Synthesis of an active hybrid growth factor (GF) in bacteria: transforming GF-α/vaccinia GF fusion protein

A hybrid gene encoding for a polypeptide consisting of the first 33 N-terminal arnino acid (aa) residues of transforming growth factor-→ (TGF-α) and a C terminus consisting of 20 aa residues of vaccinia growth factor (VGF) was chemically synthesized and expressed as a fusion protein in Escherichia coli. The primary structure of the hybrid gene product maintained the same positioning of the three disulfide bonds found in each parent molecule thus conserving the first two loop regions of TGF-α and the third loop region of VGF. After cleavage with CNBr its renatured biological activity was found to be comparable to TGF-a and VGF with respect to binding to the epidermal growth factor receptor, stimulation of DNA synthesis and induction of anchorage-independent growth of NRK cells in the presence of TGF-β. Thus, we suggest that similar domains can be interchanged within the same family of molecules and equivalent functionality maintained.     

Differential effects of epidermal growth factor, transforming growth factor-alpha, and vaccinia virus growth factor in the positive regulation of IFN-gamma production

We have recently shown that epidermal growth factor (EGF) is capable of positive regulation of IFN-gamma production, thus establishing a functional relationship between nonhemopoietic growth factors and the immune system. In order to study this relationship further, EGF and the EGF-related growth factors transforming growth factor-alpha (TGF-alpha) and vaccinia virus growth factor (VGF), which stimulate cellular proliferation via binding to the EGF receptor, were studied for their functional and physicochemical effects on IFN-gamma production. In contrast to the positive signal of purified murine EGF and recombinant human EGF (both at 1 nM), neither synthetic TGF alpha nor recombinant VGF were capable of restoring competence for IFN-gamma production by Th cell-depleted spleen cell cultures. TGF-alpha and VGF, in molar excess, also failed to block the helper signal of EGF for IFN-gamma production. Thus TGF-alpha and VGF failed to functionally compete for the EGF receptor in the murine spleen cell system. Both TGF-alpha and VGF stimulated murine 3T3 cell proliferation at concentrations similar to those of EGF, and thus their failure to provide help for IFN-gamma production was not due to a general lack of biologic activity. Binding studies with 125I-EGF suggest that the EGF receptor on murine lymphocytes is not constitutively expressed, but inducible by the T cell mitogen staphylococcal enterotoxin A. TGF-alpha did not compete with 125I-EGF for the induced receptor. The data suggest that lymphocytes express a novel inducible EGF receptor that differs from that expressed on cells such as 3T3 fibroblasts.     

Epidermal growth factor contains both positive and negative determinants for interaction with ErbB-2/ErbB-3 heterodimers

Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha are potent activators of the ErbB-1 receptor, but, unlike TGF-alpha, EGF is also a weak activator of ErbB-2/ErbB-3 heterodimers. To understand the specificity of EGF-like growth factors for binding to distinct ErbB members, we used EGF/TGF-alpha chimeras to examine the requirements for ErbB-2/ErbB-3 activation. Here we show that in contrast to these two wild-type ligands, distinct EGF/TGF-alpha chimeras are potent activators of ErbB-2/ErbB-3 heterodimers. On the basis of differences in the potency of these various chimeras, specific residues in the linear N-terminal region and the so-called B-loop of these ligands were identified to be involved in interaction with ErbB-2/ErbB-3. A chimera consisting of human EGF sequences with the linear N-terminal region of human TGF-alpha was found to be almost as potent as the natural ligand neuregulin (NRG)-1beta in activating 32D cells expressing ErbB-2/ErbB-3 and human breast cancer cells. Binding studies revealed that this chimera, designated T1E, has high affinity for ErbB-2/ErbB-3 heterodimers, but not for ErbB-3 alone. Subsequent exchange studies revealed that introduction of both His2 and Phe3 into the linear N-terminal region was already sufficient to make EGF a potent activator of ErbB-2/ErbB-3 heterodimers, indicating that these two amino acids contribute positively to this receptor binding. Analysis of the B-loop revealed that Leu26 in EGF facilitates interaction with ErbB-2/ErbB-3 heterodimers, while the equivalent Glu residue in TGF-alpha impairs binding. Since all EGF/TGF-alpha chimeras tested have maintained high binding affinity for ErbB-1, it is concluded that the diversity of the ErbB signaling network is determined by specific amino acids that facilitate binding to one receptor member, in addition to residues that impede binding to other ErbB family members.     

Tissue-Specific Processing of the Neuroendocrine Protein VGF

Abstract: VGF is a neuroendocrine-specific gene product that is up-regulated by nerve growth factor in the PC12 cell line. In rat neuroendocrine tissues two polypeptides of 90 and 80 kDa were detected by an antiserum to an N-terminal domain of VGF (from residues 4 to 240). In parallel, an antiserum directed against the C-terminal nonapeptide of VGF (from residues 609 to 617) revealed several additional posttranslational products. Peptides of apparent molecular sizes of 20, 18, and 10 kDa were prominent in nerve tissues and the hypophysis but absent in the adrenal medulla, and their relative abundance varied in distinct regions of the CNS. In PC12 cells VGF was proteolytically processed only after nerve growth factor treatment, and primary cultures of rat cerebellar granule cells accumulated the low-molecular-weight forms of VGF during in vitro maturation. In these cells the specific cleavages of VGF occurred in a postendoplasmic reticulum compartment; the processed forms were enriched in the secretory vesicles and were preferentially secreted upon cell membrane depolarization. Distinct differential distribution in the CNS and in vitro release of such posttranslational products indicate that these species may represent biologically relevant forms of VGF that play a role in neuronal communication.     

The three-dimensional structure of the first EGF-like module of human factor IX: comparison with EGF and TGF-alpha.

The three-dimensional structure of the first epidermal growth factor (EGF)-like module from human factor IX has been determined in solution using two-dimensional nuclear magnetic resonance (in the absence of calcium and at pH 4.5). The structure was found to resemble closely that of EGF and the homologous transforming growth factor-alpha (TGF-alpha). Residues 60-65 form an antiparallel beta-sheet with residues 68-73. In the C-terminal subdomain a type II beta-turn is found between residues 74 and 77 and a five-residue turn is found between residues 79 and 83. Glu 78 and Leu 84 pair in an antiparallel beta-sheet conformation. In the N-terminal region a loop is found between residues 50 and 55 such that the side chains of both are positioned above the face of the beta-sheet. Residues 56-60 form a turn that leads into the first strand of the beta-sheet. Whereas the global fold closely resembles that of EGF, the N-terminal residues of the module (46-49) do not form a beta-strand but are ill-defined in the structure, probably due to the local flexibility of this region. The structure is discussed with reference to recent site-directed mutagenesis data, which have identified certain conserved residues as ligands for calcium.     

Growth inhibition by vaccinia virus growth factor

Vaccinia virus growth factor (VGF), a highly glycosylated 77-residue epidermal growth factor (EGF)-like polypeptide encoded in vaccinia poxvirus, is reported to play an important role in stimulating growth of uninfected cells to facilitate virus infection. We have chemically synthesized the unglycosylated forms of VGF and VGF19-69, a shortened VGF analog consisting of 51 residues and comprising the EGF-homologous region (position 19-69) of VGF. Both synthetic forms of VGFs were purified to homogeneity and vigorously characterized by various criteria, including the Cf-252 ion fission fragment mass spectrometry, amino acid sequencing, and enzymatic digestion to confirm the disulfide linkages. Synthetic VGFs exhibited high affinity binding to the EGF receptors in A431, NRK 49F, NRK clone 3, and NIH 3T3 cells, but, unlike the glycosylated form, showed contrasting mitogenic activities in various cells in vitro. Synthetic VGFs showed low levels of mitogenic and colonogenic activities in NRK clone 49F cells and NIH 3T3 cells, full agonist activities in human keratinocytes and Swiss 3T3 cells, and partial agonist activities in NRK clone 3 cells. Our results suggest that the unglycosylated form of VGF is an EGF antagonist to selected cells and that the production of unglycosylated form of VGF by the cytolytic vaccinia virus may serve as a mechanism whereby inhibition of growth and metabolism of selected host cells may be used to facilitate the propagation of the virus infection.     

TGF-alpha-driven tumor growth is inhibited by an EGF receptor tyrosine kinase inhibitor.

The simultaneous presence of the EGFR and its ligand TGF-alpha in human tumor tissues suggests that autocrine TGF-alpha stimulation drives tumor growth. Here we show that autocrine TGF-alpha stimulation does cause increased tumor growth in vivo, an effect that was proven to be mediated via EGFR activation, and that this TGF-alpha/EGFR autocrine loop was accessible to an EGFR specific tyrosine kinase inhibitor. Clones of the EGFR expressing glioma cell line U-1242 MG were transfected with TGF-alpha cDNA using a tetracycline-inhibitory system for gene expression. TGF-alpha expression was inhibited by the presence of tetracycline, and subcutaneous tumors forming from cell lines injected into nude mice could be inhibited by feeding mice tetracycline. We confirmed that TGF-alpha mRNA and protein were present in these tumors and that, subsequently, the endogenous EGFR was activated. Tumor growth could be inhibited by an EGFR specific tyrosine kinase inhibitor of the type 4-(3-chloroanilino)-6,7-dimethoxy-quinazoline, administered daily by intraperitoneal injection, thereby interrupting the autocrine loop.     

Nucleotide Sequence and Regulatory Studies of VGF, a Nervous System-Specific mRNA That Is Rapidly and Relatively Selectively Induced by Nerve Growth Factor

A nervous system-specific mRNA that is rapidly induced in PC12 cells to a greater extent by nerve growth factor (NGF) than by epidermal growth factor treatment has been cloned. The polypeptide deduced from the nucleic acid sequence of the NGF33.1 cDNA clone contains regions of amino acid sequence identity with that predicted by the cDNA clone VGF, and further analysis suggests that both NGF33.1 and VGF cDNA clones very likely correspond to the same mRNA (VGF). In this report both the nucleic acid sequence that corresponds to VGF mRNA and the polypeptide predicted by the NGF33.1 cDNA clone are presented. Genomic Southern analysis and database comparison did not detect additional sequences with high homology to the VGF gene. Induction of VGF mRNA by depolarization and phorbol 12-myristate 13-acetate treatment was greater than by serum stimulation or protein kinase A pathway activation. These studies suggest that VGF mRNA is induced to the greatest extent by NGF treatment and that VGF is one of the most rapidly regulated neuronal mRNAs identified in PC12 cells.     

Substitution of lysine for arginine at position 42 of human transforming growth factor-alpha eliminates biological activity without changing internal disulfide bonds.

Transforming growth factor-alpha (TGF-alpha) is a growth-promoting protein that binds to the epidermal growth factor (EGF) receptor. To identify critical residues that govern TGF-alpha-EGF receptor binding, we prepared site-specific substitution mutants of TGF-alpha. Mutant proteins were tested in receptor-binding and mitogenesis assays. Semiconservative substitutions at positions 4, 12, 18, and 45 decreased biological activity 2.1- to 14-fold. The conservative substitution of lysine for arginine at position 42 completely eliminated biological activity. Amino acid composition analysis of proteolytic fragments from TGF-alpha and the Lys-42 mutant indicated that these proteins contained the same disulfide bonds. These studies suggest that arginine 42 may be a contact point for TGF-alpha-EGF receptor interaction.     

Parathyroid hormone-related peptide can regulate the growth of human lung cancer cells, and may form part of an autocrine TGF-alpha loop.

Parathyroid hormone-related peptide (PTHrP) and transforming growth factor-alpha (TGF-alpha) were found to stimulate proliferation of human lung cancer cells (BEN-57). TGF-alpha stimulated PTHrP secretion from these cells. The polyclonal antisera raised against PTHrP significantly inhibited the growth of BEN-57 cells, and also the proliferation induced by TGF-alpha. Treatment of cells for up to 10 days with either a PTHrP receptor antagonist (PTHrP(7-34)) or PTHrP antiserum significantly inhibited the subsequent growth of these cells. We suggest that PTHrP may be a component of a complex autocrine loop involving TGF-alpha.     

Deletion of the vaccinia virus growth factor gene reduces virus virulence.

The vaccinia virus growth factor (VGF) gene encodes a polypeptide with amino acid sequence homology to epidermal growth factor (EGF) and transforming growth factor alpha and is present twice, once at each end of the virus genome within the inverted terminal repetition. Recombination procedures were used to replace more than half of both VGF genes with a beta-galactosidase cassette which served as a color indicator for isolating an unconditionally viable VGF- mutant. The VGF- mutant genotype and phenotype were confirmed by Southern blot analysis and assays for functional growth factor. The plaque-forming efficiencies of VGF- and wild-type (WT) viruses were similar in a variety of cell types containing low or high densities of EGF receptors, suggesting a lack of a specific requirement for either VGF or the EGF receptor in the initiation of virus infection. The yield of VGF- virus was similar to that of WT virus in growing BS-C-1 and Swiss 3T3 cells, but lower in resting Swiss 3T3 cells. The greatest differences between VGF- and WT virus occurred in vivo: higher doses of VGF- virus than WT virus were required for intracranial lethality in mice and for production of skin lesions in rabbits. Thus, expression of the VGF gene is important to the virulence of vaccinia virus.     

Induction of human microvascular endothelial tubular morphogenesis by human keratinocytes: involvement of transforming growth factor-alpha.

Transforming growth factor-alpha(TGF-alpha), homologous to epidermal growth factor(EGF), is closely involved in hyperproliferation of human keratinocytes. Psoriasis is a common hyperproliferative skin disease characterized by hyperproliferation of keratinocytes and abnormal development of dermal capillary networks. In this study, we have examined whether keratinocytes could enhance angiogenesis. TGF-alpha or EGF efficiently stimulated formation of tubular-like structures of human omental microvascular endothelial(HOME) cells in type I collagen gels. Human keratinocytes produced TGF-alpha. To examine whether co-cultured keratinocytes could induce tubulogenesis of HOME cells in collagen gel, we have developed a co-culture system with human keratinocytes. Surprisingly, there appeared new development of many tubular-like structures of HOME cells in collagen gels when co-cultured with keratinocytes. This keratinocytes-dependent tubulogenesis was almost completely blocked when anti-TGF-alpha-antibody was present. The TGF-alpha molecules derived from keratinocytes appeared to enhance tubulogenesis of human microvascular endothelial cells. We propose the hypothesis that secretory TGF-alpha from human keratinocytes may promote an autocrine loop to proliferate the skin keratinocytes and also a paracrine loop to induce the skin angiogenesis.     

Structure of the gene encoding VGF, a nervous system-specific mRNA that is rapidly and selectively induced by nerve growth factor in PC12 cells.

Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.     

Transforming growth factor-alpha and epidermal growth factor receptor gene expression and action during pubertal development of the seminiferous tubule.

The potential role of transforming growth factor-alpha (TGF-alpha) as a mediator of cell-cell interactions in the growth and development of the testis was examined. Developing rat testes were collected, and preparations of mesenchymal-derived peritubular cells and epithelial-like Sertoli cells were isolated from prepubertal, midpubertal, and late pubertal rat testes. The developmental expression of TGF-alpha and its receptor, the epidermal growth factor receptor (EGFR), in whole testis and isolated cell types was determined using a nuclease protection assay. TGF-alpha and EGFR gene expression were predominant early in testis development and decreased during pubertal development. TGF-alpha expression was greatest in prepubertal peritubular cells. Sertoli cell TGF-alpha expression remained relatively constant during development, with a slight decline at the later pubertal stages. EGFR gene expression was predominant in peritublar cells throughout development. A low level of EGFR expression was detected in Sertoli cells. Scatchard analysis confirmed the presence of high affinity receptors on peritubular cells; however, no functional receptors were detected on Sertoli cells from any stage of development examined. Interestingly, low-level EGFR gene expression was also detected in pachytene spermatocytes and round spermatids. TGF-alpha was found to stimulate [3H] thymidine incorporation into DNA and increase cellular proliferation of peritubular cells from each developmental stage, while having no effect on Sertoli cells. The in vivo physiological significance of TGF-alpha was evaluated in a line of transgenic mice which overexpress TGF-alpha in the mature testis. These transgenic animals had no abnormal testicular morphology or alterations in spermatogenesis. Observations demonstrate that gene expression of TGF-alpha and its receptor is high during early pubertal stages when somatic cell growth is predominant and low at late pubertal stages when somatic cell proliferation is reduced. TGF-alpha can act as an autocrine/paracrine mitogen for the mesenchymal-derived peritubular cell, while actions on the Sertoli cell population are not evident. The observation that spermatogenic cells express the EGFR gene, although the protein remains to be identified, implies that TGF-alpha may potentially mediate Sertoli-germinal cell interactions.     

Fine epitope mapping of anti-epidermal growth factor receptor antibodies through random mutagenesis and yeast surface display

Fine epitope mapping of therapeutically relevant monoclonal antibodies (mAbs) specific for the epidermal growth factor receptor (EGFR) was accomplished through random mutagenesis and yeast surface display. Using this method, we have identified key residues energetically important for the binding of EGFR to the mAbs 806, 225, and 13A9. A yeast-displayed library of single point mutants of an EGFR ectodomain fragment (residues 273-621) was constructed by random mutagenesis and was screened for reduced binding to EGFR mAbs. If an EGFR mutant showed loss of binding to a mAb, this suggested that the mutated residue was potentially a contact residue. The mAb 806 binding epitope was localized to one face of a loop comprised of EGFR residues Cys287-Cys302, which is constrained by a disulfide bond and two salt bridges. The mAb 806 epitope as identified here is not fully accessible in the autoinhibited EGFR monomer conformation, which is consistent with the hypothesis that mAb 806 binds to a transitional form of EGFR as it changes from an autoinhibited to extended monomer. The amino acids Lys465 and Ile467 were identified as energetic hot spot residues for mAb 225 binding to EGFR. These residues are adjacent to the EGFR ligand-binding site, which is consistent with the ability of mAb 225 to block binding of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) ligands. Ser468 and Glu472 were identified as energetically important for mAb 13A9 binding to EGFR, and the location of this epitope suggests that mAb 13A9 mediates observed TGF-alpha blocking effects through conformational perturbation of EGFR domain III. Combinatorial library screening of yeast-displayed mutagenic proteins is a novel method to identify discontinuous and heat-denaturable mAb binding epitopes with residue-level resolution.     

Essential elements of the capsid protein for self-assembly into empty virus-like particles of hepatitis E virus

Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.