A bacterial signal peptide directs efficient secretion of eukaryotic proteins in the baculovirus expression system.

Escherichia coli remains an organism of choice for the production of recombinant proteins required in large quantities. Whenever possible, secretion is the preferred strategy since it permits easy and efficient purification from the extracellular medium. Our efforts to use E. coli to secrete a human CD23 soluble variant fused to a pair of IgG binding domains via the Staphylococcal protein A signal peptide were unsuccessful. Surprisingly, when the same construct was expressed in the baculovirus system, efficient secretion was observed and cleavage of the signal peptide occurred at the expected site. Varying the genes in the fusions or the tags, or the topology of the gene and the tag, did not affect the high-level secretion and cleavage at the correct site. We envision that fusion of the bacterial signal sequence to eukaryotic recombinant genes will prove to be a tool of value for efficient protein secretion in insect cells using the baculovirus expression system.

Recombinant soluble trimeric CD40 ligand is biologically active.

CD40 ligand (CD40L) is expressed on the surface of activated CD4+ T cells, basophils, and mast cells. Binding of C40L to its receptor, CD40, on the surface of B cells stimulates B cell proliferation, adhesion and differentiation. A preparation of soluble, recombinant CD40L (Tyr-45 to Leu-261), containing the full-length 29-kDa protein and two smaller fragments of 18 and 14 kDa, has been shown to induce differentiation of B cells derived either from normal donors or from patients with X-linked hyper-IgM syndrome (Durandy, A., Schiff, C., Bonnefoy, J.-Y., Forveille, M., Rousset, F., Mazzei, G., Milili, M., and Fischer, A. (1993) Eur. J. Immunol. 23, 2294-2299). We have now purified each of these fragments to homogeneity and show that only the 18-kDa fragment (identified as Glu-108 to Leu-261) is biologically active. When expressed in recombinant form, the 18-kDa protein exhibited full activity in B cell proliferation and differentiation assays, was able to rescue of B cells from apoptosis, and bound soluble CD40. Sucrose gradient sedimentation shows that the 18-kDa protein sediments as an apparent homotrimer, a result consistent with the proposed trimeric structure of CD40L. This demonstrates that a soluble CD40L can stimulate CD40 in a manner indistinguishable from the membrane-bound form of the protein.

Large scale transient expression with COS cells.

We demonstrate here that transient expression with COS cells can be performed at the one litre scale for a period of more than 10 days. Cells grown in T225 flasks were transfected by electroporation, transferred into spinners, and then grown either in suspension or on microcarriers. A daily medium change significantly extented culture life and production time, compared with standard protocols.Concentrations of the product, the secreted fusion protein CD40-Fc, were comparable in microcarrier and suspension culture. Cultures were started in fetal calf serum containing medium and the subsequent production process was performed in a low protein serum free medium which allowed easy downstream processing. 10 litres of supernatant, collected from one transfected batch of cells, yielded 30 mg of purified and biologically active protein.In addition to developing a simplified protocol for generation of cells we also reduced the material (DNA, cuvettes) required for electroporation. Our results show that scale up of transient expression to the litre scale can be successfully acieved. This provides a new tool to generate milligram quantities of protein within weeks of gene cloning.

A Ca(2+)-independent protein kinase C from fission yeast.

A protein kinase C homologue of Schizosaccharomyces pombe, pkc1+, was isolated from a genomic library by screening with the Saccharomyces cerevisiae PKC1 probe. From its primary sequence and biochemical properties, we conclude that S. pombe pkc1+ encodes a phospholipid-activated Ca(2+)-independent protein kinase, homologous to the delta/epsilon classes of mammalian protein kinase C. Gene disruption experiments show that pkc1+ is not essential for cell viability; however, overexpression of the protein leads to an abnormal cell morphology and a block in cell separation following mitosis suggestive of a role in cell division. In vitro phosphorylation experiments reveal several potential pkc1+ substrates.

Growth regulation of the AML-193 leukemic cell line: evidence for autocrine production of granulocyte-macrophage colony-stimulating factor (GM-CSF), and inhibition of GM-CSF-dependent cell proliferation by interleukin-1 (IL-1) and tumor necrosis factor (TNF alpha).

The human leukemic cell line AML-193 was tested for its proliferative response to endogenously produced autocrine factors and to a variety of cytokines and colony-stimulating factors. Cells grown in the absence of GM-CSF incorporated tritiated thymidine, and this was partially reversed by adding neutralizing anti-GM-CSF antibodies to the culture medium, suggesting that it was due, at least in part, to autocrine GM-CSF production. This was confirmed by immunopurification of a GM-CSF-like activity from cell supernatant of AML-193 cells grown in serum free medium in the absence of exogenous GM-CSF. When AML-193 cells were cultured with GM-CSF in combination with other cytokines, Interleukin-1 alpha and beta (IL-1 alpha and beta), Interleukin-3 (IL-3), Interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF) and tumor necrosis factor alpha (TNF alpha), none of them affected the concentration of GM-CSF required to induce 50% of maximum proliferation (D50). However, the maximum proliferation induced by GM-CSF alone was drastically decreased by IL-1 alpha, IL-1 beta and TNF alpha. Inhibition caused by exposure of the AML-193 to IL-1 for up to 24 hr was reversible, ruling out a direct cytotoxic effect.

Granulocyte-macrophage colony stimulating factor and interleukin-3 regulate the production of an interleukin-1 inhibitor by the differentiated AML-193 leukemic cell line.

Treatment of the AML-193 leukemic cell line with phorbol myristate acetate (PMA) resulted in the loss of their ability to proliferate in response to GM-CSF or IL-3. This was not due to a change in number or affinity of GM-CSF receptors, but possibly resulted from an other cellular mechanism. The AML-193 differentiated cells acquired the ability to phagocytose glutaraldehyde-fixed E.coli in a similar fashion to mature macrophages. In addition the PMA-differentiated AML-193 cells now secreted a factor which specifically inhibited the binding of interleukin-1 (IL-1) to its receptor on the murine thymoma cell line EL-4.6.1C10. The synthesis of this inhibitor was further increased by the addition of GM-CSF or IL-3. Pulse labelling experiments showed that this activity was due to a 26 kDa protein that bound to the IL-1 receptor even in the presence of neutralizing antibodies against IL-1 alpha or IL-1 beta, and this binding was only antagonized by IL-1 alpha or IL-1 beta. In contrast, peripheral monocytes obtained from the blood of normal donors, when induced with either GM-CSF or IL-3, produced large quantities of inhibitor in the absence of PMA. This report clearly shows that a leukaemic cell line can respond to GM-CSF and IL-3 in different ways before and after in vitro differentiation.

Human granulocyte-macrophage colony-stimulating factor plus phorbol myristate acetate stimulate a promyelocytic cell line to produce an IL-1 inhibitor.

We recently described an IL-1 inhibitor found in urine of febrile patients. It is a 26-kDa glycoprotein that acts by blocking the binding of IL-1 to its receptor. In a search for a cell source for the urinary IL-1 inhibitor, we tested three promyelocytic cell lines, H-161, AML-193, and HL-60, for their ability to produce this protein. Under normal culture conditions none of these cell lines produce detectable IL-1 inhibitory activity. The H-161 cells were treated with differentiation-inducing agents, i.e., sodium butyrate, hemin, retinoic acid, DMSO, vitamin D3, and PMA alone or in combination with IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, TNF-alpha, IFN-gamma, granulocyte-CSF, macrophage-CSF, granulocyte/macrophage-CSF (GM-CSF), and Con A and tested for the production of IL-1 inhibitor. Production of IL-1 inhibitor was detected in cell supernatant, when H-161 cells were differentiated to adherent macrophage-like cells under the influence of PMA followed by a second signal provided by GM-CSF. Treatment of the other two cell lines, AML-193 and HL-60, with PMA plus GM-CSF also yielded similar IL-1 inhibitor protein. Partial purified H-161-derived IL-1 inhibitor showed specific binding to IL-1R-bearing cells and blocked the binding of IL-1 to its receptor and is thus similar to the urinary-derived molecule. We conclude the GM-CSF provides a signal to adherent macrophage-like cells to become "inhibitory macrophages" and to produce a competitive inhibitor of IL-1.

Purification and characterization of a 26-kDa competitive inhibitor of interleukin 1.

The urine of patients with fever above 39 degrees C contains an inhibitor of interleukin 1. Herein we describe the purification of the interleukin 1 inhibitor by ion-exchange chromatography, hydrophobic chromatography, gel filtration and negative immunosorption. The purified protein has a molecular weight of 26,000; its size is reduced to 24,000 upon treatment with endoglycosidase F. The IL 1 inhibitor is a competitive inhibitor and acts by binding to the IL 1 receptor. The inhibitor recognizes the murine and human IL 1 receptor with similar affinity. The purified IL 1 inhibitor has a specific activity of 3 x 10(7) to 4.5 x 10(7) units/mg as tested on the human astrocytoma (U-373) cell proliferation bioassay and murine EL4.6.1 cell IL 1 receptor-binding assay.

Production of a 26,000-dalton interleukin 1 inhibitor by human monocytes is regulated by granulocyte-macrophage colony-stimulating factor.

An interleukin 1 (IL 1) inhibitor is secreted into culture medium by a human promyelocytic cell line, H-161, upon stimulation with (PMA) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). Since the morphological characteristics of this cell line were macrophage-like, human monocytes were tested for their ability to produce similar activity using the same induction conditions. Upon induction of adherent peripheral blood monocytes with rhGM-CSF and/or PMA, an IL 1 antagonistic activity was found in the cell supernatants, as determined by IL 1 receptor binding assay, using the murine EL-4.6.1C10 cell line as the cell target. Most of the inhibition of IL 1 binding induced by PMA or by PMA/rhGM-CSF was shown to be caused by IL 1, since it was neutralized by a mixture of anti-IL 1 alpha/beta antibodies and was active in the murine thymocyte proliferation assay (LAF). The activity induced by GM-CSF alone was not neutralized by anti-IL 1 alpha/beta antibodies and showed no LAF activity. The IL 1 inhibitor activity was induced by rhGM-CSF with a D50 around 40 pg/ml. The activity was produced for more than 3 wk in the presence of GM-CSF; removal of GM-CSF was followed by a rapid decrease of IL 1 antagonistic activity. The specific binding of biosynthetically labeled IL 1 inhibitor to target cells (EL-4.6.1C10) showed a protein of 26 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This molecule shares biological and physical characteristics with the urinary IL 1 inhibitor and the promyelocytic H-161-derived IL 1 inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of protein kinase C immunoreactivity in rat retina.

A polyclonal antiserum to protein kinase C has been used to study the distribution of the enzyme antigenic sites in rat retina. The results indicate that the kinase is concentrated in photoreceptor outer segments as well as in the outer and inner plexiform layers. In identified components of retinal neuronal circuits, the kinase immunoreactivity is present in photoreceptor presynaptic terminals, in bipolar cell dendrites and axons, and probably in bipolar cell presynaptic terminals impinging on retinal ganglion cell dendrites. Thus, protein kinase C is positioned to play a role in specialized compartments of photoreceptor membrane and at both pre- and postsynaptic levels in the function of retinal neuronal circuits. Label in the nucleus is observed in retinal ganglion cells, but not bipolar or horizontal cells and probably not in amacrine cells. A role for protein kinase C in neuronal function at the level of the cell nucleus is therefore not likely to be universal, but to be determined by the particular properties of individual neuronal types.

N-terminal methionine-specific peptidase in Salmonella typhimurium.

Crude extracts of a multiply peptidase-deficient strain of Salmonella typhimurium contain an aminopeptidase that specifically removes N-terminal methionine from peptides. This activity shows pronounced specificity for the peptide's second amino acid. Methionine is removed from peptides with alanine, threonine, or glycine in this position but not when the second amino acid is leucine or methionine. The activity is stimulated by Co2+ and is inhibited by EDTA. Mutations that lead to overproduction (up to 30-fold) of the activity have been obtained by selecting for growth on Met-Gly-Gly as a methionine source. These mutations map at approximately 3 map units, phage P22 cotransducible with leu. The overproducer mutations are dominant to wild type, and duplication of the wild-type allele of the locus leads to a gene dosage effect on peptidase levels. This suggests that the locus of the overproducer mutations may be the structural gene for the peptidase. NaDodSO4/PAGE shows an increased level of a single protein (34 kDa) in the overproducer mutant. This protein is highly enriched in a purified preparation of the peptidase. The specificity of this enzyme suggests that it is involved in the cleavage of methionine from newly synthesized peptide chains. This activity can specifically remove methionine from the N terminus of a completed protein. Treatment of purified, unprocessed (N-terminal methionine) interleukin 1 beta with the purified peptidase results in removal of N-terminal methionine with no additional alterations. N-terminal processing of at least this protein can occur after translation is complete. We propose to call this enzyme peptidase M (methionine-specific aminopeptidase).

Immunochemical characterization of protein kinase C in rat liver nuclei and subnuclear fractions.

A doublet of immunoreactive bands has been identified in rat liver nuclei, nuclear matrix and lamina by means of a polyclonal antibody against protein kinase C. The two polypeptides show an apparent molecular weight of 77 and 74 kDa on SDS-polyacrylamide gels, and appear to be tightly bound nuclear components, resistant to detergent and high salt extraction. Given the complexity of the genes encoding for protein kinase C, these two forms of the enzyme might be translational products specifically located in the nucleus, involved in the transduction to the genomic apparatus of regulatory signals generated by growth factors and tumor promoters.

Immunocytochemical localization of protein kinase C in identified neuronal compartments of rat brain.

Polyclonal antisera to the phospholipid/Ca2+-dependent protein kinase have been used to study the distribution of the enzyme in identified neurons of several brain regions. The results indicated that the enzyme was concentrated in synaptic terminals of mossy fibers, Golgi II neurons and Purkinje neurons in the cerebellum, and in granule cell terminals in the hippocampus. These synapses have different physiological properties and utilize different neurotransmitters. Electron microscopic results indicated that the enzyme was concentrated in presynaptic terminals. Thus, the protein kinase may play a broad role in Ca2+-related events of the presynaptic terminal during neurotransmission. Light- and electron-microscopic immunocytochemical analysis also indicated that the enzyme was inside the nucleus concentrated in a region adjacent to the inner nuclear membrane, where it may play a role in the regulation of neuronal function.

Effects of selenium compounds on phospholipid/Ca2+-dependent protein kinase (protein kinase C) system from human leukemic cells.

Selenium compounds (selenium dioxide, selenious acid, and selenic acid) were found to inhibit phospholipid/Ca2+-dependent protein kinase (protein kinase C) and the phorbol ester-stimulated phosphorylation of endogenous substrate proteins from HL60 cells. Kinetic analysis indicated that selenium dioxide (SeO2) inhibited the enzyme noncompetitively with respect to phosphatidylserine (apparent Ki, 60 microM) and Ca2+ (apparent Ki, 68 microM). The inhibitory effect of SeO2 on protein kinase C was additive to that of another inhibitor of the enzyme (alkyl-lysophospholipid) when present together. SeO2 was also equally inhibitory to myosin light chain kinase, a calmodulin/Ca2+-dependent class of protein kinase. It, however, affected only very slightly cyclic adenosine 3':5'-monophosphate-dependent protein kinase. It is suggested that inhibition of Ca2+-dependent reactions might be related to the anticarcinogenic property of selenium.

Immunocytochemical evidence for phorbol ester-induced protein kinase C translocation in HL60 cells.

The ability of tumor promoting 12-O-tetradecanoylphorbol-13-acetate (TPA) to redistribute protein kinase C in human promyelocytic leukemic HL60 cells was investigated. It was found that TPA caused a rapid translocation (within 10 min) of protein kinase C from the cytosolic (soluble) fraction to the particulate (membrane) fraction, as determined indirectly by assaying for the enzyme activity or by immunoblotting of the enzyme protein in the isolated subcellular fractions. Immunocytochemical localization of the enzyme demonstrated directly that the TPA caused an enzyme translocation t the plasma membrane. These findings suggest that translocation to the plasma membrane of the enzyme may represent initial events related to the TPA effect on terminal differentiation of HL60 cells to monocytes/macrophages.

Phospholipid/calcium-dependent protein kinase (protein kinase C) system: a major site of bioregulation.

The substrate specificity determinants of protein kinase C are probed using synthetic peptides encompassing the major phosphorylation site serine 115 in bovine MBP. The results indicate that basic residues arginine 107 and 113 N-terminal to the phosphorylation site are essential for the substrate activity of the peptides. Substitutions of these basic residues by alanine cause a marked decrease in their substrate activity and the resulting peptide analogs become specific and rather potent inhibitors of protein kinase C. Leukemic cells are particularly abundant in protein kinase C and its substrate proteins, and the enzyme system has been shown to play a key role in cell growth. The agents that stimulate protein kinase C include tumor promoting phorbol esters (such as TPA) and mezerein, and the putative second messenger diacylglycerol. Many antineoplastic agents, on the other hand, inhibit the enzyme which include adriamycin, tamoxifen, alkyl-lysophospholipid, selenium, retinal and lipoidal amine CP-46, 665-1. Immunocytochemical studies of protein kinase C in leukemic cells indicate that it is localized in the plasma membrane, cytoplasm, nucleus and Golgi apparatus, and the subcellular distribution of the enzyme might be related to the phases of the cell cycle. TPA induces translocation of the enzyme to plasma membrane, suggesting an additional mode of action for the tumor promotor.

Immunological quantitation of phospholipid/Ca2+-dependent protein kinase and its fragments. Tissue levels, subcellular distribution, and ontogenetic changes in brain and heart.

Levels of phospholipid/Ca2+-dependent protein kinase (protein kinase C, 80 kDa) and its presumed proteolytic fragments were quantified in a variety of animal tissues and cultured human leukemic cell lines (HL60 and K562) using an immunoblot analysis technique. Of many tissues examined, the rat brain and HL60 cells were by far the richest sources of the 80-kDa native enzyme, with its concentration estimated to be about 2-3 microM in both tissues. The major enzyme species detected in most tissues, however, was the 67-kDa fragment; the 80-kDa native enzyme was present in a smaller amount except in spleen which contained nearly equal amounts of both enzyme species. It was also found that HL60 and K562 leukemic cells contained the 50-kDa species instead of the 67-kDa species. A study of the subcellular distribution of the 80- and 67-kDa species showed the enzyme to be localized predominantly in the soluble fraction for some tissues (e.g. heart) and nearly equally distributed between soluble and particulate fractions in others (e.g. spleen). In the brain, however, the majority of the enzyme was present in the particulate fraction, in agreement with the findings made with immunocytochemical localization of the enzyme. The total enzyme content in developing rat brain and heart increased during the first 2 to 4 weeks following birth and decreased to 60% of peak levels in the adult. The present immunological method, showing for the first time that the tissue levels of phospholipid/Ca2+-dependent protein kinase and its fragments can be quantitated, would be useful for studies on the regulation of cellular events and pathophysiology of tissues thought to be involved in this multi-functional protein phosphorylation system.

Effect of tamoxifen, a nonsteroidal antiestrogen, on phospholipid/calcium-dependent protein kinase and phosphorylation of its endogenous substrate proteins from the rat brain and ovary.

Antiestrogens (tamoxifen, clomiphene and nafoxidine) were found to inhibit phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK, or protein kinase C), whereas estrogens (estradiol and diethylstilbesterol) and the weakly estrogenic chlorotrianisene were inactive. Kinetic analysis indicated that the antiestrogens inhibited PL/Ca-PK competitively with respect to phosphatidylserine (Ki = 16-27 microM), but non-competitively with Ca2+ (Ki = 14-30 microM). Tamoxifen, but not diethylstilbesterol, also inhibited the phospholipid/Ca2+-dependent phosphorylation of various endogenous proteins from the total, solubilized fraction of the rat brain and ovary. Myosin light chain kinase, a calmodulin/Ca2+-dependent class of protein kinase, was similarly inhibited by tamoxifen; the drug, however, was without effect on cyclic AMP-dependent and cyclic GMP-dependent protein kinases. It is suggested that PL/Ca-PK, by virtue of the hydrophobic interactions required for the enzyme activation, may represent a potential site of action for the lipophilic antiestrogens, in addition to the commonly recognized intracellular estrogen receptors.

Polyclonal antibodies to phospholipid/Ca2+-dependent protein kinase and immunocytochemical localization of the enzyme in rat brain.

Antisera against phospholipid/Ca2+-dependent protein kinase (protein kinase C) were raised in rabbits. Immunospecificity of the polyclonal antibodies, as determined by immunoblot and ELISA, was shown by their reactivity to the enzyme but not to other protein kinases or any of many other proteins tested. Immunocytochemical localization of the kinase in rat brains revealed that although the enzyme was distributed broadly in different brain regions, it was highly restricted to the periphery of the nucleus of neurons in cerebral cortex and to axons and cells strongly resembling oligodendroglia in white-matter regions. Initial electron microscopy of cerebral cortex revealed that the enzyme was highly concentrated in the presynaptic terminals, and only rarely were labeled postsynaptic specialization elements seen. It is suggested that the discrete localization of the enzyme, which is distinct from that of the calmodulin/Ca2+-dependent system, may be related to certain biological and functional aspects of brain that are regulated by Ca2+ at the level of protein phosphorylation.

Phospholipid-sensitive Ca2+ -dependent protein kinase preferentially phosphorylates serine-115 of bovine myelin basic protein.

Phospholipid-sensitive Ca2+ -dependent protein kinase (PL-Ca-PK) and cyclic AMP-dependent protein kinase (A-PK) both preferentially phosphorylated serine residues of bovine myelin basic protein (MBP). Tryptic peptide maps of MBP phosphorylated by PL-Ca-PK or A-PK, however, revealed different phosphopeptides, suggesting a difference in the intramolecular substrate specificity for the two enzymes. Serine-115 of MBP, in the sequence (-Arg-Phe-Ser(115)-Trp-), was found to be a preferred and probably major phosphorylation site for PL-Ca-PK. Because serine-115 of bovine MBP corresponds to serine-113 of rabbit MBP, an in vivo phosphorylation site reported by Martenson et al. (1983), and PL-Ca-PK is present at a very high level in brain and myelin, it is suggested that the enzyme may be responsible for the in vivo phosphorylation of this and other sites in MBP.