Identification of a signal-transduction pathway shared by haematopoietic growth factors with diverse biological specificity.

The haematopoietic growth factors multi-colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2 specifically control the production and proliferation of distinct leucocyte series. Each growth factor acts on a unique surface receptor associated with an appropriate signal-transduction apparatus. In this report we identify a 68 kDa substrate which is phosphorylated after stimulation of different cell types with multi-colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2. The 68 kDa substrate is also phosphorylated in each cell line stimulated with synthetic diacylglycerol, a direct activator of protein kinase C. Interestingly, granulocyte/macrophage colony-stimulating factor does not induce phosphorylation of the 68 kDa molecule. The 68 kDa molecule that is phosphorylated after stimulation with each ligand yielded similar peptide maps after chymotryptic digestion; furthermore, the substrate was always phosphorylated on threonine residues. Phosphorylation of the same residues in the 68 kDa substrate suggests that activation of protein kinase C is one common signal-transduction event associated with the action of multi-colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2.     

Identification of pro-epidermal growth factor and high molecular weight epidermal growth factors in adult mouse urine

By use of immunoblot analysis, we demonstrate the presence of a pro-Epidermal growth factor (EGF) with an approximate molecular weight of 165 kDa in adult mouse urine. In addition, urine contains four high molecular weight EGFs with approximate molecular weights of 116, 97, 66 and 56 kDa. The 165 kDa pro-EGF as well as the 66 and 56 kDa EGFs also are detectable in mouse kidney extract. Neither urine nor kidney contain the mature EGF of 6 kDa. The 165 kDa pro-EGF is the major product synthesized in renal tissue and secreted in urine. The finding of high molecular weight EGFs in urine suggests that part of pro-EGF secreted into urine undergoes partial proteolysis distal to its site of synthesis.     

Expression of the functional soluble form of human fas ligand in activated lymphocytes.

Fas is a type I membrane protein which mediates apoptosis. Fas ligand (FasL) is a 40 kDa type II membrane protein expressed in cytotoxic T cells upon activation that belongs to the tumor necrosis factor (TNF) family. Here, we found abundant cytotoxic activity against Fas-expressing cells in the supernatant of COS cells transfected with human FasL cDNA but not with murine FasL cDNA. Using a specific polyclonal antibody against a peptide in the extracellular region of human FasL, a protein of 26 kDa was detected in the supernatant of the COS cells. The signal sequence of granulocyte colony-stimulating factor was attached to the extracellular region of human FasL. COS cells transfected with the cDNA coding for the chimeric protein efficiently secreted the active soluble form of human FasL (sFasL). Chemical crosslinking and gel filtration analysis suggested that human sFasL exists as a trimer. Human peripheral T cells activated with phorbol myristic acetate and ionomycin also produced functional sFasL, suggesting that human sFasL works as a pathological agent in systemic tissue injury.     

c-kit protein, a transmembrane kinase: identification in tissues and characterization.

The proto-oncogene c-kit encodes a transmembrane kinase which is related to the receptors for colony-stimulating factor type 1 and platelet-derived growth factor, as well as to the immunoglobulin superfamily. Antibodies specific for the kinase domain of the P80 gag-kit protein of the Hardy-Zuckerman 4 feline sarcoma virus were prepared. These kit-specific antibodies were used to identify and characterize the c-kit protein in cat brain tissue. The c-kit protein product displays an autophosphorylating activity in immune complex kinase assays, and, in turn, this activity was used to identify the c-kit protein in different tissues. In cat brain, a single 145-kilodalton (kDa) glycoprotein was detected. Its N-linked carbohydrates were found to be sensitive to digestion with the endoglycosidases (neuraminidase, endoglycosidase F, and endoglycosidase H), indicating hybrid and/or complex and high-mannose structures. A partial purification of the c-kit protein was achieved by wheat germ agglutinin affinity chromatography, and the autophosphorylating activity of the partially purified c-kit protein was characterized and found to be specific for tyrosine. The kit antibodies cross-react with the murine c-kit protein product, and variant c-kit proteins in different mouse tissues were identified, with sizes of about 145 kDa (brain), 160 kDa (spleen), and 150 kDa (testis).     

Differential processing of colony-stimulating factor 1 precursors encoded by two human cDNAs.

The biosynthesis of macrophage colony-stimulating factor 1 (CSF-1) was examined in mouse NIH-3T3 fibroblasts transfected with a retroviral vector expressing the 554-amino-acid product of a human 4-kilobase (kb) CSF-1 cDNA. Similar to results previously obtained with a 1.6-kb human cDNA that codes for a 256-amino-acid CSF-1 precursor, the results of the present study showed that NIH-3T3 cells expressing the product of the 4-kb clone produced biologically active human CSF-1 and were transformed by an autocrine mechanism when cotransfected with a vector containing a human c-fms (CSF-1 receptor) cDNA. The 4-kb CSF-1 cDNA product was synthesized as an integral transmembrane glycoprotein that was assembled into disulfide-linked dimers and rapidly underwent proteolytic cleavage to generate a soluble growth factor. Although the smaller CSF-1 precursor specified by the 1.6-kb human cDNA was stably expressed as a membrane-bound glycoprotein at the cell surface and was slowly cleaved to release the extracellular growth factor, the cell-associated product of the 4-kb clone was efficiently processed to the secreted form and was not detected on the plasma membrane. Digestion with glycosidic enzymes indicated that soluble CSF-1 encoded by the 4-kb cDNA contained both asparagine(N)-linked and O-linked carbohydrate chains, whereas the product of the 1.6-kb clone had only N-linked oligosaccharides. Removal of the carbohydrate indicated that the polypeptide chain of the secreted 4-kb cDNA product was longer than that of the corresponding form encoded by the smaller clone. These differences in posttranslational processing may reflect diverse physiological roles for the products of the two CSF-1 precursors in vivo.     

Production and characterization of biologically active human GM-CSF secreted by genetically modified plant cells

Human granulocyte-macrophage colony-stimulating factor (GM-CSF), a hemopoietic growth factor, was produced and secreted from tobacco cell suspensions. The GM-CSF cDNA was carried by a binary vector under the control of the CaMV 35S promoter and the T7 terminator. In addition, a 5'-nontranslated region from the tobacco etch virus (TEV leader sequence) was fused to the N-terminal end of the GM-CSF transgene. For ease of purification, a 6-His tag was added to the 3' end of the GM-CSF cDNA. Addition of the TEV leader sequence increased protein production more than twofold compared to non-TEV controls. Initial batch cultivation studies indicated a maximum of 250 microg/L extracellular and 150 microg/L intracellular GM-CSF. Western blot analysis detected multiple peptides with masses from 14 to 30 kDa in the extracellular medium. The plant-produced GM-CSF was biologically active and could be bound to a nickel affinity matrix, indicating that both the receptor-binding region and the 6-His tag were functional. The batch production of GM-CSF was compared with the production of other recombinant proteins secreted by transformed tobacco cells. The recovery of secreted GM-CSF was increased by the addition of stabilizing proteins and by increasing salt in the growth medium to physiological levels.     

Cloning of a gene encoding a major secreted polypeptide of Listeria monocytogenes and its potential use as a species-specific probe

A gene, designated msp, that encodes a major secreted polypeptide with a molecular mass of approximately 60 kilodaltons (kDa) was cloned from Listeria monocytogenes 10403. DNA hybridization analysis indicated that the msp gene was highly conserved among 15 independent L. monocytogenes isolates and that each of 5 isolates tested secreted a 60-kDa polypeptide that was immunologically related to the msp gene product. DNA sequences related to msp were not detected in any other Listeria species or in strains of Bacillus cereus, Bacillus thuringiensis, Streptococcus pyogenes, or Streptococcus pneumoniae when standard stringent DNA hybridization conditions were used. Under nonstringent conditions, related sequences were detected in Listeria ivanovii, Listeria seeligeri, and Listeria innocua, and immunoblot analysis indicated that these strains secreted polypeptides of about 60 kDa that were immunologically related to the msp gene product. The possibility of using the msp gene as a probe for the detection of L. monocytogenes and the potential functions of the msp gene product are discussed.     

Cloning of a gene encoding a major secreted polypeptide of Listeria monocytogenes and its potential use as a species-specific probe.

A gene, designated msp, that encodes a major secreted polypeptide with a molecular mass of approximately 60 kilodaltons (kDa) was cloned from Listeria monocytogenes 10403. DNA hybridization analysis indicated that the msp gene was highly conserved among 15 independent L. monocytogenes isolates and that each of 5 isolates tested secreted a 60-kDa polypeptide that was immunologically related to the msp gene product. DNA sequences related to msp were not detected in any other Listeria species or in strains of Bacillus cereus, Bacillus thuringiensis, Streptococcus pyogenes, or Streptococcus pneumoniae when standard stringent DNA hybridization conditions were used. Under nonstringent conditions, related sequences were detected in Listeria ivanovii, Listeria seeligeri, and Listeria innocua, and immunoblot analysis indicated that these strains secreted polypeptides of about 60 kDa that were immunologically related to the msp gene product. The possibility of using the msp gene as a probe for the detection of L. monocytogenes and the potential functions of the msp gene product are discussed.     

Highly effective production of biologically active,secreted, human granulocyte-macrophage colony-stimulating factor by recombinant vaccinia virus

The recombinant vaccinia virus strain VV-GMCSF-S1/3, which contains an insertion of full-length DNA copy of messenger RNA of human granulocyte-macrophage colony-stimulating factor (GM-CSF) in the structural part of the viral thymidine kinase gene, was obtained. The expression of the GM-CSF gene as a part of the recombinant virus is under the control of the native vaccinia virus promoter р7.5K; this results in the production of a mature form of the secreted protein with a molecular mass of 32 kDa. The biological activity of GM-CSF was evaluated by stimulation of the proliferation of cytokine-dependent human TF-1 erythroleukemia cells. The secretion level of biologically active human GM-CSF in the system of recombinant vaccinia virus/mammalian cells was 1–40 μg/mL of culture medium. The recombinant strain VV-GMCSF-S1/3 can be used as a producer of the glycosylated mature form of human GM-CSF, as well as a vector for the construction of oncolytic viruses and multivalent vaccine preparations.     

Identification of a high molecular weight macrophage colony-stimulating factor as a glycosaminoglycan-containing species.

Chinese hamster ovary cells transfected with a 4.0-kilobase macrophage colony-stimulating factor (M-CSF) cDNA express two different M-CSF species; one has an apparent molecular weight of 85,000 and is identified as a homodimer of a 43-kDa subunit, and the other has an indeterminate structure greater than 200 kDa. In this study, we investigated the structure of the high molecular weight M-CSF by immunochemical procedures. The high molecular weight M-CSF was easily purified, since it bound tightly to DEAE-Sephacel and eluted at a characteristically high salt concentration. The high molecular weight M-CSF migrated as a diffuse band of over than 200,000 on nonreducing sodium dodecyl sulfate-polyacrylamide gels. Analysis of the same samples under reducing conditions revealed that the larger species consisted of a heteromer of the 43- and 150-200-kDa M-CSF subunits. Digestion of the 150-200-kDa M-CSF subunit with chondroitinase, which degrades the chondroitin sulfate glycosaminoglycan chain, yielded a 100 kDa band. This species was secreted instead of 150-200-kDa species when the cells were cultured in the presence of beta-D-xyloside, which inhibits the elongation of the chondroitin sulfate glycosaminoglycan chain in proteoglycans, providing additional evidence for the existence of a chondroitin sulfate chain in the 150-200-kDa M-CSF subunit. Removal of O- and N-linked carbohydrate from the 150-200-kDa subunit yielded a polypeptide chain with a larger molecular mass (approximately 45 kDa) than that of the 43-kDa subunit (approximately 25 kDa). Collectively, these results indicate that the 150-200-kDa M-CSF subunit is a proteoglycan with a core protein that may be an alternatively processed form of M-CSF.     

Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells

The synthesis, processing, and secretion of factor VIII expressed from heterologous genes introduced into Chinese hamster ovary cells has been studied. The results show factor VIII to be synthesized as a primary translation product of approximately 230 kDa that can be detected in the lumen of the endoplasmic reticulum. In this compartment, the majority of the factor VIII is in a complex with a resident protein of the endoplasmic reticulum, binding protein, and may never appear in the medium. Some factor VIII transits the endoplasmic reticulum to the Golgi apparatus, where it is cleaved to generate the mature heavy and light chains. In the absence of von Willebrand factor in the medium, the secreted heavy and light chains are unassociated and subsequently degraded. In the presence of von Willebrand factor in the medium, the heavy and light chains are secreted as a stable complex and activity accumulates linearly with time. The utilization and complexity of asparagine-linked carbohydrate present on the secreted recombinant-derived factor VIII and human plasma-derived factor VIII were compared and found to be very similar. In both cases, the asparagine-linked carbohydrate moieties on the heavy chain are primarily of the hybrid or complex-type. In contrast, the factor VIII from both sources contains a high-mannose type of asparagine-linked carbohydrate on the light chain.     

Alpha-factor-leader-directed secretion of recombinant human-insulin-like growth factor I from Saccharomyces cerevisiae. Precursor formation and processing in the yeast secretory pathway

A synthetic gene coding for human-insulin-like growth factor I (IGFI) was fused to the leader sequence of yeast prepro-alpha-factor and expressed in Saccharomyces cerevisiae under the control of a glyceraldehyde-3-phosphate dehydrogenase promoter fragment. Recombinant IGFI was found inside yeast cells and secreted into the medium. The secreted IGFI migrated on SDS gels with the same electrophoretic mobility as authentic IGFI, i.e. at about 7.5 kDa. HPLC analysis of secreted IGFI revealed the presence of the correctly folded, genuine molecule as well as an isomeric byproduct of equal molecular mass but with two of the three disulfide bonds interchanged. Inside exponentially growing cells the 7.5-kDa IGFI was also found, along with up to four additional IGFI-related polypeptides of higher molecular mass. By endoglycosidase F treatment the three polypeptides between 19-26 kDa were converted to a single peptide of 17 kDa. Since this peptide also reacted with an anti-alpha-factor antibody, it represents most likely the unglycosylated alpha-factor--IGFI fusion precursor. Pulse-chase experiments established the precursor nature of the intracellular higher-molecular-mass IGFI species. Conversion of the primary translation product to the differently glycosylated IGFI precursor proteins and into the mature form occurred very rapidly, within 2 min. Rapid maturation was, however, not followed by an equally rapid secretion of the mature form into the medium: only after 30-40 min did IGFI appear outside the cells. We therefore postulate the presence of an as yet undefined Golgi or post-Golgi bottleneck representing a major obstacle in secretion of recombinant IGFI from S. cerevisiae cells.     

Noncytolytic inhibition of X4 virus by bulk CD8(+) cells from human immunodeficiency virus type 1 (HIV-1)-infected persons and HIV-1-specific cytotoxic T lymphocytes is not mediated by beta-chemokines

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) mediate immunologic selection pressure by both cytolytic and noncytolytic mechanisms. Non cytolytic mechanisms include the release of beta-chemokines blocking entry of R5 HIV-1 strains. In addition, CD8(+) cells inhibit X4 virus isolates via release of as yet poorly characterized soluble factors. To further characterize these factors, we performed detailed analysis of CTL as well as bulk CD8(+) T lymphocytes from six HIV-1-infected individuals and from six HIV-1-seronegative individuals. Kinetic studies revealed that secreted suppressive activities of HIV-1-specific CTL and bulk CD8(+) T lymphocytes from all HIV-1-infected persons are significantly higher than that of supernatants from seronegative controls. The suppressive activity could be blocked by monensin and brefeldin A, was heat labile, and appeared in a pattern different from that of secretion of chemokines (MDC, I-309, MIP-1alpha, MIP-1beta, and RANTES), cytokines (gamma interferon, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor), and interleukins (interleukin-13 and interleukin-16). This suppression activity was characterized by molecular size exclusion centrifugation and involves a suppressive activity of >50 kDa which could be bound to heparin and a nonbinding inhibitory activity of <50 kDa. Our data provide a functional link between CD8(+) cells and CTL in the noncytolytic inhibition of HIV-1 and suggest that suppression of X4 virus is mediated through proteins. The sizes of the proteins, their affinity for heparin, and the pattern of release indicate that these molecules are not chemokines.     

Purification and first characterization of the secreted and cellular 52-kDa proteins regulated by estrogens in human-breast cancer cells

An estrogen-regulated 52-kDa glycoprotein secreted by MCF7 breast cancer cells was first purified from serum-free conditioned medium by concanavalin-A--Sepharose (ConA--Sepharose). The 13% pure protein was then used to obtain monoclonal antibodies to the 52-kDa protein [Garcia et al. (1985) Cancer Res. 45, 709-716]. Using ConA--Sepharose and monoclonal antibody affinity chromatographies, the secreted 52-kDa protein was finally purified to homogeneity as verified by silver staining of sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and one single N-terminal amino acid. The purification factor was approximately 1400 and the yield 40%. The same two-step procedure, applied to MCF7 cell extracts, yielded four immunologically related proteins of 52 kDa, 48 kDa, 34 kDa and 17 kDa, which were purified 1250-fold with a yield of 30%. These components were further separated by high-performance liquid chromatography gel filtration under denaturing conditions. The final products were homogeneous on the basis of silver-stained SDS-PAGE and gel filtration. However, isoelectrofocusing showed that the pI of the secreted 52-kDa protein and the cellular 34-kDa protein varied from 5.5 to 6.5. Amino acid analysis of the secreted and the related cellular 34-kDa protein is given. Western immunoblotting, pulse chase studies and post-translational studies indicate that the 52-kDa protein is the precursors of a lysosomal enzyme which is partially secreted and partially processed into smaller cellular forms.     

Interaction between TFF1, a gastric tumor suppressor trefoil protein, and TFIZ1, a brichos domain-containing protein with homology to SP-C

TFF1 is a gastric tumor suppressor that protects gastric epithelial cells from damage but can promote invasive properties of tumor cells. Antibodies were raised against correctly folded TFF1 protein. These showed that the 6.67 kDa secreted trefoil protein is present as an approximately 25 kDa complex in normal human gastric mucosa. The TFF1 complex was immunopurified from human gastric mucosa and shown to comprise two proteins joined by a disulfide bond. Both were identified by amino-terminal sequencing and MALDI TOF mass spectrometry. The TFF1 protein partner is a previously unknown protein that we have called TFIZ1 for trefoil factor interactions(z) 1. TFIZ1 is expressed and secreted in normal gastric mucosa. TFIZ1 mRNA was cloned from gastric mucosa and sequenced. TFIZ1 is an 18.31 kDa protein and contains an approximately 100 amino acid brichos domain and homology with smart00019.10, SF_P. This is the first demonstration that a member of the trefoil factor family of proteins is bound covalently to a brichos domain-containing protein. The apparent molecular mass of the TFF1:TFIZ1 heterodimer is remarkably close to the theoretical molecular mass of 24.98 kDa. In conclusion, the heterodimer comprises one molecule each of TFF1 and TFIZ1, and the disulfide bond between TFF1 and TFIZ1 is the most important factor stabilizing the heterodimer.     

Selective expression of nonsecreted triple-helical and secreted single-chain recombinant collagen fragments in the yeast Pichia pastoris

High-level recombinant expression systems for the production of stable triple-helical human collagens and collagen fragments have been developed in the yeast Pichia pastoris. Collagen fragments are secreted as single-chain polypeptides by the yeast alpha-mating factor pre-pro sequence, but secretion of full-length triple-helical procollagen molecules has not been achieved despite the use of the same secretory signal. We studied here the effects of the secretory signal and the conformation and size of the collagen polypeptide on its secretion in P. pastoris. Unlike the collagen signal sequence, the alpha-mating factor pre-pro sequence led to efficient secretion of single-chain 45 and 9 kDa type I collagen fragments. The efficiency was dependent on the length of the collagen polypeptide, as secretion of single-chain full-length 90 kDa alpha1(I) polypeptides was less efficient than that of the 45 kDa fragment. Furthermore, the conformation of the collagen polypeptides had a marked effect on secretion, as induction of trimerization of the 45 and 9 kDa fragments by either the C propeptide or the small trimerizing domain foldon led to an accumulation of triple-helical molecules inside the cells despite the presence of the alpha-mating factor pre-pro sequence. Our results show that P. pastoris is a suitable host for the development of tailored expression systems aimed at selective production of nonsecreted triple-helical and secreted single-chain collagen fragments of varying lengths for specific purposes.     

Recombinant human serum amyloid P component from Pichia pastoris: production and characterization

Human serum amyloid P component (SAP) was expressed in the methylotrophic yeast Pichia pastoris. SAP cDNA was placed under control of regulatory sequences derived from the alcohol oxidase gene (AOX1), and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. Recombinant SAP (r-SAP) was produced in a bioreactor with computer controlled fed-batch mode and purified by use of a C-terminal histidine tag. The yield of purified r-SAP was 3-4mg from 1L supernatant and 5-6mg from 1L cell paste, indicating that the majority of the produced SAP was not secreted. Treatment of the cell paste with EDTA increased the yield further by about 30%. The N-terminal of r-SAP purified from the supernatant showed non-complete cleavage of the alpha-mating factor signal sequence. Purified r-SAP, analyzed under native conditions, was shown to be a decamer, like purified human SAP (h-SAP), with monomers of 27kDa. Each monomer had one N-glycosylation site, positioned at the same site as for h-SAP. r-SAP bound to antibodies produced against h-SAP. Furthermore, r-SAP bound to ds DNA and influenza A virus subunits in a Ca(2+)-dependent manner and inhibited influenza A virus hemagglutination. These results indicate that r-SAP produced in P. pastoris has the same biological activity as purified h-SAP.     

Differential synthesis of two interleukin-1 receptor antagonist variants and interleukin-8 by peripheral blood neutrophils

With a short lifespan and containing only few ribosomes and endoplasmic reticulum structures, neutrophils are thought to have a limited capacity for protein synthesis. We here show that peripheral blood polymorphonuclear neutrophils (PMN) are able react to stimulants with differential production of two interleukin (IL)-1 receptor antagonist (IL-1ra) isoforms, secreted IL-1ra (sIL-1ra) and the 16kDa intracellular form of IL-1ra (icIL-1ra3), as well as IL-8. Neutrophils of a high purity and with a low degree of preactivation upregulate mRNA and de novo synthesize protein of both IL-1ra variants and IL-8 in response to granulocyte-macrophage colony-stimulating factor and lipopolysaccharide. The cytokines are differentially regulated and distributed in two intracellular compartments. In comparison with peripheral blood mononuclear cells (PBMC), PMN produce distinctly more sIL-1ra but significantly less IL-8. This may indicate an anti-inflammatory role, enabling PMN to antagonize proinflammatory signals. It is therefore possible that PMN play an important role in immune regulation by counteracting a dysregulation of the inflammatory process.