Disulfide pairing of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli.

The kringle-2 domain of tissue plasminogen activator, cloned and expressed in Escherichia coli (Wilhelm, O.G., Jaskunas, S.R., Vlahos, C.J., and Bang, N.U. (1990) J. Biol. Chem. 265, 14606-14611), was internally radiolabeled using [35S]methionine-cysteine. Following refolding and isolation, the labeled polypeptide was further purified by reverse-phase high performance liquid chromatography. The purified kringle-2 domain was digested with thermolysin, and the resulting peptides were purified by high performance liquid chromatography. Five major peptides containing 35S were obtained. Amino acid sequence analysis showed that these peptides represented various cleavage products containing one or more of the following disulfides: Cys180-Cys261, Cys201-Cys243, Cys232-Cys256 (sequence numbering based on Pennica et al. (Pennica, D., Holmes, W.E., Kohr, W.J., Hakins, R.N., Vehar, G. A., Ward, C.A., Bennett, W.F., Yelverton E., Seeburg, P.H., Heynecker, H.L., Goeddel, E.V., and Collen, D. (1983) Nature 301, 214-221)). These results confirm that the refolding methodology used produced kringle-2 with the predicted disulfide linkage and, thus, yielded material suitable for structural and functional studies.     

Processing and secretion of tumor necrosis factor alpha in endotoxin-treated Mono Mac 6 cells are dependent on phorbol myristate acetate.

Lipopolysaccharide (LPS, endotoxin) is a potent stimulator of tumor necrosis factor alpha (TNF alpha) synthesis and secretion in mouse macrophage tumor cells (Golenbock, D. T., Hampton, R. Y., Qureshi, N., Takayama, K., and Raetz, C. H. R. (1991) J. Biol. Chem. 266, 19490-19498). In contrast, addition of LPS (10 ng/ml) to human monomyelocytic (Mono Mac 6) cells induces very little production of TNF alpha, as judged by immunoassay of the growth medium. When 30 ng/ml 4-beta-phorbol-12-myristate 13-acetate (PMA) is added together with LPS, large amounts of TNF alpha are secreted. PMA alone is inactive. Maximal TNF alpha levels in the medium are achieved at 1 ng/ml of LPS. Protein kinase C inhibitors, such as H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine), staurosporine, and sphingosine, reduce TNF alpha secretion stimulated by PMA. The effect of PMA has been investigated at each stage of TNF alpha biogenesis. Treatment of Mono Mac 6 cells with LPS alone results in rapid, transient, and full expression of TNF alpha mRNA. Concomitant addition of PMA does not increase TNF alpha mRNA synthesis any further, but it prolongs the half-life of TNF alpha mRNA about 3-fold. However, mRNA stabilization does not account for the striking effect of PMA on TNF alpha secretion. Analysis of TNF alpha synthesis and secretion by immunoprecipitation indicates that LPS alone is fully effective in stimulating the formation of the intracellular 26-kDa TNF alpha precursor. LPS alone is not sufficient to allow processing of the precursor and secretion of mature 17-kDa TNF alpha. The rate of TNF alpha secretion observed immediately after the addition of PMA to LPS-pretreated cells is similar to the maximum rate from LPS/PMA-treated cells, but without the lag observed in cells after being exposed to LPS and PMA simultaneously. In summary, PMA is required for the completion of TNF alpha precursor processing and secretion in LPS-treated human Mono Mac 6 cells, whereas murine RAW cells are able to complete the terminal steps of TNF alpha processing in the absence of PMA.     

Molecular cloning of murine p68, a Ca2+-binding protein of the lipocortin family

A plasmid cDNA library prepared from a T-lymphocyte clone of murine strain B10.A origin was screened by cross-species DNA hybridisation using a partial human p68 cDNA clone, identified as containing coding sequences for previously determined amino-acid sequences. The longest p68 cDNA insert from this library and a full-length cDNA insert from a second similar library were fully sequenced. A comparison of the derived amino-acid sequence with that of human p68 revealed extensive homology (95% overall). Homology at the nucleotide level was 89% in the open reading frame and 85% and 50% in the 5' (33 nucleotides) and 3' (347 nucleotides) non-coding regions respectively. Eight segments of internal homology were observed, each containing a highly conserved consensus region of 17 amino acids correlating with that described for several membrane associated calcium-binding proteins [Geisow, M. J., Fritsche, U., Hexham, J. M. & Johnson, T. (1986) Nature (Lond.) 320, 636-638]. These results provide further evidence that p68 is a member of the same gene family as p32,p36 and lipocortin I and demonstrate an unusually high level of inter-species sequence conservation of p68 between mouse and human.     

Novel mutants of human tumor necrosis factor with dominant-negative properties

Tumor necrosis factor (TNF) is a polyfunctional cytokine, one of the key mediators of inflammation and innate immunity. On the other hand, systemic or local TNF overexpression is typical of such pathological states as rheumatoid arthritis, psoriasis, Crohn’s disease, septic shock, and multiple sclerosis. Neutralization of TNF activity has a marked curative effect for some diseases; therefore, the search for various TNF blockers is a promising field of protein engineering and biotechnology. According to the previously developed concept concerning the possibility of designing dominant-negative mutants, the following TNF variants have been studied: TNFY87H + A145R, TNFY87H + A96S + A145R, and TNFV91N + A145R. All of these form inactive TNF heterotrimers with the native protein. The ability of mutants to neutralize the effect of TNF was investigated. The addition of mutants to the native protein was shown to provide a concentration-dependent suppression of TNF cytotoxicity against the mouse fibroblast cell line L929. Thus, novel inhibitors of human TNF can be engineered on the basis of these muteins.     

A polymerase chain reaction strategy to identify and clone cyclic nucleotide phosphodiesterase cDNAs. Molecular cloning of the cDNA encoding the 63-kDa calmodulin-dependent phosphodiesterase.

Multiple isozymes of cyclic nucleotide phosphodiesterases (PDEs) are expressed simultaneously in mammalian tissues. To identify and clone these PDEs, a polymerase chain reaction (PCR) strategy was developed using degenerate oligonucleotide primers designed to hybridize with highly conserved PDE DNA domains. Both known and novel PDEs were cloned from rat liver, the mouse K30a-3.3 lymphoma cell line, and a human hypothalamus cDNA library, demonstrating that these PCR primers can be used to amplify the cDNA of multiple PDE isozymes. One unique mouse PDE clone was found to encode a polypeptide identical with the corresponding portion of the bovine brain 63-kDa calmodulin-dependent PDE as reported in the companion article (Bentley, J. K., Kadlecek, A., Sherbert, C. H., Seger, D., Sonnenburg, W. K., Charbonneau, H., Novack, J. P., and Beavo, J. A. (1992) J. Biol. Chem. 267, 18676-18682). This mouse clone was used as a probe to screen a rat brain cDNA library for a full-length clone. The conceptual translation of the nucleotide sequence of the resulting rat clone has an open reading frame of 535 amino acids and maintains a high degree of homology with the bovine 63-kDa calmodulin-dependent PDE, indicating that this protein is likely to be the rat homolog of the 63-kDa calmodulin-dependent PDE. Expression of the full-length clone in Escherichia coli yielded a cGMP hydrolyzing activity that was stimulated severalfold by calmodulin. Northern blot analysis demonstrated that the mRNA encoding this PDE is highly expressed in rat brain and also in the S49.1 T-lymphocyte cell line. These data demonstrate that the PCR method described is a viable strategy to isolate cDNA clones of known and novel members of different families of PDE isozymes. Molecular cloning of these PDEs will provide valuable tools for investigating the roles of these isozymes in regulation of intracellular concentrations of the cyclic nucleotides.     

Molecular cloning of rat intestinal mucin. Lack of conservation between mammalian species

We have prepared antisera to deglycosylated rat intestinal mucin and used it to obtain immunoreactive clones from a rat jejunum cDNA library. Four of these clones were sequenced, and all were found to be partial cDNAs that contained 18-base pair tandem repeats characteristic of a mucin. These cDNAs encoded a repetitive peptide with a consensus sequence of TTTPDV. Thus, they bear little resemblance to either of the two human intestinal mucin cDNAs isolated previously (Gum, J. R., Byrd, J. C., Hicks, J. W., Toribara, N. W., Lamport, D. T. A., and Kim, Y. S. (1989) J. Biol. Chem. 264, 6480-6487 and Gum, J. R., Hicks, J. W., Swallow, D. M., Lagace, R. E., Byrd, J. C., Lamport, D. T. A., Siddiki, B., and Kim, Y. S. (1990) Biochem. Biophys. Res. Commun. 171, 407-415). One of these rat mucin clones, designated RMUC 176, was chosen for further analysis. This clone recognized a band of approximately 9 kilobases when used to probe RNA blots. A strong hybridization band was present using rat small intestine and colon RNA but was not detectable when RNA isolated from heart, liver, or kidney was tested. The RMUC 176 clone and the two previously isolated human intestinal mucin cDNA clones were used to probe blots prepared from BamHI-digested DNA of various species. Here, the human probes detected fragments present only in human and chimpanzee DNA, whereas the RMUC 176 clone recognized fragments only in rat and mouse DNA. Thus, the repetitive portions of intestinal mucin genes are apparently not well conserved between phylogenetically distant species.     

Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

We have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase [Van den Bosch, H., Aarsman, A. J., DeJong, G. N., & Van Deenen, L. M. (1973) Biochim. Biophys. Acta 296, 94-104].     

Cloning and sequence determination of a complementary DNA related to human liver microsomal cytochrome P-450 S-mephenytoin 4-hydroxylase

A cDNA sequence related to the human cytochrome P-450 responsible for S-mephenytoin 4-hydroxylation (P-450MP) has been isolated from a human liver bacteriophage lambda gt11 library with antibodies specific for P-450MP. The total length of the cDNA is 2.5 kilobases (kb), of which there is a 1.6-kb EcoRI fragment coding for all but five amino acids corresponding to the N-terminus of the protein and including a small noncoding region at the 3' end. This 1.6-kb fragment has been sequenced and used as a probe to analyze human genomic DNA and liver RNA. The sequence shows extensive sequence similarity with that of rabbit liver cytochrome P-450 progesterone 21-hydroxylase [Tukey, R. H., Okino, S., Barnes, H., Griffin, K. J., & Johnson, E. F. (1985) J. Biol. Chem. 260, 13347-13354], and this cDNA, like the rabbit clone, appears to be part of a multigene family. At least two liver mRNA species, 2.2 kb and 3.5 kb, hybridize to the cDNA sequence. The cloning of this gene should aid in analyzing the molecular basis for the genetic polymorphism of S-mephenytoin 4-hydroxylation reported in humans.     

Preferential retention of paternal alleles in human retinoblastoma: evidence for genomic imprinting

The origins of the initial mutations in sporadic retinoblastoma were explored using polymorphic markers from chromosome 13q. The paternal chromosome was maintained in 3 of 3 informative bilateral tumors which had undergone reduction to homozygosity for regions of this chromosome. The paternal chromosome was maintained in 7 of 8 informative unilateral tumors which likewise demonstrated a reduction of homozygosity. These data are in contrast to previously published studies of chromosome retention in unilateral retinoblastoma [Dryja, T. P., Mukai, S., Petersen, R., Rapaport, J. M., Walton, D., and Yandel, D. W. Nature (Lond.), 339: 556-558, 1989; Zhu, Z., Dunn, J. M., Phillips, R. A., Goddard, A. D., Paton, K. E., Becker, A., and Gallie, B. L. Nature (Lond.), 340: 312-313, 1989] and provide the first evidence that genomic imprinting may play a role in this disease.     

The molecular cloning and characterization of murine ferritin heavy chain, a tumor necrosis factor-inducible gene

Ferritin is a ubiquitous and highly conserved protein which plays a major role in iron homeostasis. We have identified and sequenced a full-length cDNA for murine ferritin heavy chain. The isolated cDNA is 819 nucleotides in length. It includes 546 nucleotides which encode a protein of 182 amino acids, a 5' noncoding sequence of 120 nucleotides, and a 3'-noncoding region of 153 nucleotides. The sequence displays a high degree of homology to human ferritin H, and includes a portion of the iron-responsive element conserved in chick, frog, and human ferritin. Tumor necrosis factor (TNF), a cytokine which mediates elements of the stress response, induces expression of ferritin H mRNA. Both mouse TA1 adipocytes and human muscle cells increase expression of ferritin H mRNA 4-6-fold after 48 h exposure to TNF. This increase occurs both prior and subsequent to differentiation of adipocytes and muscle cells, and is accompanied by an increase in the synthesis of the ferritin H subunit. These findings suggest a novel role for TNF in iron metabolism.     

Expression of human cation-independent mannose 6-phosphate receptor cDNA in receptor-negative mouse P388D1 cells following gene transfer

We recently reported the cDNA cloning, sequence, and expression of the human cation-independent mannose 6-phosphate receptor (hCI-MPR) (Oshima, A., Nolan, C. M., Kyle, J. W., Grubb, J. H., and Sly, W. S. (1988) J. Biol. Chem. 263, 2553-2562). The sequence of the hCI-MPR was virtually identical to that of the human insulin-like growth factor II receptor cDNA (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). To test the role of the putative bifunctional receptor in intracellular sorting of acid hydrolases, we studied its effect on lysosomal enzyme transport following gene transfer to receptor-negative cells. Receptor-negative mouse P388D1 cells were transfected with a cDNA construct containing the entire coding sequence of hCI-MPR under the control of the mouse metallothionine I promoter. Stable transformants were isolated and characterized. The expressed hCI-MPR was localized in membranes including the plasma membrane, bound mannose 6-phosphate containing ligands, and mediated endocytosis which could be specifically blocked by mannose 6-phosphate. We next measured the effect of the expressed hCI-MPR on intracellular and secreted acid hydrolases. The intracellular activity of the lysosomal marker enzymes beta-glucuronidase and beta-hexosaminidase increased up to 2-fold following transformation. In addition, expression of the receptor greatly reduced the fraction of acid hydrolases secreted. These phenotypic changes in the transformed cell lines support the proposed role of the cation-independent mannose 6-phosphate receptor in intracellular sorting and targeting of lysosomal enzymes.     

The 10,400- and 14,500-dalton proteins encoded by region E3 of adenovirus function together to protect many but not all mouse cell lines against lysis by tumor necrosis factor.

We have reported that the E3 14,700-dalton protein (E3 14.7K protein) protects adenovirus-infected mouse C3HA fibroblasts against lysis by tumor necrosis factor (TNF) (L. R. Gooding, L. W. Elmore, A. E. Tollefson, H. A. Brady, and W. S. M. Wold, Cell 53:341-346, 1988). We have also observed that the E1B 19K protein protects adenovirus-infected human but not mouse cells against TNF lysis (L. R. Gooding, L. Aquino, P. J. Duerksen-Hughes, D. Day, T. M. Horton, S. Yei, and W. S. M. Wold, J. Virol. 65:3083-3094, 1991). We now report that, in the absence of E3 14.7K, the E3 10.4K and E3 14.5K proteins are both required to protect C127 as well as several other mouse cell lines against TNF lysis. The 14.7K protein can also protect these cells from TNF in the absence of the 10.4K and 14.5K proteins. This protection by the 10.4K and 14.5K proteins was not observed in the C3HA cell line. These conclusions are based on 51Cr release assays of cells infected with virus E3 mutants that express the 14.7K protein alone, that express both the 10.4K and 14.5K proteins, and that delete the 14.7K in combination with either the 10.4K or 14.5K protein. The 10.4K protein was efficiently coimmunoprecipitated together with the 14.5K protein by using an antiserum to the 14.5K protein, suggesting that the 10.4K and 14.5K proteins exist as a complex in the infected mouse cells and consistent with the notion that they function in concert. Considering that three sets of proteins (E3 14.7K, E1B 19K, and E3 10.4K/14.5K proteins) exist in adenovirus to prevent TNF cytolysis of different cell types, it would appear that TNF is a major antiadenovirus defense of the host.     

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.     

Adenovirus E3 14.7K protein functions in the absence of other adenovirus proteins to protect transfected cells from tumor necrosis factor cytolysis.

A 14,700-kDa protein (14.7K) encoded by the E3 region of adenovirus has been shown to protect adenovirus-infected mouse C3HA cells from lysis by tumor necrosis factor (TNF) (L. R. Gooding, L. W. Elmore, A. E. Tollefson, H. A. Brady, and W. S. M. Wold, Cell 53:341-346, 1988). These infected cells are sensitized to TNF by expression of the adenovirus E1A proteins (P. Duerksen-Hughes, W. S. M. Wold, and L. R. Gooding, J. Immunol. 143:4193-4200, 1989). In this study we show that 14.7K suppresses TNF cytolysis independently of adenovirus infection. Mouse C3HA and C127 cells were transfected with the 14.7K gene controlled by the mouse metallothionein promoter, and permanent 14.7K-expressing cell lines were tested for sensitivity to TNF cytolysis. Transfected cells which were sensitized to TNF either by inhibitors of protein synthesis, microfilament-destabilizing agents, or adenovirus infection were found to be resistant to TNF cytolysis. Two monoclonal antibodies were isolated and used to quantitate 14.7K in transfected and infected cells. Enzyme-linked immunosorbent assay (ELISA) analysis with these monoclonal antibodies and 14.7K immunoblots showed that 14.7K expression can be induced with cadmium in C3HA and C127 transfectants. The 14.7K induction correlated with a dose-dependent decrease in sensitivity to TNF cytotoxicity. The 14.7K protein does not substantially alter cell surface TNF receptor numbers or affinity on C3HA mouse fibroblasts, as determined by Scatchard analysis of 125I-TNF binding. The 14.7K protein also does not alter TNF signal transduction in general, because TNF induction of cell surface class I major histocompatibility complex molecules on 14.7K transfectants was unmodified. Our findings indicate that the adenovirus 14.7K protein functions as a specific inhibitor of TNF cytolysis in the absence of other adenovirus proteins and thus is a unique tool to study the mechanism of TNF cytotoxicity.     

The expression of human tumor necrosis factor in E. coli

cDNA of human natural TNF (n-TNF) obtained by stimulating human leukemic B cell line (Ball-1) with Sendai virus was cloned. Valine-started-TNF (V-TNF) gene was constructed from the cDNA and expressed in E.coli HB101 under the control of a trp promoter by the induction of 3-indoleacrylic acid. The expression level of V-TNF clone was about 10% of the total E.coli protein. On the other hand, the expression level of glutamine started-TNF (Q-TNF) gene having the same SD-ATG sequence which was constructed from V-TNF gene was as low as about 1/20 of that of V-TNF. The nucleotide sequence around ATG (-4 approximately +12) of Q-TNF gene was randomly changed without modifying the coded amino acid sequence, resulting to obtain high expression clones as similar TNF protein yield as that of V-TNF. These clones possessed A residue rich sequence around the initiation codon ATG. These results show that some correlation might exist between the high expression rate and A residue rich sequence around the initiation codon.     

Sendai virus induces high levels of tumor necrosis factor mRNA in human peripheral blood leukocytes.

Sendai virus induces human peripheral blood leukocytes to produce high levels of tumor necrosis factor (TNF) mRNA. TNF mRNA can represent as much as 0.6% of the total mRNA. Kinetic studies indicate that the level of TNF mRNA peaks about 2 hours before that of IFN-alpha mRNA produced in the same system. Although the peak levels of TNF and IFN-alpha mRNA were similar, TNF in the culture supernatants was at a 200 fold lower level than IFN-alpha. Cloning and sequence analysis of TNF cDNA isolated from peripheral blood leukocytes RNA showed that normal human cells in response to Sendai virus produce TNF identical to that previously isolated and cloned from tumor-derived cell lines. A bacterial expression system was used to produce the cloned TNF at a maximum level of 2 X 10(6) units per ml of culture.