Enhanced protein C activation and inhibition of fibrinogen cleavage by a thrombin modulator.

A modulator of the enzymatic activity of human thrombin, designated LY254603, was identified that enhances the thrombin-catalyzed generation of the anticoagulant factor activated protein C, yet inhibits thrombin-dependent fibrinogen clotting. By means of mutant substrates, it was shown that LY254603 mediates the change in enzymatic substrate specificity through an alteration in thrombin's S3 substrate recognition site, a mechanism that appeared to be independent of allosteric changes induced by either sodium ions or by thrombomodulin. This compound may represent the prototype of a class of agents that specifically modulates the balance between thrombin's procoagulant and anticoagulant functions.

Post-translational modifications of proteins: some problems left to solve.

Three major questions regarding the post-translational modification of amino acid side chains in proteins are briefly considered: (1) What are the biological functions of the reactions, (2) what is the specificity of the processing reactions in selecting only a few or sometimes even only one residue for modification, and (3) how do we solve the uniqueness of the processing steps in the production of recombinant proteins? The answers to these questions are not obvious at this time.

Inhibition of DNA-dependent transcription by the leader RNA of vesicular stomatitis virus: role of specific nucleotide sequences and cell protein binding.

The leader RNA transcript of vesicular stomatitis virus inhibits transcription of the adenovirus major late promoter and virus-associated genes in a soluble HeLa cell transcription system. We examined the specific nucleotide sequence involved and the potential role of leader-protein interactions in this inhibition of RNA polymerase II- and III-directed transcription. Using synthetic oligodeoxynucleotides homologous to regions of the leader RNA molecule, we extend our previous results (B.W. Grinnell and R.R. Wagner, Cell 36:533-543, 1984) that suggest a role for the AU-rich region of the leader RNA or the homologous AT region of a cloned cDNA leader in the inhibition of DNA-dependent transcription. Our results indicate that a short nucleotide sequence (AUUAUUA) or its deoxynucleotide homolog (ATTATTA) appears to be the minimal requirement for the leader RNA to inhibit transcription by both RNA polymerases, but sequences flanking both sides of this region increase the inhibitory activity. Nucleotide changes in the homologous AT-rich region drastically decrease the transcriptional inhibitory activity. Leader RNAs from wild-type virus, but not from a 5'-defective interfering particle, form a ribonuclease-resistant, protease-sensitive ribonucleoprotein complex in the soluble HeLa cell extract. Several lines of evidence suggest that the leader RNA specifically interacts with a 65,000-dalton (65K) cellular protein. In a fractionated cell extract, only those fractions containing this 65K protein could reverse the inhibition of DNA-dependent RNA synthesis by the plus-strand vesicular stomatitis virus leader RNA or by homologous DNA. In studies with synthetic oligodeoxynucleotides homologous to leader RNA sequences, only those oligonucleotides containing the inhibitory sequence were able to bind to a gradient fraction containing the 65K protein.

Activation of the adenovirus and BK virus late promoters: effects of the BK virus enhancer and trans-acting viral early proteins.

We have examined the activation of the adenovirus major late promoter (MLP) by the cis-acting enhancer element of the human polyomavirus BK and by the trans-acting simian virus 40 (SV40) T antigen and adenovirus E1A proteins. By using chloramphenicol acetyltransferase expression vectors, we found that the MLP (pLP-CAT) was trans-activated in human and monkey kidney cells expressing the SV40 T antigen. In addition, the MLP could be cis-activated by the BK virus enhancer in both human and monkey kidney cells; approximately 20 times more chloramphenicol acetyltransferase was produced from expression vectors containing a hybrid promoter (BL), in which the BK enhancer was upstream of the MLP, than from expression vectors containing the MLP alone. This same level of enhancement of the MLP by the BK enhancer was observed in cells expressing the T antigen of SV40. However, in the 293 cell line, greater enhancement of MLP activity (70-fold) was observed with the BK enhancer sequence. In contrast, MLP activity in the 293 cell line was unchanged by the SV40 enhancer. In cotransfection experiments, MLP activity, augmented by the BK enhancer, could be further stimulated with a plasmid coding for the E1A gene products. By creating deletion mutants, we determined that the high-level activation of the hybrid BL transcriptional unit by the E1A proteins requires both MLP sequences and an intact BK virus enhancer. On the other hand, activation of the BL transcriptional unit by the T antigen did not require an intact enhancer sequence. Our results suggest that the SV40 T antigen and E1A proteins trans-activate the BL promoter by different mechanisms. We also demonstrate in cotransfection experiments that the BK late promoter is activated 45-fold by the SV40 T antigen.

Negative regulation of the human polyomavirus BK enhancer involves cell-specific interaction with a nuclear repressor.

We have examined the cell type-specific regulation of the human BK virus (BKV) enhancer. This enhancer functions efficiently in cis to activate expression from the adenovirus major late promoter in the human kidney cell line, 293, and in a monkey kidney cell line, MK2, but not in the HeLa cell line. In gel retardation migration assays, specific BKV enhancer-protein complexes could be observed by using nuclear extracts prepared from each cell line. Moreover, a unique DNA-protein complex was observed by using the HeLa cell nuclear extracts. By DNase footprint analysis, four binding regions for HeLa cell nuclear proteins were defined within the BKV enhancer repeat region. Two of the protected regions encompassed nuclear factor 1 or CCAAT transcription factor binding sites. These nuclear factor 1 sites also were protected by nuclear proteins from the 293 and MK2 cell lines. The other two protected sites encompassed a region of symmetry which included a sequence similar to the simian virus 40 TC enhancer motif and to a conserved sequence present upstream or within the introns of several cellular genes. These two sites were not protected by either the 293 or MK2 nuclear proteins. Competition studies in transfected cells indicated that the reduced activity of the BKV enhancer in the HeLa cell line was due to negative regulation. Further, we have demonstrated that binding of a nuclear factor(s) to the HeLa cell-specific site is involved in the repression of enhancer activity.

E1A-induced enhancer activity of the poly(dG-dT).poly(dA-dC) element (GT element) and interactions with a GT-specific nuclear factor.

The alternating sequence poly(dG-dT).poly(dA-dC) is a highly repeated sequence in the eucaryotic genome. We have examined the effect of trans-acting early viral proteins on the ability of the GT element to stimulate transcription of the adenovirus major late promoter (MLP). We find that the GT element alone does not activate expression from the MLP in either the presence or absence of another enhancer element. However, in the presence of the E1A gene products of either adenovirus type 5 or 2, the GT element activated expression from the MLP. The stimulatory activity of the GT element in the presence of E1A had the properties of an enhancer element, and the trans-activating effect on the GT element was additive in conjunction with the E1A-responsive BK virus enhancer. We also have demonstrated that a specific nuclear factor(s) binds to the GT element. However, the E1A protein(s) do not affect the initial factor interaction(s) with the GT element. Overall, our data demonstrate that trans modulation of promoter activity can be mediated through the GT element.

Charge reversal at the P3' position in protein C optimally enhances thrombin affinity and activation rate.

We have examined the properties of several human protein C (HPC) derivatives with substitutions for acidic residues near the thrombin cleavage site, including changing the P3' Asp to Asn (D172N), Gly (D172G), Ala (D172A), or Lys (D172K). The rate of thrombin-catalyzed activation of D172N, D172G, and D172A was increased 4-9-fold compared to wild-type HPC, primarily due to a reduction in the inhibitory effect of calcium and a resulting increase in affinity for free alpha-thrombin. There was no significant increase in activation rate or affinity with these 3 derivatives in the absence of calcium, confirming that P3' Asp affects calcium dependency in the native protein C molecule. With charge reversal at P3' (D172K), there was a 30-fold increase in activation rate in the presence of calcium, but unlike the other derivatives, there was a substantial effect (5-fold) on the activation rate and affinity for free alpha-thrombin in the absence of calcium. Thus, protein C affinity for thrombin appears to be influenced by a combination of calcium-dependent and -independent effects of the acidic P3' residue.

17beta-estradiol, but not raloxifene, decreases thrombomodulin in the antithrombotic protein C pathway.

Raloxifene is a nonsteroidal selective estrogen receptor modulator (SERM) that mimics the effects of estrogen on some plasma lipids and may have direct effects on the vascular wall. The objective of this study was to determine the effects of 17beta-estradiol, raloxifene, and LY139,478 (a related benzothiophene SERM) on the anticoagulant protein C pathway. In human vascular endothelial cells activated with interleukin-1 (IL-1), we demonstrated decreased thrombomodulin-dependent protein C activation. 17beta-estradiol reduced the anticoagulant properties of both unstimulated and IL-1-activated endothelial cells by decreasing thrombomodulin expression. In contrast, raloxifene and LY139,478 enhanced the anticoagulant properties of both unstimulated and IL-1-activated endothelial cells through upregulation of thrombomodulin. Regulation of the protein C pathway via thrombomodulin on vascular endothelium may be a novel mechanism by which SERMs could potentially confer cardioprotective effects and reduce the thrombotic risk associated with HRT in compromised patients.

Amplification of multicistronic plasmids in the human 293 cell line and secretion of correctly processed recombinant human protein C.

We have constructed multicistronic vectors containing the cDNAs for murine dihydrofolate reductase (DHFR), hygromycin phosphotransferase (HyPR), and human protein C (HPC), an antithrombotic factor. Using a sequential selection protocol with hygromycin (Hy) and methotrexate (MTX), we demonstrate the selective amplification of the murine dhfr cDNA in the adenovirus-transformed human kidney cell line 293, and the coamplification of the cDNA for HPC. Such recombinant 293 cell lines secreted HPC at levels as high as 25 micrograms/10(6) cells/day. In addition, we found that the complex vitamin K-dependent posttranslational modification of gamma-carboxylation of glutamate was not limiting at these high secretion levels, although the proteolytic processing of the protein was slightly reduced. Further, the HPC secreted from the gene-amplified cell lines had full anticoagulant activity when compared to plasma-derived HPC.

A rapid and versatile method for the detection and isolation of mammalian cell lines secreting recombinant proteins.

The secreted-protein agarose diffusion immunoassay (SADI) described here is an efficient and time-saving method for identifying cell lines secreting recombinant protein products. In addition to significantly reducing the labor involved in identifying clones of interest, it also increases the possibility of detecting rare transformants by allowing the screening of a large number of clones simultaneously. The recovery of viable cell lines following the technique is nearly 100%. We demonstrate that the assay can be used to differentiate between clones producing two different protein products and that it is possible to use the technique to identify and isolate the high-producing recombinant clones in a population. Given an available antiserum, it should be possible to adapt this assay for any secreted protein of interest.

High-level expression of secreted proteins from cells adapted to serum-free suspension culture.

We have developed a host cell/vector system based on the use of adenovirus-transformed cells and a promoter, designated GBMT, capable of being activated by the Ela tumor antigen produced in these cells. GBMT-based vectors were constructed with hygromycin phosphotransferase and murine dihydrofolate reductase as selective markers. We demonstrate their utility in two adenovirus-transformed cell lines, human kidney 293 and Syrian hamster AV12-664. Further, we describe methods and conditions for the direct adaptation of isolated recombinant clones to serum-free suspension growth conditions. For exemplary purposes, we describe the generation of stable recombinant 293 cell lines with single-copy integrated vectors secreting the highly complex clotting factor human protein C at levels as high as 20 mg/l in serum-free suspension culture. In addition, using the AV12-664 cell line with GBMT and direct dominant selection of the dhfr gene, we have isolated clones secreting a tissue plasminogen activator derivative at levels of about 40 mg/l under serum-free suspension conditions. The distinct advantages of this vector/host cell system are 1) the direct selection of stable clones expressing relatively high levels of recombinant protein, eliminating the need for the tedious stepwise gene amplification process and 2) the direct adaptation to serum-free suspension culture.

Unlike thrombin, protein C and activated protein C do not affect vascular tone.

Because plasma levels of protein C (PC) or activated protein C (APC) are altered in certain diseases associated with vascular dysfunction, and APC has therapeutic potential in preventing microvascular coagulation in severe sepsis, potential vascular effects of PC and APC were compared to those of the vasoactive peptide, thrombin. Thrombin was a more potent relaxant agonist than contractile agonist in aorta. Unlike thrombin, cumulatively administered APC (10(-9)-10(-7) M) did not exert vascular effects in rat or rabbit aorta. Noncumulative challenge of PC (10(-7) M) and APC (8 x 10(-8) M) also did not contract rat or rabbit aortae, either with or without endothelium. Likewise, the same concentrations of PC and APC also did not relax norepinephrine-induced (10(-7) M) vascular tone in either rat or rabbit aortae. Thus, in contrast to thrombin, PC and APC failed to modulate vascular tone, suggesting that the therapeutic use of APC is unlikely to be accompanied by any direct effects on vascular motility.

Use of vectors to confer resistance to antibiotics g418 and hygromycin in stably transfected cell lines.

The development of dominant selection markers to identify eukaryotic cells that have undergone a gene transformation event has greatly facilitated molecular genetic studies in higher eukaryotic cells. Selection schemes based on resistance to antibiotic cytotoxicity (1,2) will be described in this chapter. Other schemes-for example, based on resistance to inhibition of DNA synthesis by methotrexate (1) or mycophenolic acid (1)-are described in other chapters of this book. Prior to the development of dominant selection markers, the use of recessive markers, such as thymidine kinase (TK) or hypoxanthine-guanine phosphoribosyl transferase (HGPT) was limited to a handful of mutant cell lines that were TK (-) or HGPT(-) (5,6). If one wished to transfect a wild-type cell line, one had first to select a recessive mutant derivative cell line and characterize it before proceeding with the experiments of interest. Such restrictions posed a significant barrier to molecular genetic analyses in higher eukaryotic cells.

Characterization and novel purification of recombinant human protein C from three mammalian cell lines.

Human Protein C (HPC), an antithrombotic factor with potential clinical utility, is a vitamin K-dependent protein that has several complex post-translational modifications. In an effort to define the functional roles of these modifications, recombinant HPC (rHPC) was expressed in and characterized from 3 adenovirus-transformed cell lines. The rHPC in crude culture medium from the 3 cell lines displayed anticoagulant activities that were either higher, slightly lower or much lower than that of plasma HPC. The rHPC from each cell line was purified and characterized using a novel, but simple chromatographic method, termed "pseudo-affinity", capable of resolving molecules differing by only very slight modifications. We demonstrate the critical dependence of full gamma-carboxylation on the function of this protein. In addition, our data indicate that both the gamma-carboxyglutamate and glycosyl contents affect the functional activities of rHPC.

Nucleotide sequence and secondary structure of VSV leader RNA and homologous DNA involved in inhibition of DNA-dependent transcription.

We have analyzed the nucleotide sequences and secondary structure required for the transcriptional inhibitory activity of the plus-strand leader RNA of vesicular stomatitis virus (VSV) in a reconstituted HeLa cell transcription system using the adenovirus-2 late promoter (LP) and virus-associated (VA) genes as templates. The New Jersey serotype (VSVNJ) leader and the leader of the Indiana serotype (VSVInd) both contain cleavage sites for the double-strand-specific ribonuclease V1, and these sites are consistent with the presence of a predicted AU-rich stem-loop structure. Studies in which the secondary structure was perturbed with the intercalating agent proflavin suggested that a stem-loop structure enhances the efficiency of transcription inhibition in the VSVNJ leader. Experiments using leader RNA fragments, a VSVInd cDNA derived from the 3' end of the genome, and synthetic oligodeoxynucleotide homologous to regions of the VSV leader indicated that the AU(AT)-rich center region of the VSV leader molecule is sufficient to inhibit DNA-dependent transcription directed by both polymerase II and III, but flanking nucleotide sequences are important for more efficient inhibition of transcription.

5' sequence of vesicular stomatitis virus N-gene confers selective translation of mRNA.

The infection of cells by vesicular stomatitis virus results in the rapid inhibition of host-cell protein synthesis, but not of viral protein synthesis. To determine if this translational selectivity might be conferred by the viral mRNA, we constructed a plasmid (pUCLN beta-4) containing the 5' end of the viral nucleocapsid (N)-gene, including the ribosome binding site, fused in frame with the gene encoding beta-galactosidase, and compared it to a control plasmid (pMC1924) containing the cellular rabbit beta-globin gene 5' end fused with the beta-galactosidase encoding gene. Both plasmids contained identical promoter and 3' nontranslated regions and expressed similar levels of beta-galactosidase in the indicator cell line 293. In cells transfected with either plasmid, viral infection resulted in a approximately 70% decrease in protein synthesis by five hours. The level of beta-galactosidase from cells transfected with pMC1924 also decreased concomitantly with the decrease in total protein synthesis. However, the level of beta-galactosidase from cells transfected with pUCLN beta-4 was not affected by viral infection. Our data suggest that sequences in the 5' end of the viral mRNA allow for the selective translation of the viral message in the presence of an inhibited translational machinery.