Secretion of biologically active recombinant fibrinogen by yeast.

Fibrinogen (340 kDa) is a plasma protein that plays an important role in the final stages of blood clotting. Human fibrinogen is a dimer with each half-molecule composed of three different polypeptides (A alpha, 67 kDa; B beta, 57 kDa; gamma, 47 kDa). To understand the mechanism of fibrinogen chain assembly and secretion and to obtain a system capable of producing substantial amounts of fibrinogen for structure-function studies, we developed a recombinant system capable of secreting fibrinogen. An expression vector (pYES2) was constructed with individual fibrinogen chain cDNAs under the control of a Gal-1 promoter fused with mating factor F alpha 1 prepro secretion signal (SS) cascade. In addition, other constructs were prepared with combinations of cDNAs encoding two chains or all three chains in tandem. Each chain was under the control of the Gal-1 promoter. These constructs were used to transform Saccharomyces cerevisiae (INVSC1; Mat alpha his3-delta 1 leu2 trp1-289 ura3-52) in selective media. Single colonies from transformed yeast cells were grown in synthetic media with 4% raffinose to a density of 1 x 10(8) cells/ml and induced with 2% galactose for 16 h. Yeast cells expressing all three chains contained fibrinogen precursors and nascent fibrinogen and secreted about 30 micrograms/ml of fibrinogen into the culture medium. The B beta and gamma chains, but not A alpha, were glycosylated. Glycosylation of B beta and gamma chains was inhibited by treatment of transformed yeast cells with tunicamycin. Intracellular B beta and gamma chains, but not the A alpha chains in secreted fibrinogen, were cleaved by endoglycosidase H. Carbohydrate analysis indicated that secreted recombinant fibrinogen contained N-linked asialo-galactosylated biantennary oligosaccharide. Recombinant fibrinogen yielded the characteristic plasmin digestion products, fragments D and E, that were immunologically indistinct from the same fragments obtained from plasma fibrinogen. The recombinant fibrinogen was shown to be biologically active in that it could form a thrombin-induced clot, which, in the presence of factor XIIIa, could undergo gamma chain dimerization and A alpha chain polymer formation.

Carboxy-terminal-extended variant of the human fibrinogen alpha subunit: a novel exon conferring marked homology to beta and gamma subunits.

Similarities between the N-terminal regions of the three subunits of the clotting protein fibrinogen--(alpha beta gamma)2--suggest that they evolved from a common progenitor. However, to date no human alpha chain has been found with the strong C-terminal homology shared by the beta and gamma chains. Here we examine the natural product of a novel fibrinogen alpha chain transcript bearing a separate open reading frame that supplies the missing C-terminal homology to the other chains. Additional splicing leads to the use of this extra sequence as a sixth exon elongating the alpha chain by 35%. Since the extended alpha chain (alpha E) is assembled into fibrinogen molecules and its synthesis is enhanced by interleukin-6, it suggests participation in both the acute phase response and normal physiology.

Regulation of fibrinogen assembly. Transfection of Hep G2 cells with B beta cDNA specifically enhances synthesis of the three component chains of fibrinogen.

Previous studies indicated that synthesis of B beta chain may be a rate-limiting factor in the production of human fibrinogen since Hep G2 cells contain surplus pools of A alpha and gamma but not of B beta chains, and fibrinogen assembly commences by the addition of preformed A alpha and gamma chains to nascent B beta chains attached to polysomes. To test whether B beta chain synthesis is rate limiting Hep G2 cells were transfected with B beta cDNA, and its effect on fibrinogen synthesis and secretion was measured. Two sets of stable B beta cDNA-transfected Hep G2 cells were prepared, and both cell lines synthesized 3-fold more B beta chains than control cells. The B beta-transfected cells also synthesized and secreted increased amounts of fibrinogen. Transfection with B beta cDNA not only increased the synthesis of B beta chain but also increased the rate of synthesis of the other two component chains of fibrinogen and maintained surplus intracellular pools of A alpha and gamma chains. Transfection with B beta cDNA did not affect the synthesis of albumin, transferrin, or anti-chymotrypsin and had a small inhibitory effect on the synthesis of C-reactive protein. Taken together these studies demonstrate that increased B beta chain synthesis specifically causes increased production of the other two component chains of fibrinogen and that unequal and surplus amounts of A alpha and gamma chains are maintained intracellularly.

A phosphoglycoprotein associated with taxol resistance in J774.2 cells.

Taxol is an inhibitor of cell replication that promotes the assembly of microtubules in vitro and in cells. In the murine macrophage-like cell line J774.2, a taxol-resistant subline (J7/TAX-50) has been developed in vitro by growing the cells in increasing drug concentrations. These cells, which are approximately 800-fold resistant to taxol, display some cross-resistance to colchicine, vinblastine, puromycin, doxorubicin, and actinomycin D but remain sensitive to bleomycin. Binding of radiolabeled drug to the resistant cells is reduced by approximately 90%. Resistant cells grown in the absence of drug for 10 days (J7/TAX-0D10), 1 month (J7/TAX-0D30), and 8 months (J7/TAX-0D240) regain a major portion (27, 92, and 99%, respectively) of their sensitivity to the drug. However, binding of the drug to the J7/TAX-0D30 and J7/TAX-0D240 cells is increased to only 20 and 70%, respectively, of that measured with sensitive cells. Analysis of plasma membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining reveals the presence of a prominent protein with an approximate molecular weight of 135,000 in the resistant line that is essentially absent from the parental line and from both of the J7/TAX-0D30 and J7/TAX-0D240 lines. Although this protein can be seen in J7/TAX-0D10, its quantity is diminished. The Mr 135,000 protein is also observed in the resistant cells when they are labeled with [3H]leucine, [35S]methionine, [3H]glucosamine, or [32P]orthophosphate. Plasma membranes from colchicine- or vinblastine-resistant J774.2 cells also contain prominent phosphoglycoproteins, with approximate molecular weights of 145,000 and 150,000, respectively. Partial purification of the Mr 135,000 glycoprotein by agarose-bound ricinus communis agglutinin I-agarose affinity chromatography indicates that it accounts for approximately 4 to 5% of total membrane protein. A Mr 100,000 phosphoglycoprotein, present in the membranes of J774.2 cells is essentially absent in J7/TAX-50 cells after labeling with [3H]glucosamine or [32P]orthophosphate. Phosphoamino acid analysis of the Mr 135,000 and 100,000 phosphoglycoproteins reveals that the phosphorylation sites are serine and threonine residues. There appears to be a good correlation between the presence of the Mr 135,000 phosphoglycoprotein in plasma membranes and resistance to taxol.

Value of serum ferritin as an index of iron deficiency in elderly anaemic patients.

Serum ferritin was correlated with bone marrow iron stores in 35 elderly anaemic patients. All 19 patients with low serum ferritin had iron deficiency as assessed on bone marrow examination, whereas six patients with low or absent bone marrow iron stores had serum ferritin within normal range. A low serum ferritin invariably indicates iron deficiency and has a better correlation than low serum iron. Serum ferritin should be included in the initial investigation of the anaemic elderly.

Characterization of the association of radiolabeled bleomycin A2 with HeLa cells.

The association of [3H]bleomycin A2 and Cu(II):[3H]bleomycin A2 with HeLa cells has been characterized. Under the conditions of our experiments, approximately 0.1% of the total drug in the medium associates with HeLa cells. Both forms of the drug bind to HeLa cells in a specific and saturable manner, with a Km of 20 microM and a Vmax of 2.5 pmol/min/10(6) cells. Scatchard analysis of the specific binding data demonstrates a single set of high-affinity binding sites. Cytotoxic activities of both forms of the drug are similar, with a 50% lethal dose of 0.5 microM at 48 hr. The specific binding in HeLa cells of either the labeled metal-free drug or its copper complex is reversible by a 100-fold excess of either unlabeled drug. Interaction of the drug with cells is temperature sensitive but is unaffected by metabolic poisons, suggesting that this process is not energy dependent. Isolation of DNA from HeLa cells incubated with the drug indicates that 1 mol of either [3H]bleomycin A2 or Cu(II):[3H]bleomycin A2 binds per 10(8) nucleotides. Further studies with the radiolabeled drug are required to define precisely the mechanisms involved in bleomycin uptake and compartmentalization within the cell.

Chemical synthesis of radiolabeled bleomycin A2 and its binding to DNA.

A method for the preparation of biologically active [3H]- and [13C]bleomycin A2 is described. Demethyl Cu(II):bleomycin A2, isolated after pyrolysis of Cu(II):bleomycin A2, was methylated with either [3H]-or [13C]methyl iodide, which resulted in Cu(II):bleomycin A2 labeled in the dimethylsulfonium moiety. Copper was removed by treatment with dithizone in chloroform, and structures were verified by thin-layer chromatography and 1H and 13C nuclear magnetic resonance spectroscopy. Copper-free [3H]-and [13C]bleomycin A2 are active in the degradation of DNA in vitro. Gel exclusion chromatograhy and equilibrium dialysis were used to determine the apparent equilibrium constants for binding of [3H]bleomycin A2 and Cu(II):[3H]bleomycin A2 to calf thymus DNA, noncovalently associated polydeoxyguanylate:polydeoxycytidylate, and noncovalently associated polydeoxyadenylate:polydeoxythymidylate. In 2.5 mM sodium phosphate buffer, pH 7.0, binding data obtained by gel filtration with calf thymus DNA reveal an apparent equilibrium constant for [3H]bleomycin A2 of 5.7 X 10(5)/mol and for Cu(II):[3H]bleomycin A2 of 3.9 X 10(5)/mol. One molecule of [3H]bleomycin A2 binds for every 3.7 base pairs in DNA, and one molecule of Cu(II):[3H]bleomycin A2 binds for every 2.8 base pairs in DNA. Analysis of binding data with calf thymus DNA, noncovalently associated polydeoxyguanylate:polydeoxycytidylate, and noncovalently associated polydeoxyadenylate:polydeoxythymidylate obtained by equilibrium dialysis reveals, in each instance, 2 types of binding sites for both the copper and metal-free form of the antibiotic. For those sites in calf thymus DNA with tighter binding affinity, the apparent equilibrium constant for [3H]bleomycin A2 was 6.8 X 10(5)/mol and for the Cu(II):[3H]bleomycin A2 complex, 4.4 X 10(5)/mol. As seen with calf thymus DNA, the affinity of [3H]bleomycin A2 is slightly greater than that of Cu(II):[3H]bleomycin A2 for the synthetic DNAs, although more of the copper form of the drug binds to these polymers.