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Query: UNIPROT:P04141 (granulocyte-macrophage colony-stimulating factor)
 6,790 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human granulocyte-macrophage colony-stimulating factor (GM-CSF), a hemopoietic growth factor, was produced and secreted from tobacco cell suspensions. The GM-CSF cDNA was carried by a binary vector under the control of the CaMV 35S promoter and the T7 terminator. In addition, a 5'-nontranslated region from the tobacco etch virus (TEV leader sequence) was fused to the N-terminal end of the GM-CSF transgene. For ease of purification, a 6-His tag was added to the 3' end of the GM-CSF cDNA. Addition of the TEV leader sequence increased protein production more than twofold compared to non-TEV controls. Initial batch cultivation studies indicated a maximum of 250 microg/L extracellular and 150 microg/L intracellular GM-CSF. Western blot analysis detected multiple peptides with masses from 14 to 30 kDa in the extracellular medium. The plant-produced GM-CSF was biologically active and could be bound to a nickel affinity matrix, indicating that both the receptor-binding region and the 6-His tag were functional. The batch production of GM-CSF was compared with the production of other recombinant proteins secreted by transformed tobacco cells. The recovery of secreted GM-CSF was increased by the addition of stabilizing proteins and by increasing salt in the growth medium to physiological levels.
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PMID:Production and characterization of biologically active human GM-CSF secreted by genetically modified plant cells. 1083

The stoichiometry of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor complex is still unresolved. We have utilised a sensitive, functional assay for receptor homodimerisation to show that GM-CSF induces dimerisation of the common signalling subunit, hbeta(c). We generated a chimeric cytokine receptor in which the extracellular and transmembrane domains of hbeta(c)are fused to the cytoplasmic domain of erythropoietin receptor (EPO-R). Given that to induce EPO-R activation and mitogenic signalling there is a requirement for formation of a specific homodimeric complex, we reasoned that the cytoplasmic domain of EPO-R could be utilised as a highly sensitive reporter for functional homodimer formation. We show that, in the presence of a cytoplasmically truncated GM-CSF alpha-subunit, the hbetac-EPO receptor chimera transduces a mitogenic signal in BaF-B03 in response to GM-CSF. This is consistent with formation of a hbeta(c)homodimer following GM-CSF binding and implies that ligand stimulation induces formation of a higher order complex that contains the hbeta(c)homodimer.
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PMID:GM-CSF binding to its receptor induces oligomerisation of the common beta-subunit. 1123 32

F1 antigen (Caf1) of Yersinia pestis is assembled via the Caf1M chaperone/Caf1A usher pathway. We investigated the ability of this assembly system to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit in Escherichia coli. Despite correct processing of a chimeric protein composed of a modified Caf1 signal peptide, mature human interleukin-1beta (hIL-1beta), and mature Caf1, the processed product (hIL-1beta:Caf1) remained insoluble. Coexpression of this chimera with a functional Caf1M chaperone led to the accumulation of soluble hIL-1beta:Caf1 in the periplasm. Soluble hIL-1beta:Caf1 reacted with monoclonal antibodies directed against structural epitopes of hIL-1beta. The results indicate that Caf1M-induced release of hIL-1beta:Caf1 from the inner membrane promotes folding of the hIL-1beta domain. Similar results were obtained with the fusion of Caf1 to hIL-1beta receptor antagonist or to human granulocyte-macrophage colony-stimulating factor. Following coexpression of the hIL-1beta:Caf1 precursor with both the Caf1M chaperone and Caf1A outer membrane protein, hIL-1beta:Caf1 could be detected on the cell surface of E. coli. These results demonstrate for the first time the potential application of the chaperone/usher secretion pathway in the transport of subunits with large heterogeneous N-terminal fusions. This represents a novel means for the delivery of correctly folded heterologous proteins to the periplasm and cell surface as either polymers or cleavable monomeric domains.
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PMID:Secretion of recombinant proteins via the chaperone/usher pathway in Escherichia coli. 1128 37

The c-fes locus encodes a 93-kDa non-receptor protein tyrosine kinase (Fes) that regulates the growth and differentiation of hematopoietic and vascular endothelial cells. Unique to Fes is a long N-terminal sequence with two regions of strong homology to coiled-coil oligomerization domains. We introduced leucine-to-proline substitutions into the coiled coils that were predicted to disrupt the coiled-coil structure. The resulting mutant proteins, together with wild-type Fes, were fused to green fluorescent protein and expressed in Rat-2 fibroblasts. We observed that a point mutation in the first coiled-coil domain (L145P) dramatically increased Fes tyrosine kinase and transforming activities in this cell type. In contrast, a similar point mutation in the second coiled-coil motif (L334P) was without effect. However, combining the L334P and L145P mutations reduced transforming and kinase activities by approximately 50% relative to the levels of activity produced with the L145P mutation alone. To study the effects of the coiled-coil mutations in a biologically relevant context, we expressed the mutant proteins in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid leukemia cell line TF-1. In this cellular context, the L145P mutation induced GM-CSF independence, cell attachment, and spreading. These effects correlated with a marked increase in L145P protein autophosphorylation relative to that of wild-type Fes. In contrast, the double coiled-coil mutant protein showed greatly reduced kinase and biological activities in TF-1 cells. These data are consistent with a role for the first coiled coil in the negative regulation of kinase activity and a requirement for the second coiled coil in either oligomerization or recruitment of signaling partners. Gel filtration experiments showed that the unique N-terminal region interconverts between monomeric and oligomeric forms. Single point mutations favored oligomerization, while the double point mutant protein eluted essentially as the monomer. These data provide new evidence for coiled-coil-mediated regulation of c-Fes tyrosine kinase activity and signaling, a mechanism unique among tyrosine kinases.
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PMID:A point mutation in the N-terminal coiled-coil domain releases c-Fes tyrosine kinase activity and survival signaling in myeloid leukemia cells. 1150 60

Box1 and 2 (box1/2) are conserved cytoplasmic motifs located in the membrane proximal region of cytokine receptors, including the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor common betac. Deletion of box1/2 abrogated all the examined activities of GM-CSF, and this phenomenon is explained by the loss of binding by Jak2. To test if a molecule other than Jak2 interacting with the box1/2 region plays a role in GM-CSF receptor signal transduction, we screened for molecules interacting with the box1/2 region by a pull-down assay using recombinant purified protein of GST fused with the betac box1/2 region and a Ba/F3 cell lysate. The mouse homologue of Mad2 protein, which plays an important role in the M phase of the cell cycle, was revealed to associate with the box1/2 region specifically. Peptides corresponding to the box1 sequence also bound to Mad2, and mutation of the box1 decreased the Mad2 interaction. Deletion analysis indicated that interaction with box1/2 occurred through the C-terminal portion of Mad2. Mad2 is known to change affinity for binding partners cell cycle dependently. Binding affinity of Mad2 to box1/2 increased in the late M phase, suggesting the possibility that GM-CSF participates in regulation of the M phase check point through interaction with Mad2.
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PMID:Cell cycle-dependent interaction of Mad2 with conserved Box1/2 region of human granulocyte-macrophage colony-stimulating factor receptor common betac. 1155

Human papillomavirus (HPV) vaccines have the potential to prevent cervical cancer by preventing HPV infection or treating premalignant disease. We previously showed that DNA vaccination with the cottontail rabbit papillomavirus (CRPV) E6 gene induced partial protection against CRPV challenge and that the vaccine's effects were greatly enhanced by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). In the present study, two additional strategies for augmenting the clinical efficacy of CRPV E6 vaccination were evaluated. The first was to fuse a ubiquitin monomer to the CRPV E6 protein to enhance antigen processing and presentation through the major histocompatibility complex class I pathway. Rabbits vaccinated with the wild-type E6 gene plus GM-CSF or with the ubiquitin-fused E6 gene formed significantly fewer papillomas than the controls. The papillomas also required a longer time to appear and grew more slowly. Finally, a significant proportion of the papillomas subsequently regressed. The ubiquitin-fused E6 vaccine was significantly more effective than the wild-type E6 vaccine plus GM-CSF priming. The second strategy was to vaccinate with multiple CRPV early genes to increase the breadth of the CRPV-specific response. DNA vaccines encoding the wild-type CRPV E1-E2, E6, or E7 protein were tested alone and in all possible combinations. All vaccines and combinations suppressed papilloma formation, slowed papilloma growth, and stimulated subsequent papilloma regression. Finally, the two strategies were merged and a combination DNA vaccine containing ubiquitin-fused versions of the CRPV E1, E2, and E7 genes was tested. This last vaccine prevented papilloma formation at all challenge sites in all rabbits, demonstrating complete protection.
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PMID:Ubiquitin-fused and/or multiple early genes from cottontail rabbit papillomavirus as DNA vaccines. 1209 75

Typically, multiple cytokines act in concert to mediate a desired immunological response, and thus more effective therapeutics may be achieved by combining several cytokines with potentially synergistic activities. We have developed a series of bi-functional cytokine fusion proteins which, when additionally linked to an intact antibody (or the Fc portion of an antibody) in a variety of configurations, can be specifically targeted. We focus here mainly on the synergizing cytokine combination interleukin-2/interleukin-12 (IL-2/IL-12), but also demonstrate the utility of this approach with interleukin-4/granulocyte-macrophage colony-stimulating factor (IL-4/GM-CSF). Cytokine activity was retained in constructs where the cytokines were fused in tandem at the carboxyl terminus of the Fc or antibody heavy (H) chain, as well as in constructs where one cytokine was fused at the carboxyl terminus of the H chain while the second cytokine was fused to the amino terminus of either the H or light (L) chain variable region. Even in such constructs, antigen binding of the antibody-cytokine fusion proteins could be maintained. In the context of bi-functional fusion proteins, hetero-dimeric IL-12 could be expressed either in a single-chain form, or maintained as a heterodimer in which the p40 subunit was fused to IL-2. These IL-12/IL-2 bi-functional fusion proteins were shown to induce extremely high levels of interferon-gamma (IFN-gamma), similar to the synergy normally seen with the combined application of the individual cytokines. In addition, these bifunctional molecules were shown to have striking anti-tumor activity as either gene therapy or as an antibody cytokine(s) fusion protein, and may provide a useful approach to the treatment of cancer.
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PMID:Bi-functional cytokine fusion proteins for gene therapy and antibody-targeted treatment of cancer. 1220 6

Two mouse-specific polyepitope protein antigens comprising different combinations of sequences chosen from the mouse fertility antigens zona pellucida proteins 1 and 3 (ZP1 and ZP3), prolactin, proliferin, granulocyte-macrophage colony-stimulating factor (GM-CSF), sperm protein SP56 and T-helper cell-stimulating epitopes were produced in bacterial protein expression systems. The recombinant proteins were fused to maltose binding protein (MBP) and used to immunize female mice; their effects on fertility were assessed. Controls were immunized with either MBP only or phosphate buffered saline (PBS). One antigen construct (MBP-polyepitope B), containing mouse-specific epitopes for ZP1, ZP3, SP56 and proliferin, significantly reduced the fertility of female BALB/c mice. Fertility in this group was decreased by > 40% compared with the MBP control and the number of viable embryos was decreased by > 60%. This construct will now be used to produce the antigen in a recombinant murine cytomegalovirus for assessment as a potential mouse-specific anti-fertility vaccine for use in the control of mice in the field.
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PMID:Mouse-specific immunocontraceptive polyepitope vaccines. 1222 Jan 59

Activation of human interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors, ectopically expressed in FDCP-mix multipotent cells, stimulates self-renewal or myeloid differentiation, respectively. These receptors are composed of unique alpha subunits that interact with common beta(c) subunits. A chimeric receptor (hGM/beta(c)), comprising the extracellular domain of the hGM-CSF receptor alpha subunit (hGM Ralpha) fused to the intracellular domain of hbeta(c), was generated to determine whether hbeta(c) activation is alone sufficient to promote differentiation. hGM-CSF activation of hGM/beta(c), expressed in the presence and absence of the hbeta(c) subunit, promoted maintenance of primitive phenotype. This indicates that the cytosolic domain of the hGM Ralpha chain is required for differentiation mediated by activation of the hGM Ralpha, beta(c) receptor complex. We have previously demonstrated that the alpha cytosolic domain confers signal specificity for IL-3 and GM-CSF receptors. Bioinformatic analysis of the IL-3 Ralpha and GM Ralpha subunits identified a tripeptide sequence, adjacent to the conserved proline-rich domain, which was potentially a key difference between them. Cross-exchange of the equivalent tripeptides between the alpha subunits altered receptor function compared to the wild-type receptors. Both the mutant and the corresponding wild-type receptors promoted survival and proliferation in the short-term but had distinct effects on developmental outcome. The mutated hGM Ralpha promoted long-term proliferation and maintenance of primitive cell morphology, whereas cytokine activation of the corresponding hIL-3 Ralpha mutant promoted myeloid differentiation. We have thus identified a region of the alpha cytosolic domain that is of critical importance for defining receptor specificity.
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PMID:Identification of primary structural features that define the differential actions of IL-3 and GM-CSF receptors. 1238 14

We developed a method to generate dendritic cells (DCs) from mouse embryonic stem (ES) cells. We cultured ES cells for 10 days on feeder cell layers of OP9, in the presence of granulocyte-macrophage colony-stimulating factor in the latter 5 days. The resultant ES cell-derived cells were transferred to bacteriologic Petri dishes without feeder cells and further cultured. In about 7 days, irregularly shaped floating cells with protrusions appeared and these expressed major histocompatibility complex class II, CD11c, CD80, and CD86, with the capacity to stimulate primary mixed lymphocyte reaction (MLR) and to process and present protein antigen to T cells. We designated them ES-DCs (ES cell-derived dendritic cells), and the functions of ES-DCs were comparable with those of DCs generated from bone marrow cells. Upon transfer to new dishes and stimulation with interleukin-4 plus tumor necrosis factor alpha, combined with anti-CD40 monoclonal antibody or lipopolysaccharide, ES-DCs completely became mature DCs, characterized by a typical morphology and higher capacity to stimulate MLR. Using an expression vector containing the internal ribosomal entry site-puromycin N-acetyltransferase gene or a Cre-lox-mediated exchangeable gene-trap system, we could efficiently generate ES cell transfectants expressing the products of introduced genes after their differentiation to DCs. ES-DCs expressing invariant chain fused to a pigeon cytochrome C epitope presented the epitope efficiently in the context of E(k). We primed ovalbumin (OVA)-specific cytotoxic T lymphocytes in vivo by injecting mice with ES-DCs expressing OVA, thus demonstrating immunization with ES-DCs genetically engineered to express antigenic protein. The methods may be applicable to immunomodulation therapy and gene-trap investigations of DCs.
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PMID:Generation and genetic modification of dendritic cells derived from mouse embryonic stem cells. 1240 78


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