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D Cosman

Publications and source records attributed to D Cosman.

At least 91 records · Page 5Linked to original sources
PubMed1988

cDNA expression cloning of the IL-1 receptor, a member of the immunoglobulin superfamily.

Interleukin-1 alpha and -1 beta (IL-1 alpha and IL-1 beta) are cytokines that participate in the regulation of immune responses, inflammatory reactions, and hematopoiesis. A direct expression strategy was used to clone the receptor for IL-1 from mouse T cells. The product of the cloned complementary DNA binds both IL-1 alpha and IL-1 beta in a manner indistinguishable from that of the native T cell IL-1 receptor. The extracellular, IL-1 binding portion of the receptor is 319 amino acids in length and is composed of three immunoglobulin-like domains. The cytoplasmic portion of the receptor is 217 amino acids long.

Amino Acid Sequence↗
PubMed1988

Generation of biologically active interleukin-1 beta by proteolytic cleavage of the inactive precursor.

Interleukin-1 beta (IL-1 beta) is derived from an inactive precursor by proteolytic cleavage. To study IL-1 beta processing, we expressed the precursor in Escherichia coli, partially purified it, and used it as a substrate for various potentially relevant protease preparations. The precursor alone was virtually inactive, but incubation with membranes from human monocytes or myeloid cell lines yielded a 500-fold increase in IL-1 bioactivity. Western blot analysis of the incubated material showed that the 31,000-Da precursor is broken down to three major products, ranging from 17,400 to about 19,000 Da. The most active of these products is the smallest one, and it co-migrates during electrophoresis with mature IL-1 beta. Four purified known proteases were also tested for their effect on precursor IL-1 beta, and none of these products co-migrated with the mature protein. Chymotrypsin and Staphylococcus aureus protease yielded slightly larger products, which were highly active. Elastase and trypsin yielded substantially larger products, and these had little IL-1 activity. The products of three of the known proteases were identified by NH2-terminal sequencing. These results show conclusively that proteolysis of precursor IL-1 beta generates biological activity and that the cleavage must occur close to the mature NH2 terminus.

Base Sequence↗
PubMed1988

Human macrophage-colony stimulating factor: alternative RNA and protein processing from a single gene.

Macrophage-colony stimulating factor (M-CSF, CSF-1) has been reported to be required for the proliferation and differentiation of macrophages from hematopoietic progenitor cells. Recently, two human M-CSF cDNA clones were isolated encoding proteins of 256 and 554 amino acids. We report here the isolation of a third M-CSF cDNA that encodes a protein of 438 amino acids. The coding regions for the three cDNA clones share a common amino-terminus of 149 amino acids and a common carboxyl-terminus of 75 amino acids including a membrane spanning region. In addition, we isolated a genomic clone of human M-CSF. When each of the cDNA clones or the genomic clone were transfected into COS-7 monkey kidney cells, biologically active M-CSF was expressed as judged by the ability of transfected cell supernatants to stimulate proliferation and colony formation of murine bone marrow cells, as well as formation of monocytic colonies from human bone marrow cells. Surprisingly, proliferation of human bone marrow cells was not induced by recombinant human M-CSF. Analysis of the M-CSF proteins released by COS-7 cells revealed that monomer subunit proteins of 44 or 28 kDa were produced. In addition, we found that the membrane spanning region, present in all three forms of M-CSF cDNA, was not required for the synthesis of a biologically active protein. However, when the membrane spanning region was present in the three M-CSF cDNAs, cell surface associated forms of M-CSF could be readily detected.

Animals↗
PubMed1988

Bovine GM-CSF: molecular cloning and biological activity of the recombinant protein.

The lymphokine granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates the growth and differentiation of granulocytes and macrophages from bone marrow progenitors, and regulates biological functions expressed by mature cells of these lineages. In order to isolate a bovine GM-CSF cDNA, a cDNA library, generated from the BT2 bovine T cell line, was screened with a human GM-CSF cDNA probe. A cDNA clone was isolated with an insert of 783 bp, that would encode a protein of 143 amino acids, with a predicted mol. wt of 16,160. Bovine GM-CSF exhibits a high degree of sequence homology with mouse and human GM-CSF at both the nucleotide and amino acid levels. Comparison of GM-CSF amino acid sequences from the three species indicates that the bovine GM-CSF precursor contains a putative 17 amino acid signal sequence, cleavage of which would yield a 14,250 mol. wt protein. The cDNA was inserted into a mammalian expression vector and transfected into COS-7 monkey kidney cells. Biologically active bovine GM-CSF was secreted as judged by a bovine bone marrow proliferation assay. Bovine GM-CSF was weakly active in both human and mouse bone marrow proliferation assays. In contrast, human GM-CSF was weakly active on bovine but not murine mouse bone marrow cells and mouse GM-CSF was only active on murine bone marrow cells.

Amino Acid Sequence↗
PubMed1988

Cloning, sequence and expression of bovine interleukin 1 alpha and interleukin 1 beta complementary DNAs.

Interleukin 1 (IL-1) is a cytokine which mediates a variety of immunoregulatory and inflammatory activities. Using human IL-1 alpha and IL-1 beta probes, cDNAs for the corresponding bovine genes were isolated from an alveolar macrophage library. The open reading frames of the bovine IL-1 alpha and IL-1 beta cDNAs encode proteins of 268 and 266 amino acids, respectively, each with a predicted mol. wt of approx. 31,000. Both forms of bovine IL-1 exhibit a high degree of sequence homology with IL-1 gene products from other mammalian species. Based upon comparisons with human IL-1 amino acid sequences, the post-translationally processed, mature forms of bovine IL-1 would occur as 17-18,000 mol. wt proteins. Sequences encoding mature bovine IL-1 alpha and IL-1 beta were inserted into E. coli expression plasmids and biologically active proteins were synthesized as judged by the ability of the recombinant proteins to induce proliferation of bovine thymocytes. Both IL-1 alpha and IL-1 beta exist as single genomic copies. In addition, bovine IL-1 beta mRNA is approx. 10-fold more abundant than IL-1 alpha mRNA in stimulated alveolar macrophages.

Amino Acid Sequence↗
PubMed1988

Stably transmitted triple-promoter retroviral vectors and their use in transformation of primary mammalian cells.

Retroviral vectors were constructed which coexpressed three inserted genes from independent transcriptional promoters in singly infected cells. Several such triple-promoter vectors were constructed with various combinations of oncogenes and selectable drug resistance genes. All expressed three mRNAs of the expected size in infected cells. One vector expressing the v-Ha-ras, v-myc, and neo genes was characterized in detail. This retrovirus did not undergo rearrangement during the process of infection, as judged by Southern analysis, and infection of primary rat embryo fibroblasts demonstrated that ras-myc-cotransformed cells could be selected in G418. This demonstration that retroviral vectors can be used to express three cistrons independently increases their value as gene transfer vehicles, particularly for studies involving oncogene cooperation in primary cells.

Cell Line↗
PubMed1988

Human interleukin-3 and granulocyte-macrophage colony stimulating factor: site-specific mutagenesis and expression in yeast.

The two human colony stimulating factors, interleukin-3 and granulocyte-macrophage colony stimulating factor, have been molecularly cloned and expressed as secreted proteins in yeast. In both cases, non-glycosylated and glycosylated forms of the molecules were produced. Removal of N-linked glycosylation sites from the genes by site-directed mutagenesis prevented addition of most of the sugar residues, but revealed a low level of residual O-linked glycosylation on a portion of the molecules. No difference in specific biological activity was found between the different forms of the proteins. It was found that a significant proportion of human granulocyte-macrophage colony stimulating factor was degraded by the yeast KEX2 protease that was cleaving after the dibasic sequence Arg-Arg at positions 23-24 of the mature protein. Site-specific mutagenesis was employed to change this sequence to Leu-Arg, and this change resulted in greatly increased expression levels of full length protein and biological activity.

Bone Marrow↗
PubMed1988

Production of recombinant human colony stimulating factors in yeast.

Efficient yeast expression and purification systems for production of recombinant human GM-CSF, IL-3 and G-CSF have been established. Though yeast-derived production of recombinant CSFs (through the use of secretion based system) allows for generation of native molecules which can then be readily separated from fermentation broth, in many instances, natural cDNAs have had to be altered to allow for efficient expression, as well as production of a less heterogeneous product. In the case of CSFs described herein, beneficial mutations (made through site-directed mutagenesis) have included elimination of potential N-linked glycosylation sites, removal of KexII protease recognition sites (notably alterations in dibasic sequences) and elimination of extraneous cysteine residues which might complicate isolation of a homogeneous product due to intermolecular disulfide bonding.

Amino Acid Sequence↗
PubMed1988

Human macrophage colony stimulating factor (M-CSF): alternate RNA splicing generates three different proteins that are expressed on the cell surface and secreted.

Human macrophage colony stimulating factor (M-CSF) cDNA clones were isolated from a pancreatic carcinoma cell line. Three different classes of M-CSF precursor protein (256, 554 and 438 amino acids in length) were predicted to be encoded by these cDNAs. Two of these, that we designate M-CSF alpha and M-CSF beta have already been described. The third, M-CSF gamma represents a novel class of M-CSF cDNA. All three precursors share a 32 amino acid signal sequence and the first 149 amino acids of the mature protein. At this position, M-CSF beta and gamma have insertions of 298 and 182 amino acids relative to M-CSF alpha. The first 182 amino acids of these insertions are shared between M-CSF beta and gamma. All three precursors share the C-terminal 75 amino acids that encode the transmembrane and cytoplasmic domains. Expression of all three cDNAs in COS-7 monkey kidney cells gave rise to soluble M-CSF activity, associated with proteins of subunit molecular weight 44 Kda (beta and gamma) or 28 Kda (alpha). In addition, M-CSF proteins could be detected on the surface of the transfected cells by indirect immunofluorescence. When the transmembrane and cytoplasmic domains of M-CSF alpha were removed by introducing a stop codon after amino acid 190, no membrane-bound M-CSF could be detected, but the truncated protein was secreted efficiently and was biologically active. This suggests that all three forms of M-CSF can exist as cell surface proteins, anchored by their hydrophobic transmembrane domains, and can be processed to soluble forms by proteolytic digestion. Although all soluble forms of M-CSF were biologically active in murine bone marrow colony and proliferation assays, they showed greatly reduced or no activity in similar assays using human bone marrow.

Amino Acid Sequence↗
PubMed1987

The interleukin-1 receptor binds the human interleukin-1 alpha precursor but not the interleukin-1 beta precursor.

Both IL-1 alpha and IL-1 beta are initially translated as approximately Mr 30,000 polypeptides and processed to approximately Mr 17,500 prior to, or during, release from macrophages. The current study utilizes an in vitro transcription-translation system to produce these four forms of IL-1 directly from cloned cDNAs, in order to investigate the relative receptor binding and biological activities of the proteins. The data show that the initial translation product from IL-1 beta mRNA must be processed in order to bind to the IL-1 receptor and hence express biological activity. By contrast, the initial translation product of IL-1 alpha mRNA can bind to the IL-1 receptor without further proteolytic processing. These data suggest that the IL-1 biological activity previously associated with proteins having Mr values in the range 30,000-40,000 is due to IL-1 alpha gene products.

Binding, Competitive↗
PubMed1987

Expression, purification and characterization of recombinant murine granulocyte-macrophage colony-stimulating factor and bovine interleukin-2 from yeast.

Expression and secretion of two lymphokines, murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) and bovine interleukin-2 (BoIL-2), to levels of 50-60 mg per liter were achieved by placing these cDNAs in a Saccharomyces cerevisiae expression vector that utilized the yeast alcohol dehydrogenase-2 promoter and alpha-factor leader peptide. These lymphokines were purified to homogeneity by direct application of the crude yeast medium to reversed-phase high-performance liquid chromatography. Despite the fact that both lymphokines contain at least one N-glycosylation site and have identical N-terminal residues (Ala-Pro-Thr), recombinant (R) GM-CSF was found to be heterogeneously glycosylated by yeast while RBoIL-2 was secreted without glycosylation. Additionally, approximately 40% of the RGM-CSF was found to be proteolytically cleaved after the second amino acid residue, while RBoIL-2 was found to be intact.

Animals↗
PubMed1987

Determination of the minimum polypeptide lengths of the functionally active sites of human interleukins 1 alpha and 1 beta.

Interleukin 1 (IL-1) is a two-member family of proteins (IL-1 alpha and IL-1 beta) that mediates a diverse series of immune and inflammatory responses. These two proteins have only 26% amino acid homology yet bind to the same receptor. It is of importance to define the active sites of these molecules in order to understand their receptor interactions and the mechanisms involved in their multiple biological functions. We report here the localization of the biologically active portions within the initial polypeptide translation products. An in vitro transcription and translation system was used to generate specific fragments of each of the IL-1 molecules, which then were assayed for receptor binding capability and biological activity. Using this system, we have demonstrated that core sequences of amino acids for IL-1 beta (numbers 120-266) and 140 amino acids for IL-1 alpha (numbers 128-267) must be left intact to retain full biological activity and further that the biological activities of the IL-1 polypeptides parallel their receptor binding capabilities.

Amino Acid Sequence↗
PubMed1987

Expression and purification of native human granulocyte-macrophage colony-stimulating factor from an Escherichia coli secretion vector.

The human granulocyte-macrophage colony stimulating factor (GM-CSF) was expressed and purified from a high-level Escherichia coli secretion vector. A cDNA fragment encoding mature GM-CSF was fused with the aid of a synthetic oligonucleotide to the E. coli outer membrane signal peptide (ompA) of the secretion expression vector pIN-III-ompA3. The primary construction, designated pLB5001, is under transcriptional control of the tandem lipoprotein promoter (lppP) lactose promoter-operator (lacPO), and is regulated by the lactose repressor. Upon induction, a polypeptide of MW = 14,600 was produced which had GM-CSF activity in a human bone marrow colony assay. The linker sequence between the ompA signal peptide and the amino terminus of the mature GM-CSF was removed by oligonucleotide-directed site-specific mutagenesis to produce GM-CSF with an authentic amino terminus. The resulting construct, designated pLB5001-4, expressed authentic GM-CSF with a specific activity similar to that observed for the pLB5001 specified GM-CSF. Both versions of GM-CSF were associated with the membrane fraction after osmotic shock, and were purified to homogeneity by DEAE-Sephacel chromatography, followed by reversed-phase HPLC. Amino acid sequencing from the amino terminus of the purified GM-CSF established that the ompA signal peptide was cleaved at its normal processing site in both cases.

Amino Acid Sequence↗
PubMed1987

Nature and specificity of lymphokine independence induced by a selectable retroviral vector expressing v-src.

A murine retroviral vector, LSNLsrc, has been constructed and examined for its ability to induce growth factor independence in cells normally dependent on interleukin 2 (IL-2) or interleukin 3 (IL-3) for growth. The LSNLsrc vector coexpressed the v-src gene of Rous sarcoma virus and the neo gene from transposon Tn5, allowing infected cells to be selected on the basis of G418 resistance. The murine cell lines CTLL-2 and FD.C/1, which are dependent for growth on IL-2 and IL-3, respectively, were both readily infected with the LSNLsrc virus. LSNLsrc-infected, G418-resistant cultures of FD.C/1 cells were able to give rise to IL-3-independent progeny, but all G418-resistant CTLL-2 cells retained normal IL-2 dependence. The induction of IL-3 independence by v-src was not a direct event, since limiting dilution analysis of the LSNLsrc-infected FD.C/1 cells showed that most of them were IL-3 dependent, despite expression of v-src mRNA and active pp60v-src kinase. However, clones selected from this population in the presence of IL-3 were able to undergo a subsequent progression event and generate IL-3-independent progeny. The generation of factor-independent variants in the clonal cultures was a rare event, as witnessed by the death of most of the cells in each clone when IL-3 was withdrawn. Together, these data indicate that a secondary event, in addition to v-src expression, was required to generate IL-3-independent growth. No evidence was found for an autocrine mechanism of transformation involving IL-2, IL-3, interleukin 4, or granulocyte-macrophage colony-stimulating factor.

Cell Division↗
PubMed1986

Cloning, sequence, and expression of bovine interferon-gamma.

Bovine interferon-gamma (IFN-gamma) sequences have been isolated by screening a cDNA library with a human IFN-gamma cDNA probe. The cDNA library was constructed from RNA isolated from concanavalin A-stimulated bovine lymph node cells. The open reading frame predicts that the bovine IFN-gamma precursor is composed of 166 amino acids with a predicted m.w. of 19,393. Alignment of the amino acid sequence with human IFN-gamma indicates that mature bovine IFN-gamma is composed of 143 amino acids with a predicted m.w. of 16,858. It has an amino acid homology of 63% with human IFN-gamma, and 47% with murine IFN-gamma. Biologically active bovine IFN-gamma was synthesized in an Escherichia coli expression system.

Amino Acid Sequence↗
PubMed1986

Phosphorylation of the human interleukin-2 receptor and a synthetic peptide identical to its C-terminal, cytoplasmic domain.

The recombinant human interleukin-2 (IL-2) receptor was expressed in mouse mammary epithelial cells following the transfection of these cells with an expression vector containing the human IL-2 receptor cDNA. The recombinant IL-2 receptor in these cells was rapidly phosphorylated in response to phorbol myristate acetate (PMA), but its phosphorylation could not be detected in the absence of PMA or upon addition of human IL-2. The C-terminal, cytoplasmic peptide domain of the IL-2 receptor, Gln-Arg-Arg-Gln-Arg-Lys-Ser-Arg-Arg-Thr-Ile, was synthesized and used as a substrate for protein kinase C. The Km for phosphorylation of the peptide by protein kinase C was 23 microM. The stoichiometry of phosphorylation was 1 mol of phosphate/mol of peptide and serine was the predominant amino acid phosphorylated. Because this peptide was a good substrate for protein kinase C in vitro, it was possible that the same serine (serine 247) was also phosphorylated in the receptor in the cell. The IL-2 receptor gene in the expression vector was therefore altered by site-directed mutagenesis to code for an IL-2 receptor containing an alanine in the place of serine 247. The IL-2 receptor expressed by these cells was not phosphorylated in the presence of PMA. These data suggest that protein kinase C, in response to PMA, phosphorylates the C-terminal serine residue (serine 247) in the human IL-2 receptor.

Amino Acid Sequence↗