibroblast growth factor and its interaction with heparin.

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo.     

Subunit structure of a high-affinity receptor for type beta-transforming growth factor. Evidence for a disulfide-linked glycosylated receptor complex

The structure of high-affinity receptors for type beta-transforming growth factor (beta TGF) has been examined by affinity labeling with 125I-beta TGF and disuccinimidyl suberate. The major receptor component labeled by 125I-beta TGF in mouse, rat, and chick fibroblasts migrated as a 280-290-kilodalton species on dodecyl sulfate-polyacrylamide electrophoresis gels in the presence of reductant, dithiothreitol. A larger (330-kilodalton) species was labeled in human fibroblasts, but comparative peptide mapping indicated a close structural relationship with receptors from mouse fibroblasts. In the absence of reductant, the affinity-labeled beta TGF receptor migrated in the gels as a larger disulfide-linked complex. The molecular mass calculated from the hydrodynamic properties of native nonreduced beta TGF receptors was 565 (mouse) or 615 kilodaltons (human). Other molecular parameters for the beta TGF receptor were: Stokes radius, 8.3-8.5 nm; sedimentation coefficient, 12.7-13.0 S; and frictional ratio, f/f0 = 1.4. The beta TGF receptor was solubilized under conditions in which the structural and ligand-binding properties of the native state were retained. beta TGF receptors solubilized from human, mouse, and chick cells interacted specifically with immobilized wheat germ agglutinin. These data suggest that the high affinity receptor for beta TGF in human, rodent, and avian fibroblasts is a disulfide-linked glycosylated 565-615-kilodalton complex with a 280-330-kilodalton subunit that contains the ligand-binding site. The oligomeric structure of the beta TGF receptor does not appear to be induced by receptor occupancy with the ligand.     

Crystal structure of human platelet-derived growth factor BB.

The crystal structure of the homodimeric BB isoform of human recombinant platelet-derived growth factor (PDGF-BB) has been determined by X-ray analysis to 3.0 A resolution. The polypeptide chain is folded into two highly twisted antiparallel pairs of beta-strands and contains an unusual knotted arrangement of three intramolecular disulfide bonds. Dimerization leads to the clustering of three surface loops at each end of the elongated dimer, which most probably form the receptor recognition sites.     

Three-dimensional structure and dynamics of a brain specific growth inhibitory factor: metallothionein-3

The brain specific member of the metallothionein (MT) family of proteins, metallothionein-3, inhibits the growth and survival of neurons, in contrast to the ubiquitous mammalian MT isoforms, MT-1 and MT-2, that are found in most tissues and are thought to function in metal ion homeostasis and detoxification. Solution NMR was utilized to determine the structural and dynamic differences of MT-3 from MT-1 and 2. The high-resolution solution structure of the C-terminal alpha-domain of recombinant mouse MT-3 revealed a tertiary fold very similar to MT-1 and 2, except for a loop that accommodates an acidic insertion relative to these isoforms. This loop was distinguished from the rest of the domain by dynamics of the backbone on the nano- to picosecond time-scale shown by (15)N relaxation studies and was identified as a possible interaction site with other proteins. The N-terminal beta-domain contains the region responsible for the growth inhibitory activity, a CPCP tetrapeptide close to the N-terminus. Because of exchange broadening of a large number of the NMR signals from this domain, homology modeling was utilized to calculate models for the beta-domain and suggested that while the backbone fold of the MT-3 beta-domain is identical to MT-1 and 2, the second proline responsible for the activity, Pro9, may show structural heterogeneity. (15)N relaxation analyses implied fast internal motions for the beta-domain. On the basis of these observations, we conclude that the growth inhibitory activity exhibited by MT-3 is a result of a combination of local structural differences and global dynamics in the beta-domain.     

Expression of neuronal growth inhibitory factor (metallothionein-III) in the salivary gland

Metallothioneins (MTs) are metal-binding proteins that have been regarded as intrinsic factors for protecting cells and tissues from metal toxicity and oxidants. Among the three major classes of MTs, MT-III is different from other MTs because it has neuronal inhibitory activity and is only expressed in the central nervous system. Recent studies, however, have confirmed that MT-III is also expressed in organs other than the brain. These findings not only indicate that MT-III has a much wider tissue distribution than was originally thought, but also suggest that it might have other unknown activities. In the present study, we examined the human salivary and thyroid glands and demonstrated that the MT-III gene is also expressed in the salivary but not in the thyroid gland. While salivary ducts showed intense immuno-reactivity with anti-MT-III, weak immunoreactivity was observed in acinar cells. This, together with the findings that some neuromodulators (i.e. nerve growth factor, etc.) exist in the salivary gland and that MT-III may participate in the transport in renal tubules, suggest that MT-III may have other functions than cytoprotection in the salivary gland.     

Platelet-derived growth factor potentiates phorbol ester-induced neuronal differentiation of human neuroblastoma cells.

Previous studies have shown that platelet-derived growth factor (PDGF) and PDGF receptors are expressed in the mammalian central nervous system and that primary cultured neuroblasts from rat hindbrain have functional PDGF beta-receptors. Here, it is shown that cultured human neuroblastoma cells express PDGF alpha- and beta-receptors, but not PDGF-A and PDGF-B chain mRNA. In contrast to alpha-receptor expression, beta-receptor expression appears to be associated with a mature neuronal phenotype. Under serum-free growth conditions, PDGF-AA and -BB induce a trophic and weak mitogenic response in SH-SY5Y neuroblastoma cells, showing that the PDGF receptors in these cells are functional. In combination with 12-O-tetradecanoylphorbol-13-acetate, all three PDGF isoforms induce sympathetic neuronal differentiation of the SH-SY5Y cells, as shown by morphology and by increased expression of the genes coding for growth-associated protein 43 and neuropeptide tyrosine, respectively. This indicates a potential role for PDGF in the development of sympathetic neurons in particular and of the nervous system in general.     

Human epidermal growth factor. High resolution solution structure and comparison with human transforming growth factor alpha.

The solution structure of the 53 amino acid peptide hormone, human epidermal growth factor (hEGF), has been determined to high resolution from nuclear magnetic resonance (n.m.r.) data. A large number of internuclear distance and dihedral restraints was obtained, including data from uniformly 15N-labelled hEGF. Dynamical simulated annealing methods using the program XPLOR were used for structure calculation. An improved protocol was developed combining efficient conformational searching at a reduced computational cost. The general fold of the calculated structures compared well with that of a derivative of the carboxy-terminally truncated hEGF determined previously. A group of 44 structures were calculated with no violations greater than 0.3 A and 3 degrees for distance and dihedral restraints, respectively. The average pairwise root mean square (r.m.s.) deviation of all backbone atoms for these structures was 2.25 A for all 53 residues, 0.92 A for the bulk of the protein, and 0.23 A for the functionally important carboxy-terminal domain. Two new helical segments containing highly conserved amino acids have been identified; one between cysteines 6 and 14 and a second at the end of the carboxy-terminal domain. New insight into the molecular architecture of the site of putative receptor binding was provided by comparing the structure of hEGF with its biologically equipotent analogue, human transforming growth factor alpha. This comparison revealed a close structural relationship between the two growth factors and provides an improved understanding of the structure/function relationships in EGF.     

Generation of inhibitory DNA aptamers against human hepatocyte growth factor

Hepatocyte growth factor (HGF), a multifunctional cytokine, can act on many cell types. It is involved in cancer growth and metastasis by enhancing the motility of cancer cells and stimulating angiogenesis. The development of effective inhibitors for HGF is an important issue in cancer therapy. In this study, we isolated DNA aptamers against human HGF using the systematic evolution of ligands by exponential enrichment method. The selected DNA aptamers had a highly conserved consensus sequence, and could be divided into two major classes (classes I and II). The consensus motif of classes I and II might contribute to the formation of a hairpin loop structure and a G-quartet structure, respectively. These DNA aptamers bound to human HGF with high affinity and specificity. The dissociation constants of typical aptamers H38-15 and H38-21, representative of the two classes, were calculated to be approximately 20 nM. H38-15 and H38-21 inhibited the biological activities of HGF including the stimulation of scattering, migration, and invasion of pancreatic cancer KP-3 cells. Furthermore, both aptamers inhibited HGF-induced tube formation by human umbilical vein endothelial cells. These results suggested that the isolated DNA aptamers will be useful as therapeutic and diagnostic reagents for cancers.     

Studies on the assembly and stability of the metal-thiolate clusters of metallothionein in dimethyl sulfoxide.

Solvent perturbation studies in 100% dimethyl sulfoxide (Me2SO) have been carried out on rabbit liver metallothionein (MT) in an effort to learn more about the factors stabilizing the three-dimensional structure and the mechanism of cluster formation. As indicated by the electronic absorption spectra of Co7-metallothionein, the reconstituted protein preserves its structural integrity in this solvent. Minor spectral differences between water and Me2SO were fully reversible. The titration of apoMT with cobalt(II) in Me2SO, followed by UV-visible-near-infrared electronic absorption, circular dichroism, magnetic circular dichroism, and EPR spectroscopy, indicate that the protein can refold in this solvent. A comparison with the previous titration data in water reveals that the first four titration steps in both solvents are identical, indicating a thermodynamically controlled folding process. However, the reversed order of the cluster completion between Me2SO and water may suggest the involvement of a kinetically controlled folding process in the last three titration steps. A new cluster form developed with approximately nine Co(II) equivalents.