Differential processing of hormone precursor. Independent production of somatostatins 14 and 28 in transfected neuroblastoma 2A cells.

Neuro 2A cells infected with a retroviral vector carrying human prosomatostatin cDNA expressed and processed correctly the precursor into somatostatins-14 and -28 [(1989) EMBO J. 8, 2911-2916]. In order to study the mechanisms by which the active hormone sequences arise, site directed mutagenesis was performed on either the dibasic (ArgLys) or monobasic (Arg) cleavage sites involved in the production of somatostatins-14 and -28, respectively. Radioimmunochemical analysis of the somatostatin-related products indicated that replacement of either Arg-2-Lys-1 by Asn-2-Asn-1 or of Arg-15 by Asn-15 resulted in the exclusive production of either somatostatin-28 or -14, respectively. Moreover only prosomatostatin[1-76] was detected and no somatostatin-28[1-12] could be measured in cell extracts. Selective suppression of either somatostatin-14 or somatostatin-28 release by mutation did not affect the level of production of the other hormone but resulted in a correlative increase of unprocessed prosomatostatin. It is concluded that in this cell type (i) somatostatin-14 is exclusively generated by dibasic cleavage at the Arg-2-Lys-1 site of the intact precursor with concomitant production of prosomatostatin[1-76], and (ii) no direct interactions between the monobasic and dibasic processing domains occur.

Exploration of the catalytic site of endopeptidase 24.11 by site-directed mutagenesis. Histidine residues 583 and 587 are essential for catalysis.

Direct comparison of the primary structure of neutral endopeptidase (NEP, EC 3.4.24.11) with that of thermolysin, a bacterial metalloendopeptidase with a similar specificity, has revealed very few similarities between the two sequences, except for two conserved short segments. In thermolysin, these segments contain several of the residues involved in catalysis, including two zinc coordinating histidines (His-142 and His-146) and a third histidine (His-231) involved in stabilizing the transition state through hydrogen bonding. The role of the corresponding histidines in NEP (His-583, His-587 and His-637) was explored by site-directed mutagenesis of NEP cDNA and expression of the mutated cDNA in COS-1 cells. Substitution of either His-583 or His-587 of NEP for Phe completely abolished the activity and Zn-directed inhibitor recognition of the recombinant enzyme, suggesting that these residues play a role similar to His-142 and His-146 of thermolysin as zinc ligands. In contrast, substitution of His-637 for a phenylalanine residue was without effect on enzyme activity.

Targeting of pro-opiomelanocortin to the regulated secretory pathway may involve cooperation between different protein domains.

The structure of pro-opiomelanocortin (POMC) can be divided into three main domains: an NH2-terminal domain formed by the NH2-terminal glycopeptide and the joining peptide, a central domain corresponding to the adrenocorticotropin sequences and a COOH-terminal domain containing the beta-lipotropin sequences. Expression of POMC in neuroendocrine cell lines such as the mouse neuroblastoma Neuro2A cells results in its targeting to the regulated secretory pathway of these cells. Intracellular targeting of proteins along non default pathways are widely believed to involve the recognition of specific structural features by a sorting machinery. To understand the nature of the signal involved in targeting prohormone to the regulated secretory pathway, we have constructed mutants of POMC in which sequences from the NH2-terminal, the central and the COOH-terminal domains were deleted and examined the sorting of these mutant POMC molecules in Neuro2A cells by immunofluorescence and immunoelectron microscopy. Our results indicate that POMC NH2-terminal glycopeptide or beta-LPH domain do not contain sufficient information for targeting to the regulated pathway since these peptides are not sorted to secretory vesicles when expressed in Neuro2A cells: Similarly, the ACTH domain does not contain essential targeting information since POMC mutants lacking these sequences were sorted to secretory vesicles. Mutant POMCs containing the sequences of more than one of the main protein domains were, however, correctly targeted to the regulated secretory pathway. Our results indicate that POMC is not targeted to the regulated secretory pathway through recognition of a unique continuous 'molecular address'.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of porcine pro-opiomelanocortin in mouse neuroblastoma (Neuro2A) cells: targeting of the foreign neuropeptide to dense-core vesicles.

Pro-opiomelanocortin (POMC) is the precursor to several pituitary hormones and neuropeptides including adrenocorticotropic hormone (ACTH) and beta-endorphin (beta-END). In neuroendocrine cells, peptide hormones and neuropeptides are targeted to the dense-core vesicles of the regulated secretory pathway. These vesicles are transported to the ends of cellular extensions where they are stored until they release their content upon external stimulation of the cell. In order to study the cellular mechanisms involved in targeting of neuropeptides, we have expressed POMC in Neuro2A cells, a cell line of neural origin. Using immunofluorescence labeling and immunoelectron microscopy we show that in Neuro2A cells POMC is packaged in dense-core vesicles which accumulate at the tips of cellular processes. Intracellular accumulation of POMC was not observed in NIH 3T3 fibroblasts. When a soluble form of an integral membrane protein, neutral endopeptidase (E.C. 3.4.24.11) (secNEP), was expressed in Neuro2A cells, the protein was found to be constitutively secreted without prior accumulation in dense-core vesicles. Our results suggest that in Neuro2A cells, targeting to the regulated secretory pathway is restricted to peptide hormones and neuropeptides and establish this cell line as a valid model for studying the molecular events involved in neuropeptide sorting into the regulated secretory pathway.

Efficacy of Cydectin moxidectin 1% injectable against experimental infections of Dictyocaulus viviparus and Bunostomum phlebotomum superimposed on natural gastrointestinal infections in calves.

Twenty male Holstein calves averaging 105 kg in weight and naturally infected with gastrointestinal nematodes and small numbers of lungworm and hookworm, were given experimental infections with the two latter species to provide adult and larval stages for anthelmintic evaluation. Following random allotment, one group of 10 calves was injected subcutaneously with moxidectin at a dosage of 0.2 mg kg-1 of body weight. A second group of 10 was injected subcutaneously with unmedicated blank vehicle at a dosage of 1 ml per 50 kg of body weight. Fecal samples were examined before treatment and at 7 and 13 days after treatment. The 20 calves were necropsied for worm recovery at 13 and 14 days after treatment. All calves were positive for lungworm and hookworm on the treatment date. Treatment was 100% effective in elimination of hookworm eggs and lungworm larvae and 99.9% in reducing total egg counts at both 7 and 13 days after treatment. Moxidectin was 100% effective (P less than 0.01) in eliminating the following 11 species of nematodes. Dictyocaulus viviparus mature and immature adults (E5), Bunostomum phlebotomum adults and L4, Ostertagia ostertagi adults and early L4, Ostertagia lyrata adult males, Haemonchus placei adults. Trichostrongylus axei adults, Cooperia spp., including Cooperia punctata, Cooperia spatulata, and Cooperia pectinata adults, Oesophagostomum radiatum adults and Trichuris discolor adults. No adverse reactions to moxidectin treatment were observed.

Efficacy of abamectin against natural infections of gastrointestinal nematodes and lungworm of cattle with special emphasis on inhibited, early fourth stage larvae of Ostertagia ostertagi.

The anthelmintic efficacy of abamectin (avermectin B1) was evaluated against gastrointestinal nematodes, including Ostertagia ostertagi inhibited larvae and lungworm, in yearling crossbred beef heifers during late spring. The calves were grazed on contaminated pasture for 10 weeks and then held under conditions free of nematode infection for 3 weeks prior to allotment and treatment on 5 June. Thirteen calves were randomly assigned to two groups of six by restricted randomization on body weights; the extra lightest calf was assigned to the non-treated control group. Group 1 calves were treated with abamectin at 200 micrograms kg-1 body weight by s.c. injection and Group 2 calves were not treated; all were killed at 14 days after treatment. Ostertagia ostertagi was present in all controls; arithmetic mean numbers of adults, developing fourth stage larvae (L4) and inhibited EL4 were 7683, 605 and 36,102, respectively. Other nematode genera present in controls in sufficient numbers for the experiment were Haemonchus placei adults, Trichostrongylus axei adults, Cooperia spp. adults, Oesophagostomum radiatum adults, Bunostomum phlebotomum adults, Dictyocaulus viviparus adults and E5 (immature adults). Abamectin was highly effective (consistently greater than 99% efficacy and P less than 0.05) in removing all nematodes present in treated calves as represented in non-treated controls, including the primary target of Ostertagia ostertagi inhibited EL4. The lowest efficacy was 93.8%, against D. viviparus E5.

Intramolecular recombination in polyomavirus DNA is a nonconservative process directed from the viral intergenic region.

Previously, we have studied intramolecular homologous recombination in polyomavirus replicons under conditions allowing only one amplifiable recombination product to be generated from a single precursor molecule. In order to detect putative reciprocal product(s), we have now constructed precursor polyomavirus replicons which contain two copies, instead of one copy, of the viral intergenic region, including the origin of replication as well as both promoters. Upon transfection of mouse cells, constructs containing directly repeated intergenic regions yielded distinct amplifiable products, in number depending upon the functional integrity of both intergenic regions. Our data indicate that of two possible reciprocal products, a given precursor molecule would yield either one or the other but never both at the same time. Most striking, however, is the observation that promoter function is required for recombination, while the origin of replication function may be needed only for amplification of the recombination product once it has been formed. The data reported here confirm and extend previous data suggesting that (i) transcription is instrumental in recombination between direct repeats and (ii) nonconservative recombination involving direct repeats relies upon two promoters of opposing polarities.

Expression of porcine pro-opiomelanocortin cDNA in heterologous monkey kidney cells. Biosynthesis and secretion of the prohormone without processing.

Pro-opiomelanocortin is the common precursor to several pituitary hormones. These include alpha-melanotropic hormone, adrenocorticotropic hormone, beta-lipotropic hormone, and beta-endorphin. The porcine pro-opiomelanocortin cDNA was inserted downstream from the early promoter of a SV40-derived expression vector and transfected into the monkey kidney COS-1 cells. Transient expression of the pro-opiomelanocortin cDNA was observed between 48 and 70 h after transfection. Analysis of pro-opiomelanocortin-related material in COS-1 cell extracts and culture medium revealed that these cells synthesize and secrete constitutively pro-opiomelanocortin without further processing into its mature hormones. Our results suggest that COS-1 cells do not contain the necessary enzymatic machinery to process complex precursors such as pro-opiomelanocortin.

Site-specific mutagenesis identifies amino acid residues critical in prohormone processing.

Peptide hormones are generally synthesized as inactive higher mol. wt precursors. Processing of the prohormone into biologically active peptides by specific proteolytic cleavages occurs most often at pairs of basic amino acids but also at single arginine residues. To study the role of protein secondary structure in this process, we used site-directed mutagenesis to modify the predicted secondary structure around the cleavage sites of human prosomatostatin and monitored the processing of the precursor after introduction of the mutated cDNAs in Neuro2A cells. Amino acid substitutions were introduced that affected the possibility of forming beta-turn structures in the immediate vicinity of the somatostatin-28 (S-28) and somatostatin-14 (S-14) cleavage sites. Infection of Neuro2A cells with a retrovirus carrying a human somatostatin cDNA resulted in the expression of prosomatostatin and its processing into S-28 and S-14, indicating that these cells have the necessary enzymes to process prohormone at both single and paired amino acid residues. Disruption of the different beta-turns had various effects on prosomatostatin processing: substitution of Ala for Pro-5 drastically decreased prosomatostatin processing and replacement of Pro-9 by Ala led to the accumulation of the intermediate maturation product [Arg-2Lys-1]-S-14. In contrast, substitution of Ala for Asn-12, Gly+2 and Cys+3 respectively had only very little effect on the proteolytic processing of prosomatostatin. Our results show that amino acids other than the basic amino acid residues are required to define the cleavage sites for prohormone proteolytic processing and suggest that higher orders of protein structure are involved in substrate recognition by the endoproteases.

Intramolecular recombination in polyomavirus DNA is controlled by promoter elements.

We show here that intramolecular homologous recombination in polyomavirus (Py) DNA depends upon discrete sequence elements of the viral regulatory region which are believed to regulate transcription initiation and exert little or no cis-control over replication. Either deleting the viral early promoter (EP) or inverting the viral late promoter (LP) strongly impairs viral DNA recombination under conditions allowing viral DNA replication to proceed undisturbed. These findings suggest that bi-directional transcription proceeding from the intergenic region favors intramolecular recombination.

Prosomatostatin processing in Neuro2A cells. Role of beta-turn structure in the vicinity of the Arg-Lys cleavage site.

Proline residues located near the processing sites of human prosomatostatin were previously shown to be important for cleavage of the precursor into somatostatin 28 and somatostatin 14 [Gomez, S., Boileau, G., Zollinger, L., Nault, C., Rholam, M. & Cohen, P. (1989) EMBO J. 8, 2911-2916]. In this study, site-directed and regional mutagenesis of the human prosomatostatin cDNA coupled with analysis by circular-dichroism and Fourier-transform-infrared spectroscopies of the native and mutated peptide sequences were used to elucidate the role of proline in proteolytic processing. Glycine was substituted for proline a position -5 and the beta-turn-promoting sequence Pro-Arg-Glu-Arg, located near the somatostatin-14 cleavage site and predicted to form a beta-turn structure, was replaced by Ser-Ser-Asn-Arg or Tyr-Lys-Gly-Arg, which have been shown by X-ray diffraction to form beta turns in other proteins. Analysis of the prosomatostatin-derived peptides produced by expression of the mutated cDNA species in Neuro2A cells indicated that while Pro-5-->Ala abolished cleavage at the dibasic site, the formation of mutants [Gly-5] prosomatostatin, [Ser-5, Ser-4, Arg-3] prosomatostatin and [Tyr-5, Lys-4, Gly-3] prosomatostatin did not affect cleavage at the dibasic site but produced modifications in both the relative proportions of the generated hormones and in precursor processing efficiency. Moreover, spectroscopical analysis showed that whereas these substitutions did not modify the presence of a beta turn structure in the corresponding peptide sequences, replacement of Pro-5-->Ala resulted in a dramatic increase in alpha-helix accompanied by the significant decrease of other structures including beta turn. The data support the hypothesis that the proline residue near the processing site for somatostatin-14 production is an important structural feature for conferring on the cleavage domain the adequate conformation for accessibility to processing enzymes and permitting production of equivalent amounts of both hormones.

Expression of neutral endopeptidase (enkephalinase) in heterologous COS-1 cells. Characterization of the recombinant enzyme and evidence for a glutamic acid residue at the active site.

Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found in the plasma membrane of many cell types. The cDNA coding for the complete primary structure of neutral endopeptidase has recently been cloned and sequenced (Devault, A. Lazure, C., Nault, C., Le Moual, H., Seidah, N. G., Chretien, M., Kahn, P., Powell, J., Mallet, J., Beaumont, A., Roques, B. P., Crine, P., and Boileau, G. (1987) EMBO J. 6, 1317-1322). Comparison of the sequence of neutral endopeptidase with that of thermolysin, a bacterial Zn-metalloendopeptidase, suggests that Glu-584 in neutral endopeptidase probably corresponds to Glu-143 in thermolysin, which is an essential amino acid involved in catalysis. To test directly the importance of Glu-584 in the catalytic activity of neutral endopeptidase by site-directed metagenesis, we have constructed an expression vector in which the rabbit kidney cDNA encoding the entire neutral endopeptidase sequence is introduced downstream from the SV40 virus early promotor. After transfection in COS-1 monkey kidney cells, this vector was found to promote the expression of a protein with biochemical and catalytic properties identical to kidney neutral endopeptidase. Oligonucleotide-directed mutagenesis of Glu-584 to either valine or aspartic acid completely abolished the enzymatic activity of the recombinant protein without changing its affinity for the substrate-related tritiated inhibitor [3H]N-[(2R,2S)-3-hydroxyamino-carbonyl-2-benzyl-1-oxopropyl]-glycine. This observation clearly identifies Glu-584 as one of the important residues responsible for the catalytic activity of the enzyme.

Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA.

Neutral endopeptidase (EC 3.4.24.11) is a major constituent of kidney brush border membranes. It is also present in the brain where it has been shown to be involved in the inactivation of opioid peptides, methionine- and leucine-enkephalins. For this reason this enzyme is often called 'enkephalinase'. In order to characterize the primary structure of the enzyme, oligonucleotide probes were designed from partial amino acid sequences and used to isolate clones from kidney cDNA libraries. Sequencing of the cDNA inserts revealed the complete primary structure of the enzyme. Neutral endopeptidase consists of 750 amino acids. It contains a short N-terminal cytoplasmic domain (27 amino acids), a single membrane-spanning segment (23 amino acids) and an extracellular domain that comprises most of the protein mass. The comparison of the primary structure of neutral endopeptidase with that of thermolysin, a bacterial Zn-metallopeptidase, indicates that most of the amino acid residues involved in Zn coordination and catalytic activity in thermolysin are found within highly honmologous sequences in neutral endopeptidase.