Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P04141 (granulocyte-macrophage colony-stimulating factor)
 6,790 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein required for the proliferation and differentiation of granulocyte and macrophage precursors. Previous investigations have identified regions in human and murine GM-CSF that are required for bioactivity. In the present study, alanine substitution mutagenesis was undertaken to define more precisely specific amino-terminal residues in murine GM-CSF that are involved in bioactivity and receptor binding. Five double alanine mutants were identified that showed at least 10-fold reductions in bioactivity (K14AK20A, K14AE21A, H15AK20A, H15AE21A, K20AE21A). Each of these mutants maintained a normal N-linked glycosylation pattern when expressed in COS-1 cells, suggesting that native polypeptide backbone conformation was preserved. The purified prokaryotic expression products of two mutants (K14AE21A and H15AE21A) had a 100-fold decrease in bioactivity and a decrease in receptor binding, indicating that the side chains of K14, H15, and E21 are required for optimal receptor binding and maximal bioactivity.
 ...
PMID:Requirement of hydrophilic amino-terminal residues for granulocyte-macrophage colony-stimulating factor bioactivity and receptor binding. 138 12

A cDNA clone (clone 71) that encodes a low-affinity receptor for murine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been isolated by direct expression. This molecule is the homologue of the human GM-CSF receptor alpha subunit, although homology between these molecules is surprisingly low (less than 35% amino acid identity). The cDNA encodes a polypeptide of 387 amino acids, which contains the conserved features of the hematopoietin receptor superfamily. When expressed in COS-7 cells, this clone encodes a protein that binds radiolabeled murine GM-CSF with low affinity. Coexpression of clone 71 with a cDNA corresponding to a low-affinity interleukin 3 (IL-3) receptor (AIC2A) did not alter the affinity of binding of either GM-CSF or IL-3. However, coexpression of clone 71 with the IL-3 receptor-related cDNA AIC2B generated high-affinity binding sites for murine GM-CSF but not murine IL-3. These studies show that clone 71 and AIC2B are capable of forming an alpha beta complex capable of binding murine GM-CSF with high affinity, while AIC2A appears not to be a component of the murine GM-CSF receptor.
 ...
PMID:Cloning of the low-affinity murine granulocyte-macrophage colony-stimulating factor receptor and reconstitution of a high-affinity receptor complex. 153 31

Nonglycosylated murine and human granulocyte-macrophage colony-stimulating factor have a molecular mass of approximately 14.5 kDa predicted from the primary amino acid sequence. The expression of both proteins in COS cells leads to a heterogeneous population of molecules that differ in the degree of glycosylation. Both human and murine molecules contain two N-linked glycosylation sites that are situated in nonhomologous locations along the linear sequence. Despite this difference both proteins show a similar size distribution among the glycosylation variants. These studies analyze the effects of introducing in the murine protein novel N-linked glycosylation sites corresponding to those sites found in the human molecule. A panel of molecules composed of various combinations of human N-linked glycosylation sites in either the presence or the absence of murine N-linked glycosylation was compared. Substitution of a proper human N-linked glycosylation consensus sequence at Asn 24 did not result in N-linked glycosylation, nor was there any considerable effect on bioactivity. Replacement of the N-linked glycosylation consensus sequence at Asn 34 results in glycosylation similar to that found in the human molecule and causes a significant decrease in bioactivity. These data suggest that the position of N-linked glycosylation is critical for maximal bioactivity in a particular species and that the changes in position of these sites in different species probably occurred during evolution in response to changes in their receptors.
 ...
PMID:Evaluation of human N-linked glycosylation sites in murine granulocyte-macrophage colony-stimulating factor. 153 71

Leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) are multifunctional cytokines with many similar activities. LIF is structurally and functionally related to another cytokine, Oncostatin M (OSM), that binds to the high-affinity LIF receptor but not to the low-affinity LIF receptor. A complementary DNA was isolated that encodes the high-affinity converting subunit of the LIF receptor. The converter conferred high-affinity binding of both LIF and OSM when expressed with the low-affinity LIF receptor and is identical to the signal transducing subunit of the IL-6 receptor, gp130. The gp130 subunit alone confers low-affinity binding of OSM when expressed in COS-7 cells. This receptor system resembles the high-affinity receptors for granulocyte-macrophage colony-stimulating factor, IL-3, and IL-5, which share a common subunit.
 ...
PMID:The IL-6 signal transducer, gp130: an oncostatin M receptor and affinity converter for the LIF receptor. 154 94

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is O-glycosylated at residues Ser9 and Thr10 during secretion by yeast and COS-1 cells [Ernst, J.F., Mermod, J.-J. and Richman, L.I. (1992) Eur. J. Biochem. 203, 663-667]. Two types of octapeptides encompassing residues 4-11 (peptide 4-11) and variants thereof, or residues 8-15 (peptide 8-15) of hGM-CSF were tested as substrates for in vitro O-glycosylation using dolichyl-phosphate- D-mannose: protein O-D-mannosyltransferase (Man-transferase) of the yeast Saccharomyces cerevisiae, or UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) of rat liver cells. Peptide 8-15 was found to be O-glycosylated at residues Ser9 and Thr10 by GalNAc-transferase and, with reduced efficiency, also by Man-transferase. Peptide 4-11 was a good substrate for yeast Man-transferase, leading to mannosylation of only Thr10, whereas it was very poorly O-glycosylated at positions Ser5 and Ser7 by GalNAc-transferase. The observed differences in peptide-acceptor activities indicate that the site of O-glycosylation depends on similar, but not identical protein structural features in yeast and mammalian cells.
 ...
PMID:Specific in vitro O-glycosylation of human granulocyte-macrophage colony-stimulating-factor-derived peptides by O-glycosyltransferases of yeast and rat liver cells. 157 99

A neutrophil chemotactic factor (human interleukin 8, human granulocyte-macrophage colony-stimulating factor)-producing cell line, named KHM-5M, was established from a patient with an undifferentiated thyroid carcinoma, neutrophilia, and malignant pleurisy with many neutrophils and a few malignant cells. The cell line was transplanted into nude rats, and the infiltration of neutrophils was observed in and around the transplanted tumor tissue. Neutrophil chemotactic activity was predicted from the clinical features and pathological findings in this case. The extreme chemotactic activity of the neutrophils was demonstrated in conditioned medium from KHM-5M cells using the modified Boyden chamber technique. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least two neutrophil chemotactic activities in conditioned medium from the cell line were observed. The levels of these activities derived from KHM-5M cells were screened by measuring conditioned medium from the COS cells, which expressed a complementary DNA library from the KHM-5M cells. Chemotactic activities (human interleukin 8, human granulocyte-macrophage colony-stimulating factor) were identified by DNA cloning. These results show that the KHM-5M cells derived from an undifferentiated thyroid carcinoma produce multicytokines and suggest that those cytokines modified some pathological features in this case.
 ...
PMID:Neutrophil chemotactic factors produced by a cell line from thyroid carcinoma. 172 17

Canine granulocyte-macrophage colony-stimulating factor (caGM-CSF) was cloned and expressed to allow further investigation of GM-CSF in a large animal model. The cDNA is 850 base pairs (bp) long and encodes a peptide of 144 amino acids. The nucleotide and amino acid sequence homology between caGM-CSF and human GM-CSF (hGM-CSF) is 80% and 70%, respectively. A mammalian expression vector pCMV/CAGM was constructed and used to transfect COS cells for expression of caGM-CSF. Supernatant from transfected COS cells enriched with caGM-CSF was shown to have significant stimulating activity in granulocyte-macrophage colony forming unit (CFU-GM) assays of canine marrow. caGM-CSF, expressed from bacteria, was used to treat seven dogs at varying doses twice daily subcutaneously (sc) for 14 to 16 days. Circulating blood neutrophils and monocytes increased significantly. The increase in circulating eosinophils was variable. Thrombocytopenia developed during administration of caGM-CSF but corrected rapidly after cessation of treatment. Evaluation of survival times of 51Cr-labeled autologous platelets suggested increased consumption as the primary reason for thrombocytopenia. A species-specific GM-CSF will be a useful tool for hematologic or immunologic studies in dogs.
 ...
PMID:Molecular cloning and in vivo evaluation of canine granulocyte-macrophage colony-stimulating factor. 186 52

A cDNA encoding ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been cloned using the polymerase chain reaction. The nucleotide sequence is approx. 93% identical to the published bovine GM-CSF-encoding sequence, 84% to the human sequence and 73% to the murine sequence. The deduced amino acid sequence of the ovine GM-CSF protein was found to be 80% identical to both the human and bovine proteins and 57% to the murine protein. Transient expression of recombinant ovine GM-CSF in COS-1 cells was obtained and its biological activity investigated in a bone-marrow colony-forming assay. Ovine GM-CSF was found to promote the formation of granulocyte-macrophage colonies as well as eosinophil, neutrophil and monocyte/macrophage colonies, an activity characteristic of GM-CSF in other species. Recombinant human GM-CSF was found to have no proliferative effect on ovine bone-marrow cells.
 ...
PMID:Cloning and expression of a cDNA encoding ovine granulocyte-macrophage colony-stimulating factor. 193 25

Contributions of alpha-helices to biological activity in murine granulocyte-macrophage colony-stimulating factor were analyzed using site-directed mutagenesis and protein expression in COS-1 cells. A series of single proline substitutions were made for residues within the four predicted alpha-helices as a means of disrupting local helical secondary structure. Mutations in three of the four helices resulted in marked reductions in bioactivity. Five mutants E21P, L56P, E60P, L63P, and L107P showed 10(2)-10(4)-fold reduction in bioactivity as well as hyperglycosylation. The same Pro substitutions made on non-N-glycosylated molecules had a similar loss in bioactivity implying that a Pro-induced structural change and not hyperglycosylation was responsible for the major decrease in bioactivity. Additional amino acid substitutions at these residues which conserved charge or hydrophobicity, or replaced the original residue with an Ala, verified that conformational changes in the protein structure were specifically due to steric constraints imposed by the Pro residue rather than loss of important side chain functions.
 ...
PMID:Single proline substitutions in predicted alpha-helices of murine granulocyte-macrophage colony-stimulating factor result in a loss in bioactivity and altered glycosylation. 200 66

A deletion mutant of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) which differs in primary structure from native GM-CSF in the carboxy-terminal 11 amino acids was prepared. Four amino acid residues are mutated and the seven terminal residues including Cys-118 are deleted. Supernatants from COS-1 cells transfected with this deletion mutant (GM-CSF(del] showed a 3000-fold decrease in the ability to stimulate bone marrow stem cells to proliferate and differentiate into granulocytes and macrophages in vitro. Northern blot analysis using poly(A)+ RNA extracted from the transfected cells showed equal accumulations of GM-CSF and GM-CSF(del). Transfection with full-length GM-CSF followed by immunoprecipitation of metabolically labeled supernatant proteins with rabbit anti-rGM-CSF antiserum yielded predominantly the 23-kDa, fully glycosylated form and small amounts of both a 29-kDa form and the 18-kDa non-N-glycosylated form. Transfection of the GM-CSF(del) mutant and immunoprecipitation revealed a large, diffuse band on sodium dodecyl sulfate--polyacrylamide gel electrophoresis with a molecular weight of about 29 kDa. Digestion of the immunoprecipitated 29-kDa species with N-glycanase converted the 29-kDa form into two forms of about 23 and 18 kDa, suggesting that the increase in molecular weight of the deletion mutant protein resulted from hyperglycosylation. Adding tunicamycin to the culture medium of cells transfected with GM-CSF(del) also yielded a single non-N-glycosylated species of about 18 kDa, but secretion was at a significantly lower level than either the 29-kDa hyperglycosylated GM-CSF(del) protein from non-tunicamycin-treated cells or the 18-kDa non-N-glycosylated full-length GM-CSF from tunicamycin-treated cells. Since very recent scanning-deletion analysis indicates that there is a critical region for activity near Cys-118 and that Cys-118 is necessary for maximal activity, we conclude that the Cys-118 residue is necessary for proper glycosylation and maximal biologic activity of GM-CSF.
 ...
PMID:Deletion of carboxy-terminal residues of murine granulocyte-macrophage colony-stimulating factor results in a loss of biologic activity and altered glycosylation. 240 51


1 2 3 4 Next >>