A receptor fusion protein for the inhibition of murine oncostatin M

Background  

Most cytokines signal through heteromeric receptor complexes consisting of two or more different receptor subunits. Fusion proteins of the extracellular parts of receptor subunits turned out to be promising cytokine inhibitors useful in anti-cytokine therapy and cytokine research.     

Concentration-dependent internalization of a cytokine/cytokine receptor complex in human hematopoietic cells

Soluble steel factor (SF) is a potent stimulator of hematopoietic progenitor cell proliferation in vitro, and cytokine combinations that include SF can support extensive expansions of hematopoietic cells. Recently, we showed that very primitive progenitor cells from normal human bone marrow require exposure to very high concentrations of cytokines to maintain their primitive status while proliferating. These cells also display higher cell-specific cytokine uptake rates than more differentiated types of hematopoietic cells. As a first step toward identifying the mechanisms involved in mediating such cytokine dose-dependent effects, we have now investigated the kinetics of SF receptor (c-kit) internalization by human Mo7e cells exposed to different extracellular concentrations of soluble SF. Transfer of Mo7e cells to a higher concentration of SF caused an initially rapid downregulation of cell surface c-kit which was accompanied by a rapid depletion of extracellular SF. Confocal microscopy showed a concomitant increase in the number and intensity of intracellular c-kit aggregates. After the first 30 min, the cells continued to deplete SF from the medium but at a much slower rate. During this period, there was a gradual recovery of expression of c-kit on the cell surface. A mathematical analysis of bulk medium to cell-surface SF-mass transport indicated that the cytokine-depletion rates measured were not likely to have significantly depleted the SF concentration in the microenvironment of the cells. Taken together, these results underscore the importance of monitoring and appropriately regulating cytokine concentrations in hematopoietic cell expansion cultures. They may also help to explain the different biological responses exhibited by primitive hematopoietic cells exposed to different types and concentrations of cytokines for periods of days.     

Characterization of an anti-apoptotic glycoprotein encoded by Kaposi's sarcoma-associated herpesvirus which resembles a spliced variant of human survivin

We have investigated the expression and function of a novel protein encoded by open reading frame (ORF) K7 of Kaposi's sarcoma-associated herpesvirus (KSHV). Computational analyses revealed that K7 is structurally related to survivin-DeltaEx3, a splice variant of human survivin that protects cells from apoptosis by an undefined mechanism. Both K7 and survivin-DeltaEx3 contain a mitochondrial-targeting sequence, an N-terminal region of a BIR (baculovirus IAP repeat) domain and a putative BH2 (Bcl-2 homology)-like domain. These suggested that K7 is a new viral anti-apoptotic protein and survivin-DeltaEx3 is its likely cellular homologue. We show that K7 is a glycoprotein, which can inhibit apoptosis and anchor to intracellular membranes where Bcl-2 resides. K7 does not associate with Bax, but does bind to Bcl-2 via its putative BH2 domain. In addition, K7 binds to active caspase-3 via its BIR domain and thus inhibits the activity of caspase-3. The BH2 domain of K7 is crucial for the inhibition of caspase-3 activity and is therefore essential for its anti-apoptotic function. Furthermore, K7 bridges Bcl-2 and activated caspase-3 into a protein complex. K7 therefore appears to be an adaptor protein and part of an anti-apoptotic complex that presents effector caspases to Bcl-2, enabling Bcl-2 to inhibit caspase activity. These data also suggest that survivin-DeltaEx3 might function by a similar mechanism to that of K7. We denote K7 as vIAP (viral inhibitor-of-apoptosis protein).     

Wild mice express an Ig V lambda gene that differs from any V lambda in BALB/c but resembles a human V lambda subgroup.

The lambda L chain locus in the inbred mouse strains commonly used in the laboratory contains a limited number of germ-line genes; only three V lambda and three functional J lambda-C lambda genes have been identified in BALB/c mice. Previous studies indicated that wild mice may have a considerably expanded number of C lambda genes, as judged by the number of DNA restriction fragments that hybridize to C lambda probes derived from BALB/c. In order to evaluate the expression of these putative lambda genes, we have determined sequences of cDNA encoding lambda-chains in hybridomas from wild mice of the subspecies Mus musculus musculus from two different geographic regions, Denmark and Czechoslovakia. Two of these hybridomas produce L chains with J and C regions that are very similar to those of BALB/c lambda 1 chains, but the V regions of these L chains are only approximately 40% identical in amino acid sequence to the known murine V lambda. Indeed, these wild mouse V lambda are closer in sequence to human V lambda than they are to BALB/c V lambda, especially to human V lambda of subgroup VI, with which they share an unusual two-residue insertion in framework 3; L chains bearing V regions of this rare human type have a marked tendency to enter into amyloid deposits. These findings suggest that similar V lambda may be widespread in mammalian populations, although analysis by Southern blotting indicates that they are not found in BALB/c mice. A third hybridoma produces a L chain whose V lambda resembles BALB/c V lambda 1. The J lambda and C lambda segments of the cDNA encoding all three hybridoma L chains are identical; evidently, of the several putative genes that hybridize to C lambda 1 probes, one is expressed preferentially.     

Characterization of an antibody Fv fragment that binds to the human, but not to the rat neurotensin receptor NTS-1

The cDNAs coding for the heavy and light chain variable domains of an antibody, recognizing the human G-protein-coupled receptor for neurotensin, NTS-1, were obtained from a hybridoma cell line, B-N6. The Fv B-N6 fragment was expressed in Escherichia coli and purified. To characterize the properties of the antibody fragment, human and rat high-affinity neurotensin receptors were expressed in E. coli in functional form, linked at their N-termini to the maltose-binding protein. Fv B-N6 was found to compete for [3H]neurotensin binding to the human neurotensin receptor, but not to the rat neurotensin receptor, with IC50 values of 1.6 microM (membrane-bound receptor) and 1.9 microM (detergent-solubilized, purified receptor). The formation of a relatively stable complex of Fv B-N6 with purified human neurotensin receptor fusion protein was also demonstrated by gel filtration experiments. The Fv B-N6 fragment will be used to isolate a high-affinity binder to the human neurotensin receptor as a valuable tool for cocrystallization and receptor structure determination.     

The expressed human hepatic receptor for low-density lipoproteins differs from the fibroblast low-density lipoprotein receptor

The role of the cellular receptor for the low-density lipoproteins (LDL) in cholesterol transport was initially defined through the study of nonhepatic cells in vitro. Since the liver is central in plasma lipoprotein metabolism, an investigation of hepatic lipoprotein receptors is important for understanding normal lipoprotein transport. Utilizing human hepatic and fibroblast membranes, the characteristics of receptors for LDL from hepatic and nonhepatic tissues were directly compared. Human hepatic membranes reversibly bound LDL within 5 min. Although both fibroblast and hepatic membranes saturably bound LDL at 37 degrees C, the fibroblast LDL receptor affinity (Kd = 2.5 X 10(-8) M) and number (5.5 X 10(12) sites/mg membrane protein) were greater than the hepatic receptor affinity (Kd = 10.8 X 10(-8) M) and number (0.5 X 10(12) sites/mg membrane protein). In contrast to the fibroblast LDL receptor which was unable to bind LDL in the presence of EDTA, the hepatic LDL receptor binding of LDL was only partially blocked by EDTA. The binding of LDL to its hepatic receptor is highly temperature-dependent, and studies utilizing both radiolabeled LDL and colloidal gold-labeled LDL indicate that little, if any, binding of LDL hepatic membranes occur at 0-4 degrees C. The hepatic membrane receptor(s) (Mr approximately equal to 270 000 and 330 000) differ from that of the fibroblast LDL receptor (Mr approximately equal to 130 000) and these proteins are present in hepatic membranes from a patient lacking the fibroblast LDL receptor. These data indicate that an expressed hepatic LDL receptor has unique properties different from those of the fibroblast LDL receptor and that the expressed protein(s) is genetically distinct from the fibroblast receptor.     

Structure of the murine erythropoietin receptor complex. Characterization of the erythropoietin cross-linked proteins.

The structure of the murine erythropoietin receptor was studied using antibodies against the intracellular part of the cloned erythropoietin receptor chain. These antibodies precipitated erythropoietin-receptor complexes from Triton X-100-solubilized cells. When the complexes were cross-linked by disuccinimidyl suberate, the 85- and 100-kDa erythropoietin-cross-linked proteins previously described were immunoprecipitated. However, these proteins were not precipitated when the complexes were denatured and reduced before immunoprecipitation. Using 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide, we observed erythropoietin cross-linking with a protein of 66 kDa in addition to the 100- and 85-kDa proteins. Only the 66-kDa erythropoietin-cross-linked protein was immunoprecipitated by anti-receptor antibodies after denaturation and reduction of the complex. Thus, our results suggest that the 85- and 100-kDa proteins previously evidenced by cross-linking are associated with the cloned chain of the receptor to form a multimeric complex but these proteins seem immunologically unrelated to the cloned chain. We observed that reducing the length of molecules able to cross-link amino groups decreased the efficiency of cross-linking with the 100-kDa protein and only the 85-kDa protein was cross-linked with erythropoietin using 1,5-difluoro-2,4-dinitrobenzene. These results suggest that the 85- and 100-kDa proteins occupate slightly different positions relative to the erythropoietin molecule bound to the receptor.     

Characterization of the soluble human granulocyte-macrophage colony-stimulating factor receptor complex.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-R) is expressed on both hematopoietic and non-hematopoietic tissues. Although the receptor has been identified by cross-linking studies as an 84,000-dalton protein, very little is known about its biochemistry. In this report, we describe a soluble binding assay for the human GM-R which allowed us to characterize the receptor complex from various sources, including plasma membranes of placenta, neutrophils, and human myeloid leukemia cell lines. Preparation of membranes as well as solubilization by Triton X-100 and N-octylglucoside resulted in a 5-10-fold lower affinity of the receptor for GM-CSF. The Kd decreased from 20 to 80 pM in intact cells to 200-500 pM in both intact and solubilized membranes. Binding in solution was rapid, specific for GM-CSF, and best fit a "one-site" model with an approximate Kd of 500 pM. The dissociation rate constant for the soluble GM-R was very similar to that of intact cells (k2 = 0.013 min-1 versus 0.017 min-1, respectively). As expected, solubilized membranes obtained from those cells expressing the highest number of GM-R (neutrophils and dimethyl sulfoxide-induced HL-60 cells; approximately 500-800 sites/cell) possessed the highest concentration of soluble GM-R (approximately 2-3 x 10(8) GM-R/micrograms). Cross-linking of 125I-GM-CSF to soluble GM-R resulted in the appearance of two specifically labeled complexes. A major 110-kDa receptor-ligand complex is found when cross-linking is performed with intact cells; both 110- and 200-kDa species are seen when cross-linking is performed with either intact membranes or soluble GM-R. These studies define methods by which intact GM-R can be solubilized and measured in solution, permitting a more complete biochemical characterization of the intact GM-R complex.     

Characterization of the purified molybdate-stabilized glucocorticoid receptor from rat liver. An in vitro transformable complex

Rat liver glucocorticoid receptor was purified in the presence of molybdate by a three-step procedure comprising protamine sulfate precipitation, affinity chromatography on a dexamethasone matrix and high-performance size-exclusion chromatography (HPSEC) on a TSK G 3000 SW column. The [3H]triamcinolone-acetonide-receptor complex was obtained in 20% yield with an overall 11 800-fold purification. The dissociation rate constant of this complex was 1.6 X 10(-4) min-1. The purified receptor sedimented at 8.3 S in high-salt and 9.4 S in low-salt sucrose gradients containing molybdate. A 7.0-nm Stokes radius was determined by HPSEC on a TSK G 4000 column in high-salt buffer. The calculated Mr was 278000. Dodecyl sulfate/polyacrylamide gel electrophoresis revealed an almost homogeneous 90 000-Mr band. Three minor bands with Mr of 78 000, 72 000 and 48 000 were also inconstantly seen. An apparent pI = 5.1 was observed for the [3H]steroid complex by isoelectric focusing in agarose gel. Furthermore high-performance ion-exchange chromatography of the purified complex on a DEAE 545 LKB column (DEAE HPLC) yielded a sharp peak eluted at a 315 mM potassium ion concentration. This peak was shown to contain almost all the 90 000-Mr protein. Moreover the purified receptor complex appeared to be transformable to a DNA-binding state after molybdate removal followed by warming 30 min at 25 degrees C in presence of 0.2% bovine serum albumin: 50-78% transformation yield could be demonstrated by DNA-cellulose chromatography. Partial transformation could also be obtained at 0 degrees C in the absence of any added protein and was followed by DEAE HPLC. The transformed complex was eluted by 180 mM potassium.     

Characterization of a human homologue of the murine peripheral lymph node homing receptor

Lymphocyte trafficking is a fundamental aspect of the immune system that allows B and T lymphocytes with diverse antigen recognition specificities to be exposed to various antigenic stimuli in spatially distinct regions of an organism. A lymphocyte adhesion molecule that is involved with this trafficking phenomenon has been termed the homing receptor. Previous work (Lasky, L., T. Yednock, M. Singer, D. Dowbenko, C. Fennie, H. Rodriguez, T. Nguyen, S. Stachel, and S. Rosen. 1989. Cell. 56:1045-1055) has characterized a cDNA clone encoding a murine homing receptor that is involved in trafficking of lymphocytes to peripheral lymph nodes. This molecule was found to contain a number of protein motifs, the most intriguing of which was a carbohydrate binding domain, or lectin, that is apparently involved in the adhesive interaction between murine lymphocytes and peripheral lymph node endothelium. In this study, we have used the murine cDNA clone to isolate a human homologue of this peripheral lymph node-specific adhesion molecule. The human receptor was found to be highly homologous to the murine receptor in overall sequence, but showed no sequence similarity to another surface protein that may be involved with human lymphocyte homing, the Hermes glycoprotein. The extracellular region of the human receptor contained an NH2 terminally located carbohydrate binding domain followed by an EGF-like domain and a domain containing two repeats of a complement binding motif. Transient cell transfection assays using the human receptor cDNA showed that it encoded a surface glycoprotein that cross reacted with a polyclonal antibody directed against the murine peripheral lymph node homing receptor. Interestingly, the human receptor showed a high degree of sequence homology to another human cell adhesion glycoprotein, the endothelial cell adhesion molecule ELAM.     

Ablation of steroid receptor coactivator-3 resembles the human CACT metabolic myopathy

Oxidation of lipid substrates is essential for survival in fasting and other catabolic conditions, sparing glucose for the brain and other glucose-dependent tissues. Here we show Steroid Receptor Coactivator-3 (SRC-3) plays a central role in long chain fatty acid metabolism by directly regulating carnitine/acyl-carnitine translocase (CACT) gene expression. Genetic deficiency of CACT in humans is accompanied by a constellation of metabolic and toxicity phenotypes including hypoketonemia, hypoglycemia, hyperammonemia, and impaired neurologic, cardiac and skeletal muscle performance, each of which is apparent in mice lacking SRC-3 expression. Consistent with human cases of CACT deficiency, dietary rescue with short chain fatty acids drastically attenuates the clinical hallmarks of the disease in mice devoid of SRC-3. Collectively, our results position SRC-3 as a key regulator of β-oxidation. Moreover, these findings allow us to consider platform coactivators such as the SRCs as potential contributors to syndromes such as CACT deficiency, previously considered as monogenic.     

Inhibition of phosphodiesterase 4 modulates cytokine induction from toll like receptor activated,but not rhinovirus infected,primary human airway smooth muscle

Background

Virus-induced exacerbations of Chronic Obstructive Pulmonary Disease (COPD) are a significant health burden and occur even in those receiving the best current therapies. Rhinovirus (RV) infections are responsible for half of all COPD exacerbations. The mechanism by which exacerbations occur remains undefined, however it is likely to be due to virus-induced inflammation. Given that phophodiesterase 4 (PDE₄) inhibitors have an anti-inflammatory effect in patients with COPD they present a potential therapy prior to, and during, these exacerbations.

Methods

In the present study we investigated whether the PDE₄ inhibitor piclamilast (10^-6 M) could alter RV or viral mimetic (5 μg/mL of imiquimod or poly I:C) induced inflammation and RV replication in primary human airway smooth muscle cells (ASMC) and bronchial epithelial cells (HBEC). The mediators IL-6, IL-8, prostaglandin E₂ and cAMP production were assayed by ELISA and RV replication was assayed by viral titration.

Results

We found that in ASMCs the TLR3 agonist poly I:C induced IL-8 release was reduced while induced IL-6 release by the TLR7/8 agonist imiquimod was further increased by the presence of piclamilast. However, in RV infected ASMCs, virus replication and induced mediator release were unaltered by piclamilast, as was also found in HBECs. The novel findings of this study reveal that although PDE inhibitors may not influence RV-induced cytokine production in ASMCs and replication in either ASMCs or HBECs, they have the capacity to be anti-inflammatory during TLR activation by modulating the induction of these chemotactic cytokines.

Conclusion

By extrapolating our in vitro findings to exacerbations of COPD in vivo this suggests that PDE₄ inhibitors may have beneficial anti-inflammatory properties when patients are infected with bacteria or viruses other than RV.     

Characterization of a novel complex from halophilic archaebacteria, which displays chaperone-like activities in vitro

We isolated a protein, P45, from the extreme halophilic archaeon Haloarcula marismortui, which displays molecular chaperone activities in vitro. P45 is a weak ATPase that assembles into a large ring-shaped oligomeric complex comprising about 10 subunits. The protein shows no significant homology to any known protein. P45 forms complexes with halophilic malate dehydrogenase during its salt-dependent denaturation/renaturation and decreases the rate of deactivation of the enzyme in an ATP-dependent manner. Compared with other halophilic proteins, the P45 complex appears to be much less dependent on salt for its various activities or stability. In vivo experiments showed that P45 accumulates when cells are exposed to a low salt environment. We suggest, therefore, that P45 could protect halophilic proteins against denaturation under conditions of cellular hyposaline stress.     

Characterization of the Ah receptor from human placental tissue

The rate of thermal inactivation of the unliganded human Ah receptor, studied by sucrose density gradient centrifugation, with respect to loss of ligand binding ability, was found to be greater than those of most rodents at 20°C, but the temperature coefficient of the rate constant was much smaller than for the rodent species. This implies that the unliganded human Ah receptor would be thermally more stable than the rodent analogs at physiological temperatures. The liganded form of the human Ah receptor was found to be less stable with respect to ligand release than the rodent receptors. These differences in behavior between human and rodent Ah receptors underline the difficulties in using rodent data in the development of receptor-based models of dioxin toxicity. Attempts to develop an alternative to sucrose density gradient centrifugation, comparable with the hydroxylapatite adsorption method used to assay rodent hepatic Ah receptor, were unsuccessful.     

Characterization of truncated alpha chain products from human, rat, and mouse high affinity receptor for immunoglobulin E

The high affinity receptor for immunoglobulin E (IgE) is a tetrameric structure (alpha beta gamma 2) consisting of non-covalently associated subunits: one IgE-binding alpha chain, one 4-fold membrane spanning beta chain, and two disulfide-linked gamma chains. Here, we have engineered alpha cDNA constructs (alpha trunc) encoding exclusively the leader peptide and the extracellular domain of the alpha subunit. Transfection of human alpha trunc into COS-7 cells resulted in the secretion of soluble IgE-binding polypeptides. By contrast, the polypeptides generated from rat and mouse alpha trunc transfections were sequestered in the endoplasmic reticulum and degraded even though they appeared to fold properly as judged by their capacity to bind IgE. Stable transfectants with human alpha trunc were obtained from a dihydrofolate reductase-deficient Chinese hamster ovary cell line. Several clones secreted substantial amounts (0.1 microgram/ml/10(6) cells) of IgE-binding polypeptides. The dissociation rate of bound IgE from this soluble truncated alpha (kappa-1 = 4.9 x 10(-6) s-1 at 25 degrees C) was characteristic of receptors on intact cells. After treatment with tunicamycin, the transfectants secreted unglycosylated 18-kDa polypeptides which could also bind IgE. These unglycosylated products had a tendency to form dimers and higher oligomers which were resistant to treatment by sodium dodecyl sulfate and reducing agents. These data demonstrate unequivocally that the extracellular domain of the alpha subunit is sufficient to mediate high affinity binding of IgE. Furthermore, posttranslational addition of carbohydrates is not required for proper folding and function of the receptor binding site. The truncated human alpha should be a suitable reagent for crystallographic analysis and for detailed analysis of the receptor binding sites.     

Characterization of the molybdate-stabilized glucocorticoid receptor from rat thymus.

The untransformed glucocorticoid receptor of rat thymus cytosol was characterized in the form of its complex with [1,2,4-3H]triamcinolone acetonide by ion-exchange chromatography and by gel filtration and sucrose-density-gradient ultracentrifugation at different ionic strengths. Molybdate (10 mM) was present throughout all experimental procedures and prevented receptor inactivation and degradation as well as transformation. At low ionic strength the molybdate-stabilized steroid-receptor complex was detected as a single highly asymmetric entity with a Stokes radius of 5.85 nm, a sedimentation coefficient of 9.6 S and an apparent molecular weight of 236 000. This form was converted into a smaller, even more asymmetric, form in increasing proportion as the ionic strength was increased. In the presence of 0.4 M-KCl, the smaller form had a Stokes radius of 4.95 nm, a sedimentation coefficient of 4.6 S and an apparent molecular weight of 95 500. It is concluded that the glucocorticoid-receptor complex exists at low ionic strengths as a homodimer or as a heterodimer in which only one subunit possesses a steroid-binding site, and that the process of dissociation into subunits brought about by increasing the ionic strength is a process distinct from, but possibly preceding, the transformation phenomenon responsible for conferring DNA-binding properties on the complex.     

Structure and activity of insect cytokine GBP which stimulates the EGF receptor

Growth-blocking peptide (GBP) is an insect cytokine that possesses diverse biological activities such as larval growth regulation, cell proliferation, and stimulation of immune cells. GBP is a 25-amino acid peptide with one disulfide bond. It has been revealed that the tertiary structure of GBP consists of an N- and C-terminal disordered region and a well-structured core. Although there is only a slight similarity between the primary structures of GBP and EGF and the molecular weight of GBP is about half that of EGF, GBP directly binds and activates the EGF receptor of human keratinocyte cells. Furthermore, the tertiary structure of the well-defined region of GBP is similar to that of the C-terminal domain of EGF. This review will focus on the tertiary structure of GBP and its activities, as compared with those of EGF.     

Characterization of Rauscher murine leukemia virus envelope glycoprotein receptor in membranes from murine fibroblasts.

Plasma membrane preparations from KA31 (mouse) cells contained receptors for the binding of Rauscher murine leukemia virus (R-MuLV) envelope glycoprotein, gp70. This binding was demonstrated by gel filtration of a mixture of the microsomal fraction of the cells and 125I-labeled gp70. A rapid and convenient assay was developed to measure the complex formation between the membrane receptors and gp70 involving specific precipitation of the complex by 3 to 4% polyethylene glycol. The complex formation was responsive to the concentrations of both the receptor and gp70 and also to changes in temperature and pH. The gp70 binding was a noncooperative, saturable process, and an association constant of 3.5 X 10(8) M-1 was estimated from the binding data. The complex formation was reversible and a near-total exchange of 125I-labeled gp70 in the complex was achieved by incubation with excess of unlabeled gp70. The complex formation was inhibited by protein denaturing agents, guanidine-hydrochloride and urea. Pretreatment of the membrane fractions with either chymotrypsin or phospholipase C led to a loss of the membrane-associated receptor activity, indicating that a lipoprotein structure was important for the receptor function, consistent with the observation that nonionic detergents strongly inhibited the complex formation.