Sites of phosphorylation in recombinant human interferon-gamma

Recombinant human interferon-gamma was phosphorylated with ATP and c-AMP-dependent protein kinase. After phosphorylation, interferon-gamma was separated from the adenosine phosphates and the kinase and analyzed by SDS-PAGE, reverse phase HPLC, and HPLC peptide mapping. Comparison of the S. aureus V8 protease maps of intact and phosphorylated interferon-gamma showed that the maps were identical except that one peptide fragment elutes earlier in the map of the phosphorylated sample. This peptide was identified as the C-terminal fragment containing two serinyl phosphorylation sites at positions 132 and 142. This phosphorylated interferon-gamma exhibited a slightly higher specific antiviral activity than the intact protein.     

Metabolic effects on recombinant interferon-gamma glycosylation in continuous culture of Chinese hamster ovary cells

Asparagine linked (N-linked) glycosylation is an important modification of recombinant proteins, because the attached oligosaccharide chains can significantly alter protein properties. Potential glycosylation sites are not always occupied with oligosaccharide, and site occupancy can change with the culture environment. To investigate the relationship between metabolism and glycosylation site occupancy, we studied the glycosylation of recombinant human interferon-gamma (IFN-gamma) produced in continuous culture of Chinese hamster ovary cells. Intracellular nucleotide sugar levels and IFN-gamma glycosylation were measured at different steady states which were characterized by central carbon metabolic fluxes estimated by material balances and extracellular metabolite rate measurements. Although site occupancy varied over a rather narrow range, we found that differences correlated with the intracellular pool of UDP-N-acetylglucosamine + UDP-N-acetylgalactosamine (UDP-GNAc). Measured nucleotide levels and estimates of central carbon metabolic fluxes point to UTP depletion as the cause of decreased UDP-GNAc during glucose limitation. Glucose limited cells preferentially utilized available carbon for energy production, causing reduced nucleotide biosynthesis. Lower nucleoside triphosphate pools in turn led to lower nucleotide sugar pools and reduced glycosylation site occupancy. Subsequent experiments in batch and fed-batch culture have confirmed that UDP-sugar concentrations are correlated with UTP levels in the absence of glutamine limitation. Glutamine limitation appears to influence glycosylation by reducing amino sugar formation and hence UDP-GNAc concentration. The influence of nucleotide sugars on site occupancy may only be important during periods of extreme starvation, since relatively large changes in nucleotide sugar pools led to only minor changes in glycosylation.     

Preparation and characterization of recombinant DNA-derived human interferon-gamma

An attempt was made to prepare a highly purified, active recombinant DNA-derived human interferon-gamma. When the protein was denatured in urea and refolded, gel filtration and sedimentation velocity experiments indicated the presence of two forms, which are different in size and are not in a rapid reversible equilibrium. The two forms could be chromatographically separated. Far-UV circular dichroic spectra indicated the presence of secondary structures for both forms. Near-UV circular dichroic spectra revealed that the smaller form is folded into a rigid tertiary structure. The antiviral activity of the two forms of interferon-gamma showed a significant difference, i.e. the smaller form was 4-8-fold more active than the larger form. A variety of experiments show that the smaller form is more active, homogeneous, soluble, and stable than the larger form.     

Reversibility of acid denaturation of recombinant interferon-gamma

It has been shown that interferon-gamma (IFN-gamma) loses activity after acid treatment and this property can be used to distinguish it from other types of interferons. Therefore, reversibility of acid denaturation of IFN-gamma was examined using the recombinant human protein. The fluorescence spectra showed that conformation of the protein is similar before and after acid treatment, suggesting reversibility of the acid denaturation. The antiviral activity of the protein was also identical in the same treatment. However, the antiviral activity was significantly reduced when it was determined by directly diluting the acidic samples into the assay medium containing high salts and serum proteins. Similar results were obtained with the recombinant murine IFN-gamma. This observation demonstrates that acid denaturation of the IFN-gamma is dependent on the way the protein is renatured, and hence that the difference in response to acid treatment between IFN-gamma and other interferons is quantitative rather than qualitative.     

Activation of bovine neutrophils by recombinant interferon-gamma

The effect of recombinant bovine interferon-gamma (r-IFN-gamma) on neutrophil functions was investigated and compared to the effects of an unpurified lymphokine preparation. Incubation of purified bovine neutrophils with r-IFN-gamma or antigen-induced lymphokine for 2.5 hr at 37 degrees C resulted in impairment of the ability of neutrophils to migrate under agarose, and an enhancement of their ability to mediate antibody-dependent and antibody-independent cell-mediated cytotoxicity against chicken erythrocytes. Neither the lymphokine preparation nor the r-IFN-gamma had any influence on Staphylococcus aureus ingestion, or iodination by neutrophils. The lymphokine preparation enhanced cytochrome c reduction by neutrophils and was weakly chemotactic, whereas the r-IFN-gamma had neither of these effects. Only 5 min of r-IFN-gamma preincubation with neutrophils were needed to trigger protein synthesis by the neutrophils resulting in inhibition of random migration. Therefore, recombinant interferon-gamma acts as a neutrophil migration inhibition factor and a neutrophil activation factor resulting in enhanced neutrophil-mediated antibody-dependent and -independent cell-mediated cytotoxicity. Many, but not all, of the in vitro effects of an unpurified lymphokine preparation on neutrophil function can be attributed to the interferon-gamma contained in the lymphokine.     

Time-dependent differential effects of natural and recombinant murine interferon-gamma on ornithine decarboxylase activity of tumor cells

Incubation of quiescent tumor cells with fetal calf serum induced ornithine decarboxylase (ODCase) activity concomitantly with mitogenic stimulation. Pretreatment of cells with highly purified natural or recombinant murine interferon-gamma (MuIFN-gamma) for 5 h caused a dose-dependent increase of ODCase activity induced by fetal calf serum (FCS). Pretreatment of target cells with IFN-gamma for 5 h in absence of FCS stimulation did not induce ODCase activity. When pretreatment of cells with natural or recombinant MuIFN-gamma was prolonged for 18 h both ODCase activity and DNA synthesis induced by FCS were suppressed. By contrast when a mixture of MuIFN-alpha and -beta was used, ODCase activity was significantly suppressed after 5 h pretreatment compared to untreated controls. These results suggest that IFN-gamma exerts a differential effect on mitogen-stimulated events depending on the dose and the time of addition.     

重组鸡γ_干扰素在昆虫细胞中的高效表达

将鸡γ-干扰素(ChIFN-γ)基因克隆到载体pFASTBAC1中,构建转移载体pFASTBAC1-ChIFN-γ,然后转化DH10Bac感受态大肠杆菌,通过位点特异性转座,将ChIFN-γ基因整合到Bacmid穿梭载体中,构建表达质粒Bacmid-ChIFN-γ;通过脂质体将表达质粒转染Sf9昆虫细胞,用间接免疫荧光试验鉴定重组鸡γ-干扰素(rChIFN-γ)的表达;通过水泡性口炎病毒感染鸡胚成纤维细胞(CEF)的细胞病变抑制试验,检测rChIFN-γ的活性。研究结果表明,感染重组杆状病毒的Sf9细胞能高效表达rChIFN-γ,且当每个细胞的感染量为1个病毒时,细胞在感染96h后,rChIFN-γ基因表达产物的活性最高,达到106~107.2U/mL。以rChIFN-γ进行对新城疫病毒(NDV)F48E8株、禽流感H5N1病毒(AIV-H5)和马立克氏病病毒(MDV)GA株的抗病毒活性试验,发现rChIFN-γ对AIV-H5和MDV GA株病毒有明显的抑制其致细胞病变作用,但对NDVF48E8株病毒在体外不能抑制其致细胞病变作用,仅能在病毒滴度上表现抑制效果。     

Fluorescence studies on denaturation and stability of recombinant human interferon-gamma

Unfolding/folding transitions of recombinant human interferon-gamma (hIFNgamma) in urea and guanidine chloride (Gn.HCl) solutions were studied by fluorescence spectroscopy. At pH 7.4 Gn.HCl was a much more efficient denaturant (midpoint of unfolding C* = 1.1 M and deltaG0 = 13.4 kJ/mol) than urea (C* = 2.8 M and deltaG0 = 11.7 kJ/mol). The close deltaG0 values indicate that the contribution of electrostatic interactions to the stability of hIFNgamma is insignificant. Both the pH dependence of the fluorescence intensity and the unfolding experiments in urea at variable pH showed that hIFNgamma remains native in the pH range of 4.8-9.5. Using two quenchers, iodide and acrylamide, and applying the Stern-Volmer equation, a cluster of acidic groups situated in close proximity to the single tryptophan residue was identified. Based on the denaturation experiments at different pH values and on our earlier calculations of the electrostatic interactions in hIFNgamma, we assume that the protonation of Asp63 causes conformational changes having a substantial impact on the stability of hIFNgamma.     

Crystallization and preliminary x-ray diffraction studies of recombinant rabbit interferon-gamma

Two different crystal forms of recombinant rabbit IFN-gamma were obtained under different crystallization conditions. The first, a tetragonal form with space group P43212 or P41212, was obtained through vapor phase equilibration using the sitting drop rods technique with ammonium citrate as the major precipitating agent. The unit cell dimensions of this crystal form are a = b = 82.1 A and C = 116.3 A. These crystals diffract to 2.8 A resolution and contain a dimer in the asymmetric unit. A second crystal form was obtained by the batch method at pH 8.0 using sodium chloride as the precipitating agent. The crystals are hexagonal, space group P6122 or P6522, and with unit cell dimensions of a = b = 58.0 A and c = 169 A. This form contains monomer in the asymmetric unit and diffracts to greater than 2.7 A resolution. Both forms appear to be eminently suitable for further analyses and crystal structure solution.     

Stimulation of microtubule assembly by recombinant human interferon-alpha and interferon-gamma

Interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) have been demonstrated to stimulate microtubule assembly measured in the in vitro assembly system. The process is substoichiometric occurring when the interferon concentrations are below that of tubulin. IFN-gamma is a more potent effector than IFN-alpha. The critical tubulin concentration describing microtubule assembly decreases from 1.5 mg/ml measured in the absence of added effector to 1.05 mg/ml and 1.3 mg/ml when measured in the presence of 2.16.10(-6) M IFN-gamma and 3.06.10(-6) M IFN-alpha, respectively.     

In vivo and in vitro activation of alveolar macrophages by recombinant interferon-gamma

In vivo administration of recombinant interferon-gamma (rIFN-gamma) was previously shown to result in activation of the microbicidal activities of peritoneal macrophages (PM phi). Because macrophages at different anatomical sites vary in their functional capacities, we considered it of interest to determine whether administration of murine rIFN-gamma, either in vitro or in vivo, can enhance the microbicidal activity of resident alveolar macrophages (AM phi) and to compare the effects of rIFN-gamma on AM phi and PM phi. After incubation in vitro with rIFN-gamma, the antimicrobial activities of both murine AM phi and PM phi were enhanced, as assessed by their ability to inhibit replication of the intracellular parasite, Toxoplasma gondii. This effect was dose dependent for AM phi over a range of 0.1 to 1 U/ml and for PM phi over a range of 0.5 to 1000 U/ml. In this assay, the minimum dosage required for in vitro activation of AM phi was one-half that required for activation of PM phi, suggesting a greater sensitivity of AM phi to the in vitro activity of rIFN-gamma. Macrophages from both anatomical sites were also activated when rIFN-gamma was administered in vivo. This effect was dose dependent over a range of 10(3) to 10(5) U/mouse. Freshly harvested AM phi and PM phi from mice injected 24 hr earlier with 10(4) U rIFN-gamma by either the i.v. or i.p. routes markedly inhibited intracellular multiplication of Toxoplasma. In contrast, AM phi and PM phi from control mice permitted fourfold to ninefold increases in numbers of intracellular Toxoplasma. The anti-toxoplasma activity of AM phi and PM phi gradually diminished over a period of 3 days when assayed at successive 24 hr periods after a single i.v. injection of rIFN-gamma. At 3 days after injection, a substantial loss of anti-toxoplasma activity was observed with PM phi as compared with controls; residual anti-toxoplasma activity was still demonstrable in AM phi at 3 days. These results demonstrate that in vitro as well as in vivo treatment with rIFN-gamma confers on AM phi an enhanced antimicrobial activity. These findings provide a rationale for evaluating rIFN-gamma in the treatment of pulmonary infections, especially those due to opportunistic pathogens against which AM phi play a major role in host defense.     

The suppressive effects of recombinant human tumor necrosis factor-alpha on normal and malignant myelopoiesis: synergism with interferon-gamma

The modulation of growth of normal and leukemic myeloid progenitor cells in soft agar cultures by recombinant human tumor necrosis factor-alpha (TNF alpha) and recombinant human interferon-gamma (IFN gamma) was investigated. TNF alpha inhibited colony formation of all colony types representing different maturational stages of normal progenitor cells committed to the myeloid lineage with different orders of sensitivity. Blast-type colonies derived from patients with acute myelogenous leukemia were more sensitive to TNF alpha inhibition than progenitor cells purified from normal bone marrow or bone marrow from patients with stable-phase chronic myelogenous leukemia. The response of most colony types to IFN gamma was poor. However, when IFN gamma was administered together with TNF alpha, synergistically enhanced antiproliferative effects were detected in all colony types tested. The antiproliferative action of IFN gamma on myelopoiesis was enhanced in culture by the presence of autologous monocytes, presumedly by inducing endogenous production of TNF alpha. However, TNF alpha seemed to act directly on the progenitor cells themselves to suppress their clonal growth, rather than involving accessory marrow elements such as monocytes and/or T lymphocytes.     

Heterogeneity of Chinese hamster ovary cell-produced recombinant murine interferon-gamma

Chinese hamster ovary cells transformed with a hybrid expression plasmid containing both the murine interferon-gamma (MuIFN-gamma) and the murine dihydrofolate reductase-coding sequences were subjected to selection in stepwise increasing concentrations of methotrexate. By this procedure the production rate of MuIFN-gamma was increased from an initial level of approximately 20,000 to approximately 500,000 antiviral units per milliliter of culture supernatant. [35S]Methionine-labeled proteins secreted by these cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without prior immunoprecipitation with polyclonal or monoclonal antibodies against splenocyte-derived MuIFN-gamma. Besides two major components of Mr 19,000 and 38,000, a multiplicity of minor components were immunoprecipitated. Cells treated with tunicamycin, an inhibitor of N-glycosylation, secrete two major components of Mr 14,000 and 27,000 and only two minor components of Mr 12,000 and 13,000. When the proteins were labeled with [35S]cysteine, a residue that is only present at the carboxyl terminus of the mature MuIFN-gamma, no minor components could be detected in the growth medium of tunicamycin-treated cells. The presented results indicate that the heterogeneity of the recombinant Chinese hamster ovary-produced MuIFN-gamma is due to at least three cumulative modifications of the Mr 14,000 MuIFN-gamma peptide: carboxyl-terminal proteolytic processing (the Mr 13,000 and 12,000 components), variations in N-glycosylation (components ranging in size from Mr 12,000 to 26,500), and dimerization (components ranging from Mr 27,000 to 50,000).