Role of PRL-3, a human muscle-specific tyrosine phosphatase, in angiotensin-II signaling.

Action of protein kinases and phosphatases contributes to myocardial hypertrophy. PRL-3, a protein tyrosine phosphatase, was identified in a cDNA library from an explanted human heart obtained from a patient with idiopathic cardiomyopathy. PRL-3 is expressed in heart and skeletal muscle, exhibiting approximately 76% identity to the ubiquitous tyrosine phosphatase PRL-1, which was reported to increase cell proliferation. PRL-3 was cloned into E. coli and purified using affinity chromatography. PRL-3 activity was determined using the substrate 6,8-difluoro-4-methylumbelliferyl phosphate, and was inhibited by vanadate and analogs. HEK293 cells expressing PRL-3 demonstrated increased growth rates versus nontransfected cells or cells transfected with the catalytically inactive C104S PRL-3 mutant. The tyrosine phosphatase inhibitor, potassium bisperoxo (bipyridine) oxovanadate V, normalizes the growth rate of PRL-3 expressing cells to that of parental HEK293 cells in a concentration-dependent manner. Using FLIPR analysis, parental HEK293 cells mobilize calcium when stimulated with angiotensin-II (AngII). However, calcium mobilization is inhibited in cells expressing wild-type PRL-3 when stimulated with AngII, while cells expressing the inactive mutant of PRL-3 mobilize calcium to the same extent as parental HEK293 cells. Western blots comparing PRL-3 transfected cells to parental HEK293 cells showed dephosphorylation of p130(cas) in response to AngII. These data suggest a role for PRL-3 in the modulation of intracellular calcium transients induced by AngII.

Expression, purification, and characterization of active recombinant prostate-specific antigen in Pichia pastoris (yeast).

BACKGROUND: Prostate-specific antigen (PSA), a member of the kallikrein family of serine proteases, is a chymotrypsin-like glycoprotein produced by the prostate epithelium. Elevated serum PSA (> 4 ng/ml) is a tumor marker for prostatic cancer and benign prostatic hypertrophy; increasing serum PSA over time is indicative of metastatic disease. It has been suggested that PSA may contribute to tumor metastasis through degradation of extracellular matrix glycoproteins, as well as cleavage of IGF binding protein-3, a modulator of IGF-1. To elucidate the role of PSA in the development and progression of prostatic cancer, it is necessary to have a reliable, cost-effective source of enzymatically active protein. Previous efforts to express recombinant PSA (rPSA) produced inactive proPSA, or mixtures of active and inactive PSA requiring activation by removal of the propeptide. We describe the expression of active recombinant mature PSA in yeast. METHODS: Stable chromosomal integration of a construct consisting of the yeast alpha-factor signal sequence preceding the mature PSA sequence resulted in secretion of rPSA. The rPSA was purified from the yeast cell culture supernatant to homogeneity by strong cation-exchange chromatography, and characterized by SDS-PAGE, Western analysis, electrospray mass spectrometry, N-glycanase digestion, N-terminal amino acid sequencing, and inactivation by a PSA-specific inhibitor. RESULTS: We report the production of active, mature rPSA in Pichia pastoris. Two forms of rPSA varying slightly in glycosylation were identified. The specific activity of the rPSA was equal to that of human seminal plasma PSA (0.56 micromol/min mg) as determined using a chromogenic substrate. CONCLUSIONS: Large-scale production of active rPSA will be useful in the exploration of PSA effects on tumor cell proliferation, migration and metastasis. In addition, a large supply of enzyme should facilitate the discovery of novel inhibitors for in vitro and in vivo evaluation, and may provide a reproducible source of rPSA for use as a standard in diagnostic testing.

LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate receptors.

The in vitro pharmacology of a structurally novel compound, LY341495, was investigated at human recombinant metabotropic glutamate (mGlu) receptor subtypes expressed in non-neuronal (RGT, rat glutamate transporter) cells. LY341495 was a nanomolar potent antagonist of 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD)-induced inhibition of forskolin-stimulated cAMP formation at mGlu2 and mGlu3 receptors (respective IC50S of 0.021 and 0.014 microM). At group I mGlu receptor expressing cells, LY341495 was micromolar potent in antagonizing quisqualate-induced phosphoinositide (PI) hydrolysis, with IC50 values of 7.8 and 8.2 microM for mGlu1a and mGlu5a receptors, respectively. Among the human group III mGlu receptors, the most potent inhibition of L-2-amino-4-phosphonobutyric acid (L-AP4) responses was seen for LY341495 at mGlu8, with an IC50 of 0.17 microM. LY341495 was less potent at mGlu7 (IC50 = 0.99 microM) and least potent at mGlu4 (IC50 = 22 microM). Binding studies in rat brain membranes also demonstrated nanomolar potent group II mGlu receptor affinity for LY341495, with no appreciable displacement of ionotropic glutamate receptor ligand binding. Thus, LY341495 has a unique range of selectivity across the mGlu receptor subtypes with a potency order of mGlu3 > or = mGlu2 > mGlu8 > mGlu7 >> mGlu1a = mGlu5a > mGlu4. In particular, LY341495 is the most potent antagonist yet reported at mGlu2, 3 and 8 receptors. Thus, it represents a novel pharmacological agent for elucidating the function of mGlu receptors in experimental systems.

Group III human metabotropic glutamate receptors 4, 7 and 8: molecular cloning, functional expression, and comparison of pharmacological properties in RGT cells.

Cloning and expression in a stable mammalian cell line co-transfected with a glutamate transporter (RGT cells) were used as tools for studying the functions and pharmacological properties of group III metabotropic glutamate receptors (mGluRs). Complementary DNAs (cDNAs) encoding the human mGluR4, human mGluR7, and human mGluR8 were isolated from human cerebellum, fetal brain or retinal cDNA libraries. The human mGluR4, mGluR7 and mGluR8 receptors were 912, 915 and 908 amino acid residues long and share 67-70% amino acid similarity with each other and 42-45% similarity with the members of mGluR subgroups I and II. The human mGluR4 and mGluR7 had amino acid identity of 96% and 99.5% with rat mGluR4 and 7, respectively, whereas the human mGluR8 has 98.8% amino acid identity with the mouse mGluR8. The nucleotide and amino acid sequences in the coding region of human mGluR4 and mGluR7 were found to be identical to the previously published sequences by Flor et al. and Makoff et al. Following stable expression in RGT cells, highly significant inhibitions of forskolin stimulation of cAMP production by group III agonists were found for each receptor. The relative potencies of the group III agonist L-AP4 varied greatly between the group III clones, being mGluR8>mGluR4 >> mGluR7. The reported group II mGluR agonist L-CCG-I was a highly potent mGluR8 agonist (EC50=0.35 microM), with significant agonist activities at both mGluR4 (EC50=3.7 microM) and mGluR7 (EC50=47 microM). The antagonist potency of the purported group III mGluR antagonist MPPG also varied among the receptors being human mGluR8 >> mGluR4 = mGluR7. The expression and second messenger coupling of human group III mGluRs expressed in the RGT cell line are useful to clearly define the subtype selectivities of mGluR ligands.

Effects of the M1 agonist xanomeline on processing of human beta-amyloid precursor protein (FAD, Swedish mutant) transfected into Chinese hamster ovary-m1 cells.

Complementary DNA (cDNA) encoding human beta-amyloid precursor protein familial Alzheimer's disease (FAD) Swedish mutant (beta APPSM) form was cloned into a mammalian expression vector (PK255) containing the CMV promoter. The vector was transfected into Chinese hamster ovary cells containing human muscarinic m1 receptors (CHO-m1), and clonal cells stably expressing beta APPSM were isolated. The effects of m1-receptor activation by the selective m1 agonist xanomeline and the non-selective muscarinic agonist carbachol on processing of beta APPSM to release soluble APP (APPs) and beta-amyloid peptide (A beta) were compared. Xanomeline stimulated APP release with a potency 1000-fold greater than that observed for carbachol. Concentrations of carbachol and xanomeline producing maximal effects on APPs release reduced the secretion of A beta by 28 and 46%, respectively. These results extend previous studies with xanomeline and suggest that cholinergic replacement therapy for Alzheimer's disease may reduce amyloid deposition.

Increased production of low molecular weight recombinant proteins in Escherichia coli.

A general method for obtaining high-level production of low molecular weight proteins in Escherichia coli is described. This method is based on the use of a novel Met-Xaa-protein construction which is formed by insertion of a single amino acid residue (preferably Arginine or Lysine) between the N-terminal methionine and the protein of interest. The utility of this method is illustrated by examples for achieving high-level production of human insulin-like growth factor-1, human proinsulin, and their analogs. Furthermore, highly produced insulin-like growth factor-1 derivatives and human proinsulin analogs are converted to their natural sequences by removal of dipeptides with cathepsin C.

The novel metabotropic glutamate receptor agonist 2R,4R-APDC potentiates stimulation of phosphoinositide hydrolysis in the rat hippocampus by 3,5-dihydroxyphenylglycine: evidence for a synergistic interaction between group 1 and group 2 receptors.

The mGlu receptor subtypes and second messenger pathways that mediate 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) responses in brain tissues are not fully understood. 1S,3R-ACPD differs from 3,5-dihydroxyphenylglycine (DHPG) or quisqualate in that 1S,3R-ACPD also activates group 2 mGlu receptors (mGlu2 and mGlu3) that are negatively linked to cAMP formation. To investigate the contribution of group 2 mGlu receptor activity of 1S,3R-ACPD to the phosphoinositide response in the rat hippocampus, we examined the effects of the novel group 2 mGlu receptor agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC). 2R,4R-APDC did not activate or inhibit group 1 mGlu receptors (human mGlu1 alpha and mGlu5a) or group 3 mGlu receptors (human mGlu4 and mGlu7), but potently decreased forskolin-stimulated cAMP formation in human mGlu2- and mGlu3-expressing cells. In slices of the adult rat hippocampus 2R,4R-APDC had no effect on basal phosphoinositide hydrolysis; however, it was found to greatly enhance phosphoinositide hydrolysis to DHPG or quisqualate. In the neonatal rat hippocampus, 2R,4R-APDC enhanced the potency of DHPG, while not affecting the maximal response to group 1 mGlu receptor agonists. Thus, the phosphoinositide response in the rat hippocampus to 1S,3R-ACPD is mediated by a synergistic interaction between group 1 and group 2 mGlu receptors.

(A-C-B) human proinsulin, a novel insulin agonist and intermediate in the synthesis of biosynthetic human insulin.

The hormone insulin is synthesized in the beta cell of the pancreas as the precursor, proinsulin, where the carboxyl terminus of the B-chain is connected to the amino terminus of the A-chain by a connecting or C-peptide. Proinsulin is a weak insulin agonist that possesses a longer in vivo half-life than does insulin. A form of proinsulin clipped at the Arg65-Gly66 bond has been shown to be more potent than the parent molecule with protracted in vivo activity, presumably as a result of freeing the amino terminal residue of the A-chain. To generate a more active proinsulin-like molecule, we have constructed an "inverted" proinsulin molecule where the carboxyl terminus of the A-chain is connected to the amino terminus of the B-chain by the C-peptide, leaving the critical Gly1 residue free. Transformation of Escherichia coli with a plasmid coding for A-C-B human proinsulin led to the stable production of the protein. By a process of cell disruption, sulfitolysis, anion-exchange chromatography, refolding, and reversed-phase high-performance liquid chromatography, two forms of the inverted proinsulin differing at their amino termini as Gly1 and Met0-Gly1 were identified and purified to homogeneity. Both proteins were shown by a number of analytical techniques to be of the inverted sequence, with insulin-like disulfide bonding. Biological analyses by in vitro techniques revealed A-C-B human proinsulin to be intermediate in potency when compared to human insulin and proinsulin. The time to maximal lowering of blood glucose in the fasted normal rat appeared comparable to that of proinsulin. Additionally, we were able to generate fully active, native insulin from A-C-B human proinsulin by proteolytic transformation. The results of this study lend themselves to the generation of novel insulin-like peptides while providing a simplified route to the biosynthetic production of insulin.

Expression and secretion of a functional scorpion insecticidal toxin in cultured mouse cells.

We have expressed a synthetic gene encoding the insecticidal neurotoxin of scorpion Androctonus australis (AaIT) in NIH/3T3 mouse fibroblast cells under the transcriptional control of a murine retroviral long terminal repeat. The secretion of the toxin into the culture medium was directed by the signal peptide of human interleukin-2. The recombinant AaIT produced was selectively toxic to yellow-fever mosquito larvae and harmless to mice.

Characterization of antigen receptor response elements within the interleukin-2 enhancer.

T-cell activation and induction of interleukin-2 (IL-2) expression in human T lymphocytes require both interaction of foreign antigen with the T-cell antigen receptor and protein kinase C (PKC) stimulation. Agents such as phorbol 12-myristate 13-acetate (PMA) that stimulate PKC augment the effects of antigen but are not sufficient for IL-2 activation. By analysis of deletion mutants, we identified three DNA sequences extending from -73 to -89, -217 to -255, and -263 to -279, designated IL-2 sites A, D, and E, respectively, that are required for maximal induction of IL-2 expression. One of these regions, site E, interacted with a protein (NF-IL-2E) present only in the nuclei of cells which have been stimulated. The other two sequences interacted with a protein (NF-IL-2A) that is constitutively expressed in T cells. When multiple tandem copies of either the E site or the A site were placed upstream of the gamma-fibrinogen promoter, they activated expression via this promoter in response to signals initiated at the antigen receptor but not following PMA stimulation. For this reason, we denoted them antigen receptor response elements. The uncoupling of antigen receptor and PKC requirements in these studies indicates that these signal pathways are, at least in part, distinct and integrated at the level of the gene.

Analysis of the role of cysteine residues in isopenicillin N synthetase activity by site-directed mutagenesis.

The predicted amino acid sequences of isopenicillin N synthetase from both Cephalosporium acremonium and Penicillium chrysogenum have two cysteine residues in analogous positions (Cys-106 and Cys-255 in the C. acremonium numbering). To examine the role of these cysteine residues in the activity of the C. acremonium enzyme, we used site-directed in vitro mutagenesis to change these cysteine residues to serine residues. Mutation of Cys-255 reduces specific activity approximately equal to 50%, whereas mutation of Cys-106 or mutation of both Cys-106 and Cys-255 reduces specific activity about 97%. This suggests that the cysteines are important but not essential for IPNS activity. Alkylation of IPNS also almost completely inactivated the enzyme, but residual activity could have been due to incomplete alkylation. Atomic substitution via genetic manipulation in this case is a more accurate means of assessing the role of sulfhydryl moieties in enzyme activity.

Vitamin K-dependent carboxylase: possible role of the substrate "propeptide" as an intracellular recognition site.

The liver microsomal vitamin K-dependent carboxylase catalyzes the posttranslational conversion of specific glutamate residues to gamma-carboxyglutamate residues in a limited number of proteins. A number of these proteins have been shown to contain a homologous basic amino acid-rich "propeptide" between the leader sequence and the amino terminus of the mature protein. Plasmids encoding protein C, a vitamin K-dependent protein, containing or lacking a propeptide region were constructed and the protein was expressed in Escherichia coli. The protein products were assayed as substrates in an in vitro vitamin K-dependent carboxylase system. Only proteins containing a propeptide region were substrates for the enzyme. These data support the hypothesis that this sequence of the primary gene product is an important recognition site for this processing enzyme.

Translation of a synthetic two-cistron mRNA in Escherichia coli.

A synthetic two-cistron expression system was constructed for the high-level expression of eukaryotic genes in Escherichia coli. This system was designed to overcome translational inhibition of mRNAs containing eukaryotic sequences. The first cistron in this system is a 31-base A + T-rich synthetic sequence that provides for efficient translation initiation. The second cistron contains the protein coding sequence for the eukaryotic gene. Insertion of the first cistron between the 5' untranslated region of the mRNA and the protein coding region separates the two and thereby potentially minimizes the formation of local secondary structures that might prevent ribosomes from binding and initiating translation. The 31-base cistron contains three nonsense codons (TAA), one in each of the three translational reading frames, and an 8-base Shine-Dalgarno sequence that is complementary to the 3' end of the 16S rRNA. The effects of translation of the first cistron in all three reading frames on the expression of the second cistron was examined. The most efficient expression of the second cistron seemed to occur when the stop codon that terminates translation of the first cistron is located 3' to the Shine-Dalgarno sequence and close to the AUG start codon for the second cistron. When the Shine-Dalgarno sequence was deleted from the first cistron, no detectable expression of the second cistron was observed. This two-cistron system has been used to express the gene encoding methionylalanyl bovine growth hormone with its native codons and the gene encoding methionyl human growth hormone at a level greater than 20% of total cell protein. In the case of human growth hormone, we show that the amount of gene product is not significantly diminished by placing a "functional" first cistron in front of a gene that can be expressed without a cistron.

Isolation, sequence determination and expression in Escherichia coli of the isopenicillin N synthetase gene from Cephalosporium acremonium.

The enzyme isopenicillin N synthetase (IPS) catalyses the oxidative condensation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (LLD-ACV) to isopenicillin N, which is a central reaction in the pathway to clinically important penicillins and cephalosporins. Here we report the cloning, characterization and expression in Escherichia coli of the gene encoding the IPS protein in Cephalosporium acremonium. The IPS gene was identified by purifying IPS protein, determining the first 23 amino-terminal amino acids, preparing a set of synthetic oligonucleotides encoding a portion of the determined amino-acid sequence, and probing a cosmid genome library with the mixed oligonucleotides. A cosmid hybridizing with the probe was isolated and the IPS gene was localized and sequenced. The IPS gene encodes a polypeptide of relative molecular mass (Mr) 38,416. When this open reading frame was cloned into an E. coli expression vector and inserted into E. coli, the recombinant E. coli produced a new protein co-migrating with authentic IPS as the major protein of the cell (approximately 20% of cell protein). Crude cell extracts condensed LLD-ACV to a penicillinase-sensitive molecule whose antibacterial activity indicated that it was isopenicillin N.

Rabbit muscle creatine phosphokinase. CDNA cloning, primary structure and detection of human homologues.

A cDNA library was constructed from rabbit muscle poly(A) RNA. Limited amino acid sequence information was obtained on rabbit muscle creatine phosphokinase and this was the basis for design and synthesis of two oligonucleotide probes complementary to a creatine kinase cDNA sequence which encodes a pentapeptide. Colony hybridizations with the probes and subsequent steps led to isolation of two clones, whose cDNA segments partially overlap and which together encode the entire protein. The primary structure was established from the sequence of two cDNA clones and from independently determined sequences of scattered portions of the polypeptide. The reactive cysteine has been located to position 282 within the 380 amino acid polypeptide. The rabbit cDNA hybridizes to digests of human chromosomal DNA. This reveals a restriction fragment length polymorphism associated with the human homologue(s) which hybridizes to the rabbit cDNA.

Synthesis of an ochre suppressor tRNA gene and expression in mammalian cells.

We have used site-specific mutagenesis to change the anticodon of a Xenopus laevis tyrosine tRNA gene so that it would recognize ochre codons. This tRNA gene is expressed when amplified in monkey cells as part of a SV40 recombinant and efficiently suppresses termination at both the ochre codon separating the adenovirus 2 hexon gene from a 23-kd downstream gene and the ochre codon at the end of the NS1 gene of influenza virus A/Tex/1/68. Termination at an amber codon of a NS1 gene of another influenza virus strain was not suppressed by the (Su+) ochre gene suggesting that in mammalian cells amber codons are not recognized by ochre suppressor tRNAs. Finally, microinjection into mammalian cells of both (Su+) ochre tRNA genes and selectible genes containing ochre nonsense mutations gives rise to colonies under selective conditions. We conclude that it should be possible to isolate a wide assortment of mammalian cell lines with ochre suppressor activity.

Cloning and sequencing of liver cDNA coding for bovine protein C.

cDNA coding for protein C has been cloned from a bovine liver library in plasmid vector pBR322 and its sequence has been determined. Two overlapping clones code for the entire light and heavy chains of the mature protein, as well as a previously unreported connecting dipeptide (Lys-Arg) and a 39-amino acid leader peptide region. Identification and characterization of the clones establishes the liver as a site of protein C biosynthesis. A contiguous coding region reveals that a one-chain precursor protein is made that upon limited proteolysis yields both the mature light and heavy chains. The codon for aspartic acid is found at light chain amino acid position 71, showing that the beta-hydroxyaspartic acid that exists in this position of the mature protein is the result of post-translational modification of an aspartic acid residue. Amino acid sequence homology in the amino-terminal region of the light chain with other vitamin K-dependent coagulation factors is continued into the leader peptide region.

Role of mRNA translational efficiency in bovine growth hormone expression in Escherichia coli.

The conditions necessary for high-level expression of methionyl bovine growth hormone (Met-bGH) in Escherichia coli were investigated. Plasmids were constructed that contain a thermoinducible runaway replicon and either the E. coli tryptophan or lipoprotein promoter and ribosome binding sites, which served as transcriptional and translational initiation sites for the expression of the bGH gene. The expression of Met-bGH was low with either system. However, expression levels of up to 30% of total cell protein were obtained after the introduction of additional codons 3' to the initiating AUG codon, thus altering the NH2-terminal amino acid sequence of bGH. To obtain high-level expression of Met-bGH a two-cistron system was constructed in which the codons that enhanced the expression of bGH were incorporated into the first cistron, and the coding region for Met-bGH was incorporated into the second cistron. This approach may be generally applicable to achieving high-level expression of a gene that contains NH2-terminal sequences that do not allow for its efficient expression. Analyses of the stabilities of the bGH derivatives and their transcripts in vivo suggested that the variations in the level of expression were due to variations in the efficiency of mRNA translation.