The secondary structure of human amyloid deposits as determined by circular dichroism spectroscopy

The deposition of amyloid fibrils has been associated with a diversity of pathologies including plasma cell dyscrasias, chronic inflammatory diseases, and several types of neurological diseases including Alzheimer's disease. Using circular dichroism spectroscopy, the secondary structure of a human amyloid protein deposit from a patient with plasma cell (light chain)-associated amyloidosis (amyloidosis AL) has been determined. The protein contains 52% beta structure, which is consistent with these depositions arising from the aberrant catabolism of immunoglobulin light chains, which are rich in beta sheets. The protein was also found to contain 20% alpha-helix, suggesting that partial refolding may occur during amyloidogenesis.     

Characterization of a cysteine-free analog of recombinant human basic fibroblast growth factor

Using oligo site-directed mutagenesis, we have modified our synthetic gene for human basic fibroblast growth factor (bFGF) to replace all four cysteine codons with serine codons. The corresponding protein was expressed in Escherichia coli and purified from inclusion bodies by solubilization in urea followed by a series of column chromatographies and a folding step. The resulting protein, having no cysteine residues, is unable to form either intramolecular or intermolecular disulfide bonds. The secondary and tertiary structures of the purified analog, as determined by circular dichroism and fluorescence spectroscopy, were identical within experimental error to recombinant bovine and human bFGF with unaltered amino acid sequences. Reflecting the similar conformation, the analog protein exhibited mitogenic activity on NIH 3T3 cells which was indistinguishable from the natural sequence molecule.     

Somatomedin-C and platelet-derived growth factor stimulate human fibroblast replication

Previous studies have demonstrated that serum contains mitogens, such as platelet-derived growth factor (PDGF), which may alter fibroblast responsiveness to growth factors contained in plasma. Somatomedin-C (SM-C) has been identified as one of the plasma growth factors required for mouse Balb/c 3T3 fibroblasts to initiate DNA synthesis. The present experiments were undertaken to explore the interaction between PDGF, human growth hormone (hGH), SM-C, and other growth-promoting agents in stimulating the growth of human fibroblasts. Proliferation of human dermal fibroblasts plated at low density (3,000 cells/cm²) was found to be equally stimulated by continuous exposure to either normal or somatomedin-C-deficient serum. In contrast, when confluent monolayers were sequentially exposed to PDGF, followed either by normal platelet poor plasma (PPP) or hypopituitary PPP, the cells exposed to normal PPP entered the “S” phase of the cell cycle 50% faster. This difference could be abolished by a 6-hour incubation with growth hormone (10 ng/ml) or somatomedin-C (5 ng/ml) preceding the addition of plasma. When medium containing either hGH or Sm-C was changed frequently so as to remove factors secreted by fibroblasts, only those cells exposed to exogenous somatomedin-C entered DNA synthesis. This finding is in agreement with previous findings that human fibroblasts are capable of making Sm-C in response to hGH. These findings support the hypothesis that somatomedin is required for fibroblast replication in vitro, and that growth hormone appears to stimulate replication indirectly through somatomedin production.     

The effect of recombinant human basic fibroblast growth factor rhfgf-2 on human osteoblast in growth and phenotype expression

This paper describes the studies of human recombinant basic fibroblast growth factor (rhFGF-2) for its effects on human osteoblast growth and phenotype expression. During a 24-h period of treatment, rhFGF-2 highly stimulated DNA synthesis in a dose-related fashion with a maximum stimulation of 150% for 1 ng/ml. On the other hand, rhFGF-2 decreases alkaline phosphatase activity, synthesis of type I collagen, and cumulative amount of osteocalcin. Moreover, rhFGF-2 provoked a threefold increase in the amount of intracellular cAMP. Scatchard plots show the presence of two classes of [¹²⁵I] rhFGF-2 receptors. This data suggests that rhFGF-2 which stimulate cell replication may act indirectly as an anabolic agent and stimulate some of the phenotypic expression markers.     

Stabilization of recombinant human basic fibroblast growth factor by chemical modifications of cysteine residues.

The production of recombinant human basic fibroblast growth factor (rhbFGF) in Escherichia coli cells yielded active forms of this polypeptide which, however, displayed a high degree of instability towards oxidative processes. Biochemical studies in our laboratory and those of others indicated that the reactivity of the four cysteine residues was the main cause of the observed instability. Several attempts to obtain more stable derivatives of rhbFGF were carried out by modification of the sulfhydryl groups. Among these, treatment of rhbFGF with iodoacetic acid led to the isolation of a partially carboxymethylated form (Cm-FGF). Peptide mapping analysis of the modified protein showed that two cysteines (78 and 96) were blocked by a carboxymethyl group. The remaining cysteines (34 and 101) were not modified under the conditions used and were found to be in the reduced form. Cm-FGF and unmodified rhbFGF showed similar affinity both for heparin and for high-affinity receptors. Cm-FGF was more stable than the unmodified molecule as measured by HPLC and SDS/PAGE analysis. Interestingly, Cm-FGF was more active than unmodified rhbFGF in stimulating proliferation of endothelial cells and DNA synthesis in 3T3 fibroblasts. This new derivative could represent a desirable complementation to rhbFGF for the development of more stable pharmaceutical formulations in wound healing applications.     

Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine replaced by serine

We have chemically synthesized the gene encoding bovine basic fibroblast growth factor (bFGF) and cloned it into a plasmid vector. This gene was then used as a template for site-directed mutagenesis to produce the human bFGF gene and a gene coding for an analog in which serine residues were substituted for the cysteine residues at positions 70 and 88. All three constructs were cloned and expressed in Escherichia coli and the proteins purified. The recombinant human and bovine bFGFs exhibited the potent mitogenic activity toward both fibroblasts and endothelial cells, which characterizes natural bFGF. The serine-70,88 analog and natural sequence bovine and human forms were equally active in all assays. Sulfhydryl titration of the purified recombinant bovine bFGF in 4.8 M guanidine hydrochloride indicated the presence of approximately two free sulfhydryl groups. This was consistent with the sequence analysis of peptides derived from trypsin digestion, which suggests that cysteines 70 and 88 exist in free sulfhydryl form while cysteines 26 and 93 form a disulfide bond. Circular dichroism shows that the protein has little ordered structure but is folded into a rigid tertiary configuration. Carboxymethylation of the free sulfhydryl groups resulted in no change in the mitogenic activity or conformation. These results are consistent with previous suggestions that, for tissue-derived bFGF, at least 2 of the 4 cysteines in the molecule are not involved in a disulfide bond.     

Site-directed PEGylation of human basic fibroblast growth factor

Through site-directed mutagenesis, three cysteines of human basic fibroblast growth factor (hbFGF) were replaced with serine residues, resulting in a hbFGF mutant named hbFGFSer25,69,92. The mutant with only one cysteine residue at the 87th position, whose mitogenic activity was comparable to that of wild-type hbFGF, was further coupled to polyethylene glycol with a molecular size of 5 kDa (PEG5K) via the cysteine residue to obtain another hbFGF derivative, PEG5K-hbFGFSer25,69,92. The optimal modification reaction was conducted at 4 degrees C for 4 h at a molar ratio of PEG5K to hbFGFSer25,69,92 of 20:1. The result of SDS-PAGE showed that the modification extent was up to 80%. The modified product was purified by ion exchange chromatography. Compared to the hbFGF mutant, the purified PEG5K-hbFGFSer25,69,92 still retained about 60% of the mitogenic activity of the former, which provided a good basis for further studying the bioactivity of the PEGylated protein in vivo.     

Production of a recombinant human basic fibroblast growth factor with a collagen binding domain

Summary Basic fibroblast growth factor (bFGF) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and may participate in tissue repair. Basic FGF is found in abundance in tissues such as brain, kidney, and cartilage. This study reports the expression, purification, and renaturation of a biologically active human basic fibroblast growth factor fusion protein (hbFGF-Fl) fromEscherichia coli. A prokaryotic expression vector was engineered to produce a tripartite fusion protein consisting of a purification tag, a protease-sensitive linker and collagen binding domain, and a cDNA sequence encoding the active fragment of hbFGF. The expressed hbFGF-F1 and hbFGF-F2 (it contains the collagen binding domain), located in inclusion bodies, were solubilized with 6 M guanidine-HCl and renatured by a glutathione redox system and protracted dialysis under various experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion protein on a nickel-nitrilotriacetic acid metal chelate column. The biological activity of the recombinant growth factor was demonstrated by its ability to stimulate proliferation of human vein endothelial cells, monitored by [³H]thymidine incorporation, where commercial recombinant human bFGF (rhbFGF) served as a positive control. Purified rhbFGF-F1 and rhbFGF-F2 constructs exhibited proliferative activity comparable to commercial rhbFGF. The high-affinity binding was demonstrated by the binding of [³H]collagen to the rhbFGF-F2 protein immobilized on a Ni-nitrilotriacetic acid column. The rhbFGF-F2 fusion protein bound to collagen-coated surfaces with high affinity. Taken together, these results demonstrate that biologically active rhbFGF fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for their high-yield production, purification, and renaturation from microorganisms. Furthermore, we demonstrate that the auxiliary collagen binding domain effectively targets the recombinant growth factor to type 1 collagen. These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins for specific biomedical applications.     

A novel recombinant basic fibroblast growth factor and its secretion.

Basic fibroblast growth factor (FGF-2) is a pleiotropic mitogen which plays an important role in cell growth, differentiation, migration, and survival in different cells and organ systems. Recently, several clinical applications for FGF-2 gene transfer are being evaluated in wound healing and collateral artery development to relieve myocardial and peripheral ischemia due to the ability of FGF-2 to regulate the growth and function of vascular cells. However, FGF-2 lacks a classical hydrophobic secretion signal peptide, the FGF-2 chimeras containing various signal sequences have been explored. In this study, a novel recombinant 4sFGF-2 was constructed by replacing nine residues from the amino-terminus of native FGF-2 (Met1 to Leu9) with eight amino acid residues of signal peptide of FGF-4 (Met1 to Ala8) to better increase the secretion level of FGF-2. When the recombinant FGF-2 gene, cloned into the expression vector with CMV promoter, was expressed in COS-7 cells, the recombinant 4sFGF-2 was highly secreted into the media. The secreted 4sFGF-2 showed the same biological activity as the native FGF-2 in the dose-response effects on DNA synthesis and cell growth of rat aortic smooth muscle cells (RASMCs) and NIH3T3 cells. The 4sFGF-2 also was able to activate MAPK as wild FGF-2 in RASMCs. These results indicate that a novel recombinant 4sFGF-2 may be useful as clinical applicability of angiogenic growth factor gene transfer.     

The disulfide structure of bovine pituitary basic fibroblast growth factor.

Basic fibroblast growth factor has 4 cysteine residues in its amino acid sequence, two of which are perfectly conserved within the fibroblast growth factor family of proteins suggesting a disulfide bond at this position. Furthermore, thiol titration of bovine pituitary basic fibroblast growth factor (bFGF) indicates the presence of two free thiols, which is consistent with an intramolecular disulfide. Direct analysis of natural and recombinant fibroblast growth factor proteins have not confirmed the existence of such a disulfide. Instead, the two nonconserved cysteines of bFGF purified from bovine pituitaries are S-thiolated with glutathione. Inclusion of 75 mM N-ethylmaleimide during the homogenization of the pituitaries effectively blocks the S-thiolation, demonstrating that this modification is an artifact of the purification procedure. Analysis of the N-ethylmaleimide purified bovine pituitary bFGF suggests that the natural protein is in the correct redox state when all 4 cysteines are in the reduced form.     

Molecular characterization of recombinant human acidic fibroblast growth factor produced in E. coli: comparative studies with human basic fibroblast growth factor.

Synthetic cDNA coding for human acidic fibroblast growth factor (haFGF) was expressed in E. coli under the control of the T7 promoter. The haFGF produced was purified extensively using heparin-Sepharose and phenyl-Sepharose columns. The mitogenic activity of haFGF on 3T3 and endothelial cells was significantly potentiated in the presence of heparin (10-50 micrograms/ml), while angiogenic activity was observed on chick embryo chorioallantoic membrane without exogenously added heparin. This significant potentiation of mitogenic activity was observed specifically with haFGF, not human basic fibroblast growth factor (hbFGF). Circular dichroism spectra of haFGF was not affected by the presence of heparin. The affinity of haFGF for heparin was examined using heparin affinity HPLC and was precisely confirmed to be relatively lower than that of hbFGF. These results implied that haFGF was potentiated by heparin and that this potentiation did not involve a significant change in the conformation of the haFGF molecule. The affinity of haFGF for copper was also confirmed to be higher than that of hbFGF using a copper affinity HPLC column. In addition, under acidic conditions, haFGF appeared more stable than hbFGF and was further stabilized in the presence of heparin.     

High-level expression of human acidic fibroblast growth factor and basic fibroblast growth factor in silkworm (Bombyx mori L.) using recombinant baculovirus

A hybrid of Autographa californica nuclear polyhedrosis virus and Bombyx mori nuclear polyhedrosis virus, which is infectious to both Spodoptera frugiperda and Bombyx mori, was prepared in our previous study. Two recombinant hybrid baculoviruses, carrying cDNAs of human acidic and basic fibroblast growth factors, respectively, were successfully constructed in this study, for the large-scale production of human aFGF and bFGF using silkworm as host. These recombinant viruses were used to inoculate silkworm larvae. After the infection, the recombinant proteins were not found in the hemolymph. Such nonsecretion from cells has also been observed in the established insect cell lines, Sf21 and Tn-5. Tissue distribution analysis indicated that the expressed products were mainly located in fat body and the production of the recombinant aFGF and bFGF was maximal at around 80 h postinfection. Therefore, silkworm larvae infected with recombinant viruses were dissected and fat bodies were collected for the purification of recombinant aFGF and bFGF. The expression levels in both cases were estimated to be as high as approximately 600-700 microg per larva. Furthermore, the recombinant proteins were characterized and their biological activities were evaluated by in vitro bioassay using cell culture.     

Stimulation of human synovial fibroblast DNA synthesis by platelet-derived growth factor and fibroblast growth factor. Differences to the activation by IL-1

The pronounced synovial hyperplasia often found in the joints of patients with rheumatoid arthritis could be explained partially by the action of monocyte-macrophage polypeptides (monokines). This report demonstrates that two cytokines which may be derived from monocyte-macrophage populations, namely platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), stimulate the DNA synthesis and proliferation of human synovial fibroblast-like cells cultured in low (i.e., 1%) fetal bovine serum. Epidermal growth factor, insulin-like growth factor-I, insulin-like growth factor-II (multiplication stimulating activity) and substance P were inactive. Unlike IL-1, PDGF and FGF do not also stimulate PGE2, plasminogen activator, and hyaluronic acid levels. Thus PDGF and FGF, arising from stimulated monocyte-macrophages, may play a role in the stimulation of mesenchymal cell proliferation that often accompanies chronic inflammatory arthritic disease. The synovial cells respond to a variety of cytokines in different ways suggesting multiple-signaling pathways.     

Stimulation of mouse vibrissal follicle growth by recombinant human fibroblast growth factor 20

Objectives

To explore potential effects of recombinant human fibroblast growth factor 20 (rhFGF20) in the growth of cultured mouse vibrissal follicles.

Results

The growth of cultured mouse vibrissal follicles was significantly induced by rhFGF20 in a dose dependent pattern in the in vitro vibrissal follicle organ culture model. However, too high concentration of rhFGF20 could inhibit the growth of vibrissal follicles. We further demonstrated that rhFGF20 stimulated the proliferation of hair matrix cells and activated Wnt/β-catenin signaling pathway.

Conclusions

The rhFGF20 might be a potential therapeutic agent to treat hair loss disorders.

     

Secondary structure of the hydrophobic myelin protein in a lipid environment as determined by Fourier-transform infrared spectrometry

The secondary structure of a hydrophobic myelin protein (lipophilin), reconstituted with dimyristoylphosphatidylcholine or dimyristoylphosphatidylglycerol, was investigated by Fourier-transform infrared spectroscopy. Protein infrared spectra in the amide I region were analyzed quantitatively using resolution enhancement and band fitting procedures. Lipophilin in a phospholipid environment adopts a highly ordered secondary structure which at room temperature consists predominantly of alpha-helix (approximately 55%) and beta-type conformations (36%). The secondary structure of the protein is not affected by the lipid gel to liquid crystalline phase transition. Heating of the lipid-protein complex above approximately 35 degrees C results in a gradual decrease in alpha-helical content, accompanied by an increase in the amount of beta-structures. Lipophilin dissolved in 2-chloroethanol is, compared to the protein in a lipid environment, richer in the alpha-helical conformation but still contains a sizable amount of beta-structure.     

1H, 15N, 13C and 13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy

Summary The assignments of the ¹H, ¹⁵N, ¹³CO and ¹³C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled ¹⁵N- and ¹⁵N-/¹³C-labeled FGF-2 with an isotope incorporation >95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N in the CBCANH and HNHA experiments. In addition, C and C chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic ¹H and ¹³C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H and H protons as well as ³J_H n _H coupling constants, amide exchange and ¹³C and ¹³C secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel -sheets (residues 30–34, 39–44, 48–53, 62–67, 71–76, 81–85, 91–94, 103–108, 113–118, 123–125 and 148–152) and a helix-like structure (residues 131–136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131–136 were defined as -strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128–138) instead of the -strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9–28. This is consistent with the X-ray structures of FGF-2, where the first 17–19 residues were ill defined.     

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.     

Platelet-derived growth factor or basic fibroblast growth factor induce anchorage-independent growth of human fibroblasts

Anchorage-independent growth, i.e., growth in semi-solid medium is considered a marker of cellular transformation of fibroblast cells. Diploid human fibroblasts ordinarily do not exhibit such growth but can grow transiently when medium contains high concentrations of fetal bovine serum. This suggests that some growth factor(s) in serum is responsible for anchorage-independent growth. Much work has been done to characterize the peptide growth factor requirements of various rodent fibroblast cells for anchorage-independent growth; however, the requirements of human fibroblasts are not known. To determine the peptide growth factor requirements of human fibroblasts for anchorage-independent growth, we used medium containing serum that had had its peptide growth factors inactivated. We found that either platelet-derived growth factor (PDGF) or the basic form of fibroblast growth factor (bFGF) induced anchorage-independent growth. Epidermal growth factor (EGF) did not enhance the growth induced by PDGF, or did so only slightly. Transforming growth factor beta (TGF-beta) decreased the growth induced by PDGF. EGF combined with TGF-beta induced colony formation in semi-solid medium at concentrations at which neither growth factor by itself was effective, but the combination was much less effective in stimulating anchorage-independent growth than PDGF or bFGF. This work showed that PDGF, or bFGF, or EGF combined with TGF-beta can stimulate anchorage-independent growth of nontransformed human fibroblasts. The results support the idea that cellular transformation may reduce or eliminate the need for exogenous PDGF or bFGF.