Production of recombinant salmon calcitonin by in vitro amidation of an Escherichia coli produced precursor peptide.

Salmon calcitonin (sCT) is a 32 amino acid peptide hormone that requires C-terminal amidation for full biological activity. We have produced salmon calcitonin by in vitro amidation of an E. coli produced precursor peptide. Glycine-extended sCT, the substrate for amidation, was produced in recombinant E. coli as part of a fusion with glutathione-S-transferase. The microbially produced soluble fusion protein was purified to near homogeneity by affinity chromatography. Following S-sulfonation of the fusion protein, the glycine-extended peptide was cleaved from the fusion by cyanogen bromide. The S-sulfonated peptide was recovered and enzymatically converted to the amidated peptide in a reaction with recombinant peptidylglycine alpha-amidating enzyme (alpha-AE) secreted from Chinese hamster ovary (CHO) cells. After reformation of the intramolecular disulfide bond, the sCT was purified with a step yield of 60%. The ease and speed of this recombinant process, as well as its potential for scale-up, make it adaptable to production demands for calcitonin, a proven useful agent for the treatment of post-menopausal osteoporosis. Moreover, the relaxed specificity of the recombinant alpha-AE for the penultimate amino acid which is amidated allows the basic process to be applied to the production of other amidated peptides.

Accumulation of pepstatin in cultured endothelial cells and its effect on endothelial processing.

Aspartic acid proteases have been implicated in the processing of ET-1(1-39) to ET-1(1-21). To further understand the role of this class of enzymes in ET-1 synthesis, cultured vascular endothelial cells were incubated with pepstatin, and the accumulation of the inhibitor and its effect on the processing of ET-1(1-39) was examined. Pepstatin accumulated in the cells in a time-dependent manner, to a concentration (greater than 10(-7) M) sufficient to inhibit aspartic acid proteases. Pepstatin did not alter the ratio of ET-1(1-21) to ET-1(1-39), nor did it affect the rate of secretion of either peptide. When endothelial cells were incubated with phosphoramidon under identical conditions, the secretion of ET-1(1-21) was significantly reduced with a concomitant increase in the secretion of ET-1(1-39). These results suggest that the processing of ET-1(1-39) does not involve a pepstatin-sensitive aspartic acid protease, and that the enzyme responsible for generating ET-1(1-21) is sensitive to phosphoramidon.

Rat pro-atrial natriuretic factor expression and post-translational processing in mouse corticotropic pituitary tumor cells.

Atrial natriuretic factor (ANF) is stored within atrial myocyte secretory granules as pro-ANF (ANF-(1-126] and is proteolytically processed co-secretionally C-terminal to a single basic amino acid to form ANF-(1-98) and the bioactive product ANF-(99-126). Pro-ANF is also expressed in certain non-cardiac neuroendocrine cell types (e.g. brain, adrenal). Although the relatively low levels of the peptide in these cell types have precluded detailed processing and secretion studies using cultured cells, some work with tissue extracts suggests that pro-ANF is pre-secretionally processed between or C-terminal to Arg101-Arg102 in such cells. In order to assess whether cultured non-cardiac endocrine cells process pro-ANF pre- or co-secretionally, and to establish whether both paired and single basic amino acids can serve as cleavage sites, transfection studies were carried out using the adrenocorticotropic hormone (ACTH)-producing pituitary tumor cell line AtT-20/D-16v. These cells normally cleave pro-ACTH/endorphin pre-secretionally at selected, but not all, pairs of basic amino acids to a variety of product peptides. A prepro-ANF expression plasmid was constructed and transfected into the AtT-20 cells. The resulting ANF/AtT-20 cell clone selected for this study expressed ACTH at levels similar to the untransfected wild type cells and secreted immunoreactive ANF-related material at a rate of approximately 1 fmol/min/10(5) cells, which was about 10% the rate of ACTH secretion. The rates of secretion of both ANF and ACTH could be increased 3-5-fold with a variety of known AtT-20 cell secretagogues including phorbol esters and the beta-adrenergic agonist, isoproterenol, thus indicating that both peptides were routed through regulated secretory pathways. Utilizing a combination of specific antisera directed against various regions of pro-ANF, size exclusion and reversed phase high performance liquid chromatography, and peptide mapping, it was shown that the ANF/AtT-20 cells contained and secreted the bioactive peptide ANF-(103-126) and -(1-97). These results indicate that the ANF/AtT-20 cells specifically cleave pro-ANF pre-secretionally at the same single basic site used by cardiac tissue; this single basic cleavage is apparently followed by removal of Arg98 by carboxypeptidase H. It is also apparent that the cells can cleave at the sole paired basic site in pro-ANF, which is the probable cleavage site used by neurons and some other endocrine cells that express low levels of the prohormone.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of atrial natriuretic factor-(99-126) secretion from neonatal rat primary atrial cultures by activators of protein kinases A and C.

Atrial natriuretic factor (ANF) is stored in atrial myocytes as a prohormone (ANF-(1-126] and is cosecretionally processed to the circulating ANF-related peptides, ANF-(1-98) and ANF-(99-126). Recently, we have shown that the cosecretional processing of ANF can be replicated in primary cultures of neonatal rat atrial myocytes maintained under serum-free conditions and that glucocorticoids are responsible for supporting this processing activity. Activators of protein kinase C (phorbol esters and alpha-adrenergic agonists) and of protein kinase A (cAMP analogs, forskolin, and beta-adrenergic agonists) were tested for their abilities to alter the rate of ANF secretion from the primary cultures. ANF secretion was stimulated approximately 4-fold after a 1-h incubation of the cultures with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA); maximal release occurred at about 100 nM TPA. Reversed-phase high performance liquid chromatography analysis of secreted material indicated that the cells efficiently cosecretionally processed ANF under both basal and TPA-stimulated conditions. However, incubating the cultures for more than 1 h with TPA resulted in a blunted secretory response to further TPA challenge and a 40-50% decrease in the quantity of ANF in the cells. The alpha-adrenergic receptor agonist phenylephrine was also capable of stimulating ANF secretion by about 4-fold at a half-maximal dose of about 1 microM. Phenylephrine-stimulated ANF secretion was inhibited by the alpha 1-adrenergic antagonist prazosin with half-maximal inhibition occurring at approximately 1 nM. Forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate, and N6-2(1)-O-dibutyryladenosine 3':5'-cyclic monophosphate inhibited basal, TPA- and phenylephrine-stimulated ANF secretion. The beta-adrenergic agonist isoproterenol partially inhibited phenylephrine-stimulated ANF secretion with the maximal effect occurring at 1 nM. These results indicate that ANF secretion from the neonatal rat atrial cultures is enhanced by activators of protein kinase C, and decreased by activators of protein kinase A, and that these secretory effects may be mediated through the actions of alpha- and beta-adrenergic receptors, respectively.

The secretion of atrial natriuretic factor-(99-126) by cultured cardiac myocytes is regulated by glucocorticoids.

Atrial natriuretic factor (ANF) is stored in mammalian atria primarily as ANF-(1-126), the precursor to the known circulating form of the hormone ANF-(99-126). When primary cultures of atrial myocytes were maintained in a complete serum-free medium, they contained and secreted an ANF-(1-126)-like peptide. The addition of dexamethasone to the culture medium, however, resulted in the secretion of a molecule with chromatographic characteristics identical to ANF-(99-126), although the intracellular storage form of ANF was unchanged. Radiosequencing and amino acid analysis confirmed that the cultures maintained in dexamethasone secreted authentic ANF-(99-126). Chronic exposure of the cells to dexamethasone also resulted in a significant increase in the quantity of immunoreactive ANF both contained and secreted by the cultures. Dexamethasone stimulated ANF processing and secretion by atrial cultures in a dose-dependent manner, with an approximate EC50 of 10 nM. This stimulation could be reversed by removing the glucocorticoid from the culture medium. ANF processing was also stimulated by the specific glucocorticoid receptor agonist RU 28362, and both DEX- and RU 28362-stimulated ANF processing was inhibited by the specific glucocorticoid receptor antagonist RU 38486. Ventricular cells, which possess few granules and release ANF in a constitutive fashion, were also capable of processing ANF in a glucocorticoid-dependent fashion. Medium freshly removed from atrial cultures did not convert ANF-(1-126) to ANF-(99-126) nor was exogenous ANF-(1-126) efficiently processed when added to the medium of actively processing cultures. These results indicate that the post-translational processing of ANF-(1-126) to ANF-(99-126) likely occurs within or in close association with the cardiac myocytes and is not dependent on the presence of large quantities of secretory granules. Furthermore, it is apparent that both the expression and the post-translational processing of ANF by cultured cardiac myocytes is specifically regulated by glucocorticoids.

The post-translational processing of rat pro-atrial natriuretic factor by primary atrial myocyte cultures.

Primary cultures of neonatal rat atrial myocytes were maintained in two different serum-free media for up to 25 days. Reversed-phase high performance liquid chromatography coupled with atrial natriuretic factor (ANF)-specific radioimmunoassay demonstrated that the cultures maintained in our previously described serum-free medium (Glembotski, C.C., and Gibson, T. R. (1985) Biochem. Biophys. Res. Commun. 132, 1008-1017) secreted primarily ANF-(1-126)-like material, whereas those cultures maintained in a different formulation of medium secreted mostly ANF-(99-126)-like material. Cultures that secreted ANF(99-126)-like material were biosynthetically labeled with [35S]cysteine followed by immunoprecipitation of secreted ANF and analysis by reversed-phase, size exclusion, and ion-exchange high performance liquid chromatography. The labeled ANF-(99-126)-like peptide was shown to be chromatographically indistinguishable from other synthetic peptides related to ANF-(99-126). Labeled ANF purified from extracts of the cultured cells was chromatographically indistinguishable from authentic ANF-(1-126), and could be cleaved specifically by thrombin into labeled ANF-(99-126)-like material. These results indicate that primary atrial myocytes maintained under certain serum-free conditions are capable of secreting ANF-related material that is chromatographically indistinguishable from ANF-(99-126), the known circulating form of the hormone. Additional preliminary studies suggest that the presence of glucocorticoids in the culture medium may confer ANF processing ability on cultured myocytes.

The characterization of atrial natriuretic peptide (ANP) expression by primary cultures of atrial myocytes using an ANP-specific monoclonal antibody and an ANP messenger ribonucleic acid probe.

The biochemical and morphological characteristics of primary neonatal rat atrial myocytes were examined in order to establish a model system for future studies of the biosynthesis and secretion of atrial natriuretic peptide (ANP). Preliminary studies demonstrated that the quantity of immunoactive ANP/microgram protein within rat atria increased as a function of age from 2 ng/micrograms in 19 day prenatal animals to 400 ng/micrograms in the adult. Gel filtration, reversed phase HPLC, and ion exchange HPLC indicated that there were similar quantities of immunoactive ANP in the right and left atria at various ages, and that the major molecular form of the peptide in the heart is chromatographically indistinguishable from ANP(1-126). Cultures of dissociated cells were prepared from pooled left and right atria derived from 1 day postnatal animals. A complete serum-free medium was developed which resulted in the maintenance of high levels of immunoactive ANP in the cultures. As determined by RIA, the cellular content of ANP increased in the cultures as a function of time through 7 days in vitro. The quantity of immunoactive ANP in the cultures increased approximately 2- to 3-fold between days 3 and 7. When the cultures that had been maintained for 7 days were submitted to immunocytochemistry using an ANP-specific monoclonal antibody, distinct colonies of spindle-shaped cells stained positively. In situ hybridization, utilizing an 35S-labeled ANP messenger RNA probe, demonstrated that these colonies of myocytes expressed the ANP message. Using quantitative dot-blot hybridization it was shown that the ANP mRNA level increased approximately 50-fold between days 1 and 7 in culture. These studies indicate that the serum-free culture medium allows continued accumulation of both ANP and the ANP message in culture and will provide a useful model system to characterize factors that regulate the biosynthesis and secretion of this hormone.

Characterization of the molecular forms of ANP released by perfused neonatal rat heart.

Although cultures of neonatal rat atria and ventricles have been widely used to study ANP biosynthesis and secretion, little is known regarding the circulating form of ANP in neonatal animals. To begin to address this issue, we have developed a method for perfusing isolated neonatal rat hearts. Reversed phase-HPLC analysis of the heart effluents coupled with ANP RIA demonstrated that the predominant form of ANP released was chromatographically identical to ANP(99-126). Size exclusion-HPLC confirmed that the secreted ANP was indistinguishable from ANP(99-126). This demonstrated that the neonatal rat heart can efficiently generate and secrete a peptide similar to the circulating form of ANP found in adult rats, and further justifies the use of neonatal rat atria as a source of primary cells for studies of ANP biosynthesis and secretion.

The conversion of atrial natriuretic peptide (ANP)-(1-126) to ANP-(99-126) by rat serum: contribution to ANP cleavage in isolated perfused rat hearts.

Atrial myocytes cultured for 7 days in serum-free medium secrete a 15K form of atrial natriuretic peptide (15K ANP), but isolated perfused rat hearts secrete the major circulating form of the hormone, a 3K peptide, 3K ANP. This difference was examined in the present study. 15K ANP was purified from rat atria, and sequencing analysis demonstrated that this atrial-derived ANP possessed an NH2-terminal sequence identical to that of pro-ANP; this is consistent with other reports suggesting that the major form of ANP in the atria is ANP-(1-126). Fresh rat serum was shown to cleave efficiently ANP-(1-126) to form a 3K immunoactive ANP-related peptide. Upon purification and sequencing the identity of this peptide was confirmed as ANP-(99-126); ANP-(99-126) was relatively resistant to further proteolysis by rat serum. To probe further the specificity of the serum conversion, synthetic ANP-(92-126) was used as a substrate; purification and sequencing of the immunoactive product peptide verified its identity as ANP-(99-126). Since purified thrombin and plasma kallikrein both cleaved ANP-(1-126) to 3K ANP-like material, inhibitors of these enzymes were tested for their ability to inhibit the serum cleavage of ANP-(1-126). D-Phe-Phe-Arg-Chloromethylketone (D-Phe-Phe-Arg-CMK) and D-Phe-Pro-Arg-CMK both inhibited serum ANP cleavage by over 90% at low micromolar concentration. When these inhibitors were added to the isolated heart perfusate, 3K ANP was still released by the atria, indicating that ANP processing occurs in the heart in a region not accessible to the inhibitors (i.e. intracellularly) or that the ANP-processing enzyme(s) is not inhibited by these CMK analogs and is, therefore, not related to serum-derived proteases.

Ascorbate transport by AtT20 mouse pituitary corticotropic tumor cells: uptake and secretion studies.

Ascorbate is an important cofactor in the biosynthesis of alpha-amidated endocrine and neural peptides. Peptidylglycine alpha-amidating monooxygenase (PAM) is the enzyme responsible for the generation of mature COOH-terminal alpha-amidated peptides from COOH-terminal glycine-extended peptides, and this enzyme requires ascorbate for full activity in vitro. Also, cultured intermediate pituitary lobe cells contain PAM and require ascorbate for the COOH-terminal alpha-amidation of alpha MSH. Since pituitary cells are not capable of synthesizing ascorbate, the ability of the cells to accumulate the cofactor must play an important role in the biosynthesis of alpha-amidated peptides. The AtT20 corticotropic pituitary tumor cell line also contains PAM and a potential site for COOH-terminal alpha-amidation of the pro-ACTH/endorphin-derived hinge peptide and was, thus, used for the study of cellular ascorbate transport. Radiolabeled L-[1-14C]ascorbate ([1-14C]ascorbate) was incubated with the cells under various conditions, and the accumulation of radioactivity by the cells was followed. Reverse phase HPLC was used to identify the integrity of the labeled ascorbate, both intra- and extracellular, during the course of the experiments. The uptake of [1-14C]ascorbate was saturable (Km = 31.5 microM), sodium and temperature dependent, and stereoselective. The products of ascorbate autooxidation, dehydroascorbate and 2,3-diketogulonic acid, did not inhibit [1-14C]ascorbate uptake. To study the presence of ascorbate in the secretory granules, cells were incubated with [1-14C]ascorbate and then induced to secrete with isoproterenol or 8-bromo-cAMP. A 2- to 6-fold stimulation of ACTH secretion over the basal secretion rate was observed; however, the secretion of intracellular [1-14C]ascorbate did not change significantly with stimulation, suggesting that very little of the cellular ascorbate was contained within secretory granules.

Characterization of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin.

The time dependence of the release of fibrinopeptides from fibrinogen was studied as a function of the concentration of fibrinogen, thrombin, and Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The release of fibrinopeptides during fibrin assembly was shown to be a highly ordered process. Rate constants for individual steps in the formation of fibrin were evaluated at pH 7.4, 37 degrees C, gamma/2 = 0.15. The initial event, thrombin-catalyzed proteolysis at Arg-A alpha 16 to release fibrinopeptide A (kcat/Km = 1.09 X 10(7) M-1s-1) was followed by association of the resulting fibrin I monomers. Association of fibrin I was found to be a reversible process with rate constants of 1 X 10(6) M-1s-1 and 0.064 s-1 for association and dissociation, respectively. Assuming random polymerization of fibrin I monomer, the equilibrium constant for fibrin I association (1.56 X 10(7) M-1) indicates that greater than 80% of the fibrin I protofibrils should contain more than 10 monomeric units at 37 degrees C, pH 7.4, when the fibrin I concentration is 1.0 mg/ml. Association of fibrin I monomers was shown to result in a 6.5-fold increase in the susceptibility of Arg-B beta 14 to thrombin-mediated proteolysis. The 6.5-fold increase in the observed specificity constant from 6.5 X 10(5) M-1s-1 to 4.2 X 10(6) M-1s-1 upon association of fibrin I monomers and the rate constant for fibrin association indicates that most of the fibrinopeptide B is released after association of fibrin I monomers. The interaction between a pair of polymerization sites in fibrin I dimer was found to be weaker than the interaction of fibrin I with Gly-Pro-Arg-Pro and weaker than the interaction of fibrin I with fibrinogen.