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S Benjannet

Publications and source records attributed to S Benjannet.

At least 37 records · Page 2Linked to original sources
PubMed1995

7B2 is a specific intracellular binding protein of the prohormone convertase PC2.

Biosynthetic pulse-chase analyses have previously demonstrated that the prohormone convertase PC2 is first synthesized as a precursor pro-PC2 and that zymogen activation to PC2 occurs following the slow exit of pro-PC2 from the endoplasmic reticulum (ER) and its concentration within the trans-Golgi network (TGN). The endocrine and neural protein 7B2 is first synthesized as a nonglycosylated precursor (pro-7B2), which is cleaved within the TGN by a furin-like ubiquitous convertase at the RRKRR155S site to generate 7B2. In this report, we demonstrate that within the ER, pro-7B2 binds pro-PC2 but not any of the other convertases furin, PC1, PACE4, or PC5. This specific binding is Ca2+ dependent and does not require an N-glycosylated pro-PC2. Mutagenesis of the RRKRRS sequence demonstrated that the intact hexapeptide is critical for this binding, because the latter was abolished by mutations of the RR152 and greatly diminished by mutations of either the R151 or S156 residues of pro-7B2. Once the complex is formed in the ER, it is then transported to the TGN where furin or a furin-like convertase cleaves both precursors, even when present as a complex. We also provide evidence that following zymogen cleavage, 7B2 remains bound to PC2, suggesting the presence of at least one other Ca(2+)-dependent binding site within the 7B2 sequence. Coexpression of 7B2 and PC2, although resulting in an elevation of the level of pro-PC2, did not eliminate the processing of pro-PC2 to PC2.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Maintained PC1 and PC2 expression in the AtT-20 variant cell line 6T3 lacking regulated secretion and POMC: restored POMC expression and regulated secretion after cAMP treatment.

Two variant cell lines were recently established from parent AtT-20 cells. Whereas HYA.15.10.T.2 have a reduced level of secretory granules, HYA.15.6.T.3 are completely devoid of both the regulated pathway of secretion and of dense-core secretory granules. AtT-20 cells normally express the processing enzymes PC1, PC2, furin, carboxypeptidase E, and peptidylglycine alpha-amidating monooxygenase, as well as proopiomelanocortin, chromogranin B, and 7B2. We measured the expression of these mRNAs in both variant cell lines. Although some differences in mRNA level were noted, HYA.15.10.T.2 and HYA.15.6.T.3 cell lines maintained their expression of the processing enzymes and of 7B2. Furthermore, PC1 and PC2 were shown to be functionally active in the HYA.15.6.T.3 cells. In contrast, proopiomelanocortin and chromogranin B mRNA levels were no longer detectable in HYA.15.6.T.3 cells. Interestingly, stimulation of the HYA.15.6.T.3 cells with cAMP restored proopiomelanocortin mRNA, beta-endorphin immunoreactivity, and dense-core granules. Furthermore, at the ultrastructural level, beta-lipotropin immunoreactivity was detected in granules of cAMP-induced HYA.15.6.T.3 cells. Finally, depolarization of cAMP-induced HYA.15.6.T.3 cells with 56 mM potassium chloride resulted in a marked increase in the release of beta-endorphin immunoreactivity. These observations demonstrate that cAMP restores the regulated pathway of secretion in HYA.15.6.T.3 cells, which under untreated conditions do not demonstrate regulated release. These variant cell lines are unique models to understand better the relationship of the regulated pathway and the expression of the processing enzymes.

Animals↗
PubMed1993

Differential processing of proenkephalin by prohormone convertases 1(3) and 2 and furin.

Recombinant vaccinia virus vectors were used to coexpress mouse prohormone convertase 1 (mPC1), mPC2, or human furin together with human proenkephalin in GH4C1 cells (rat pituitary somatomammotrophs) to examine the proteolytic processing of proenkephalin by these enzymes. Radioimmunoassays performed on high pressure gel permeation size-fractionated extracts obtained from GH4C1 cells and corresponding conditioned media revealed distinct profiles of immunoreactivity for products generated by each enzyme. PC1 produced intermediate sized processing products (3-10 kDa); the major immunoreactive enkephalin-containing species observed eluted at the positions of peptide B, the 5.3-kDa fragment, and free Leu5-enkephalin. PC2 exhibited a more complete processing profile. The major immunoreactive enkephalins produced were free Met5-enkephalin-Arg-Phe, free Met5-enkephalin-Arg-Gly-Leu, free Leu5-enkephalin, and free Met5-enkephalin. Thus PC2 appears to be more capable of generating active opioid units from proenkephalin than is PC1. Finally, furin cleaved proenkephalin to generate peptide B, an unidentified peak between the 18- and 5.3-kDa fragments, and a small amount of the 5.3-kDa fragment. Radiosequencing data verified that the production of the 5.3-kDa fragment by PC1 occurred as a result of a Lys-Lys cleavage. The ability of PC1 to cleave proenkephalin (but not proopiomelanocortin) at a Lys-Lys site implies that the structural context of the paired basic cleavage site may be more important in the determination of cleavage specificity than the particular pair of basic residues at the site.

Amino Acid Sequence↗
PubMed1993

Comparative biosynthesis, covalent post-translational modifications and efficiency of prosegment cleavage of the prohormone convertases PC1 and PC2: glycosylation, sulphation and identification of the intracellular site of prosegment cleavage of PC1 and PC2.

We present herein the pulse-chase analysis of the biosynthesis of the prohormone convertases PC1 and PC2 in the endocrine GH4C1 cells infected with vaccinia virus recombinants expressing these convertases. Characterization of the pulse-labelled enzymes demonstrated that pro-PC1 (88 kDa) is cleaved into PC1 (83 kDa) and pro-PC2 (75 kDa) into PC2 (68 kDa). Secretion of glycosylated and sulphated PC1 (84 kDa) occurs about 30 min after the onset of biosynthesis, whereas glycosylated and sulphated PC2 (68 kDa) is detected in the medium after between 1 and 2 h. Furthermore, in the case of pro-PC2 only, we observed that a fraction of this precursor escapes glycosylation. A small proportion (about 5%) of the intracellular glycosylated pro-PC2 (75 kDa) is sulphated, and it is this glycosylated and sulphated precursor that is cleaved into the secretable 68 kDa form of PC2. Major differences in the carbohydrate structures of PC1 and PC2 are demonstrated by the resistance of the secreted PC1 to endoglycosidase H digestion and sensitivity of the secreted PC2 to this enzyme. Inhibition of N-glycosylation with tunicamycin caused a dramatic intracellular degradation of these convertases within the endoplasmic reticulum, with the net effect of a reduction in the available activity of PC1 and PC2. These results emphasize the importance of N-glycosylation in the folding and stability of PC1 and PC2. Pulse-labelling experiments in uninfected mouse beta TC3 and rat Rin m5F insulinoma cells, which endogenously synthesize PC2, showed that, as in infected GH4C1 cells, pro-PC2 predominates intracellularly. In order to define the site of prosegment cleavage, pulse-chase analysis was performed at low temperature (15 degrees C) or after treatment of GH4C1 cells with either brefeldin A or carbonyl cyanide m-chlorophenylhydrazone. These results demonstrated that the onset of the conversions of pro-PC1 into PC1 and non-glycosylated pro-PC2 into PC2 (65 kDa) occur in a pre-Golgi compartment, presumably within the endoplasmic reticulum. In contrast, pulse labelling in the presence of Na(2)35SO4 demonstrated that the processing of glycosylated and sulphated pro-PC2 occurs within the Golgi apparatus. In order to test the possibility that zymogen processing is performed by furin, we co-expressed this convertase with either pro-PC1 or pro-PC2. The data demonstrated the inability of furin to cleave either proenzyme.

Animals↗
PubMed1993

Enzymic characterization of murine and human prohormone convertase-1 (mPC1 and hPC1) expressed in mammalian GH4C1 cells.

Prohormone convertase-1 (PC1), an endopeptidase that is structurally related to the yeast subtilisin-like Kex2 gene product, has been proposed to be involved in mammalian tissue-specific prohormone processing at pairs of basic residues. To better study this enzyme, a rat somatomammotroph cell line, GH4C1, was infected with vaccinia virus recombinants of murine PC1 (mPC1) and human PC1 (hPC1). An enzymically active form of each protein was secreted into the cell medium and partially purified by anion-exchange chromatography. The 80-85 kDa enzyme was shown to be Ca(2+)-dependent and exhibited a pH optimum of 6.0 when assayed against a synthetic fluorogenic substrate, acetyl-Arg-Ser-Lys-Arg-4-methylcoumaryl-1-amide. mPC1 and hPC1 displayed identical cleavage selectivity towards a number of fluorogenic substrates, and those incorporating an Arg at the P4 site were most favoured. Synthetic peptides, encompassing the junction between the putative pro-region and the active enzyme, and between the pro-region and the biologically active parathyroid hormone, were shown to be recognized and cleaved specifically at the pair of basic residues by both enzymes. Group-specific proteinase inhibitors such as metal ion chelators and p-hydroxymercuribenzoate, but not phenylmethanesulphonyl fluoride and pepstatin, strongly inhibit the PC1-associated activity. In addition, it is shown that an enzyme activity displaying identical properties is present in the cell medium of uninfected corticotroph AtT-20 cells and that its level is increased following stimulation of secretion by the secretagogue 8-bromo cyclic AMP.

Amino Acid Sequence↗
PubMed1992

The cDNA structure of the porcine pro-hormone convertase PC2 and the comparative processing by PC1 and PC2 of the N-terminal glycopeptide segment of porcine POMC.

The complete cDNA structure of the porcine (p) pro-protein and pro-hormone convertase PC2 (pPC2) was obtained from a cDNA library of pituitary neurointermediate lobes mRNA. The deduced amino acid sequence revealed that pPC2 exhibits a 99-97% sequence identity to the human, mouse and rat homologues. The 3' end of the 2.1 kb cDNA is the least conserved segment. On Northern blots of pars intermedia poly A+ RNA two transcripts of 3 and 5 kb were detected. Molecular analysis of the N-terminal glycopeptide products of porcine pro-opiomelanocortin (pPOMC) co-expressed with vaccinia virus recombinants of PC1 or PC2, revealed that in cells devoid or containing secretory granules both convertases can cleave pPOMC with PC1 releasing the 1-80, 1-107 and 1-148 glycopeptide fragments, and PC2 cleaving pPOMC directly into pPOMC 1-107.

Amino Acid Sequence↗
PubMed1992

Proprotein conversion is determined by a multiplicity of factors including convertase processing, substrate specificity, and intracellular environment. Cell type-specific processing of human prorenin by the convertase PC1.

Proprotein and prohormone processing at pairs of basic residues is generally thought to be both tissue- and precursor-specific and to be developmentally regulated. Furin, PC1 (also called PC3), and PC2 represent three recently discovered subtilisin-like proteinases which cleave a number of precursors at the same pairs of basic residues normally processed in vivo. Using human prorenin as a model, we show that PC1 can process it to active renin in cells containing secretory granules, such as the somatomammotroph cell line GH4, but not in cells which lack granules, such as the Chinese hamster ovary or African green monkey kidney epithelial (BSC-40) cell lines. In contrast, in both cell types, human prorenin is not activated by either PC2 or furin. Using the vaccinia virus expression system, biosynthetic labeling experiments demonstrated that PC1 and PC2 are themselves cleaved intracellularly at pairs of basic residues and that these two proenzymes are processed to different extents independent of whether the cell line contains dense core secretory granules. Furthermore, we also show that the cells mostly secrete the cleaved forms of PC1 and PC2, and that intracellularly the pro- form of PC2 predominates. Our data demonstrate that propeptide removal from these enzymes, possibly leading to their activation, is not the only criterion which governs precursor processing.

Amino Acid Sequence↗
PubMed1991

PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues.

A recombinant vaccinia virus vector was used to coexpress the two candidate mouse prohormone convertases, PC1 and PC2, together with mouse proopiomelanocortin (POMC) in the constitutively secreting cell line BSC-40 and in the endocrine tissue-derived cell lines PC12 and AtT-20, which exhibit regulated secretion. Monitoring of POMC processing demonstrated the distinct cleavage specificities of PC1 and PC2, since in the cell lines analyzed (i) PC1 cleaves POMC into corticotropin and beta-lipotropin, (ii) PC2 cleaves POMC into beta-endorphin, an N-terminally extended corticotropin containing the joining peptide, and either alpha MSH or desacetyl-alpha MSH, and (iii) PC2 cleaves POMC at the five pairs of basic residues analyzed, whereas PC1 cleaves two of them preferentially, suggesting that PC2 has a broader spectrum of activity than PC1. These data are consistent with our hypothesis on the physiological role of PC1 and PC2 as distinct proprotein convertases acting alone or together to produce a set of tissue-specific maturation products in the brain and in peripheral tissues.

Amino Acid Sequence↗
PubMed1991

Chromosomal assignments of the genes for neuroendocrine convertase PC1 (NEC1) to human 5q15-21, neuroendocrine convertase PC2 (NEC2) to human 20p11.1-11.2, and furin (mouse 7[D1-E2] region).

The chromosomal localization of the genes coding for the pro-protein and pro-hormone convertases PC1, PC2, and Furin has been achieved by in situ hybridization. The genes for PC1 and PC2 were located on human chromosomes 5q15-21 and 20p11.1-11.2, respectively. The gene for Furin was assigned to the mouse chromosome 7D1-7E2 region. These data complete the chromosomal localization of these three convertases in both human and mouse. The results confirm the regional correspondence of the human chromosomes 15 and mouse chromosomes 7, as well as between human chromosome 20 and mouse chromosome 2. Furthermore, the identification of the NEC1 locus on human chromosome 5 and mouse chromosome 13 suggests a conservation of synthenic regions between these regions of the human and mouse genomes.

Animals↗
PubMed1991

Processed forms of neuroendocrine proteins 7B2 and secretogranin II are found in porcine pituitary extracts.

The complete structure of the novel polypeptide 7B2 recently deduced from cDNA clones has been reported to be highly conserved in a variety of species. The deduced amino acid sequence of the mature protein is predicted to be 185 or 186 amino acids long. While its biological role is still unknown, its occurrence in neuroendocrine secretory granules has been largely documented. This report shows: (i) that the protein, isolated from a large quantity of porcine pituitary glands, does not correspond to the full predicted cDNA structure but, on the contrary, to a truncated form; (ii) that the latter could arise from proteolytic cleavage at position 150 following pairs of basic residues; (iii) that it contains an extra residue at position 100 which is absent in the cDNA sequence; and, finally, (iv) that it displays a higher than expected molecular weight on SDS-polyacrylamide gel electrophoresis. In addition, a copurifying peptide was identified as an NH2-terminal related fragment of the secretogranin II molecule. Protein sequencing of the latter demonstrates (i) that the correct amino terminus of mature porcine secretogranin II is an Ala residue and not the previously proposed Gln residue and (ii) that this fragment could also arise from proteolytic cleavage at a pair of basic residues located within the secretogranin II sequence.

Amino Acid Sequence↗
PubMed1991

Mammalian neural and endocrine pro-protein and pro-hormone convertases belonging to the subtilisin family of serine proteinases.

Conversion of pro-hormones and precursor proteins into biologically active peptides and proteins involves the concerted action of a number of convertases and post-translation modification enzymes. The identification of the yeast convertase kexin as a prototype processing enzyme led to the discovery of the mammalian convertase designated furin, PC1 and PC2. Whereas furin is ubiquitously expressed, PC1 and PC2 are found only in endocrine and neural tissues and cell lines. In man and mouse, the genes coding for furin, PC1 and PC2 reside on three different chromosomes. The analysis of the intracellular processing of PC1 and PC2 and the removal of their pro-segment is presented, together with a summary of the cleavage specificity of these enzymes for precursors such as pro-opiomelanocortin (POMC) and human pro-renin. The distinct tissue distribution of PC1 and PC2 and their coregulation with POMC in the pituitary neurointermediate lobe adds credence to their physiological role as convertases involved in the tissue-specific processing of precursor proteins.

Amino Acid Sequence↗
PubMed1991

Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, Furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2.

Using a 796-basepair cDNA fragment obtained from a mouse pituitary library we have screened two mouse insulinoma libraries and isolated a full-length cDNA clone (2516 basepairs; 753 amino acids), designated mPC1. The cDNA sequence of mPC1 codes for a protein containing 753 amino acids and three potential N-glycosylation sites. This cDNA encodes a putative novel subtilisin-like proteinase, exhibiting within its presumed catalytic domain 64%, 55%, and 47% amino acid sequence identity to the recently characterized candidate prohormone convertases human Furin, mouse PC2, and yeast Kex2 gene products, respectively. An identical sequence to mPC1 was derived from a cDNA library of mouse corticotroph AtT-20 tumor cells. An ArgGlyAsp tripeptide identical to the recognition sequence of integrins was observed in the structures of the mammalian PC1, PC2, and Furin. In situ hybridization results demonstrated a distinct localization of the mPC1 and mPC2 transcripts in pituitary and brain. Thus, whereas both mPC1 and mPC2 are found in the intermediate lobe of the pituitary, only mPC1 is easily detected in the anterior lobe. In extrahypothalamic regions of the brain, including cortex, hippocampus, thalamus, and spinal cord, mPC2 transcripts predominate over mPC1. Both mRNAs are found in only a fraction of hypothalamic neurons, with greater abundance of mPC1 over mPC2 in the supraoptic nucleus. The genes coding for mPC1 and mPC2 map to the murine chromosomes 13 (band 13c) and 2 (2F3-2H2 region), respectively.

Amino Acid Sequence↗
PubMed1989

Rat plasma kallikrein: purification, NH2-terminal sequencing and development of a specific radioimmunoassay.

Rat plasma kallikrein (rPK) was purified to homogeneity form plasma using affinity and high-performance liquid chromatography techniques, and subjected to NH2-terminal sequencing. The data showed that the sequenced segments of the regulatory (heavy) and catalytic (light) chains of the proteinase, respectively, display 73 and 91% sequence similarity with their counterpart in human plasma kallikrein. This sequence homology in conjunction with the determined molecular structure and inhibitor sensitivity support the identity of the isolated enzyme as plasma kallikrein. A polyclonal antiserum against rPK was obtained after immunization of rabbits with the purified enzyme, and a specific radioimmunoassay was developed. Since Tyr-iodinated rPK was not recognized by the antiserum, two alternative approaches were found to be successful. These included the use of a tracer consisting of rPK modified with either the affinity reagent 125I-labeled DTyr-Glu-Phe-Lys-Arg chloromethyl ketone or with the Bolton Hunter reagent. The usable range of the assay is between 15-150 fmol per tube. The antibody was shown to bind both monomeric and dimeric forms of rPK. Denaturation of the enzyme in sodium dodecyl sulfate does not abolish immune recognition only as long as the regulatory subunit is attached to the catalytic chain. Oxidation or reduction of rPK results in complete loss of immunoreactivity. This observation suggests that perhaps the disulfide linkage of the catalytic and regulatory polypeptides somehow helps to protect the antigenic epitope from denaturation. Alternatively, the epitope(s) recognized by the antibody spans a domain which includes both Tyr and Cys residues necessary for immune recognition.

Amino Acid Sequence↗
PubMed1989

Value of plasma NH2-terminal fragment of pro-opiomelanocortin in marking human lung cancer in various clinical settings.

Plasma concentration of amino-terminal segment of pro-opiomelanocortin (N-POMC) was measured by radioimmunoassay (RIA) in 144 patients with various forms of lung cancer during pneumonectomy, at different times of the day after being newly diagnosed and serially, throughout their treatment (surgery or chemotherapy) in order to assess its value as a biomarker in this disease. Normal volunteers, coal miners smoking but without known lung diseases, and the patients with diverse pulmonary disorders served as comparison groups. A significant transtumoral gradient of N-POMC was found at surgery in 15 of 57 (26%) patients. Subjects without lung afflictions had significantly lower N-POMC levels than patients with pulmonary diseases (benign or malignant) only when the blood was drawn before breakfast. Furthermore, fasting levels in all subgroups of patients were higher at any time of the day than nonfasting ones. Finally, N-POMC levels did not decrease significantly after successful treatment of lung cancer (by surgery or chemotherapy) but were markedly higher after relapse. These results suggest that N-POMC, despite the fact that it cannot be used to discriminate lung cancer patients from controls, is a biomarker which may predict relapse in patients successfully treated by chemotherapy for their pulmonary neoplasm.

Adult↗
PubMed1988

Correct processing and secretion of a human prostatic secretory protein (PSP94) in Escherichia coli.

The cDNA for PSP94, a cysteine-rich protein secreted by the human prostate, was unidirectionally digested with exonuclease III to generate deletion mutants with varying 5' ends. These were placed under the control of the lac promoter of the Bluescribe plasmid (pbs) to encode hybrid proteins containing the N terminus of beta-galactosidase (beta Gal) and various fragments of PSP94. Escherichia coli clones transformed by these constructs and expressing PSP94 epitopes were identified by radioimmunoassay of cellular and periplasmic extracts. One such clone (I-25) secreted most of its immunoreactive material into the periplasmic space. Nucleotide sequencing showed that a new consensus ribosome-binding site had been generated fortuitously, allowing expression of pre-PSP94 free of any beta Gal sequence. Periplasmic PSP94 is indistinguishable from the natural human protein, indicating correct processing and folding of this cysteine-rich protein in bacteria.

Amino Acid Sequence↗
PubMed1988

Two-dimensional immunoblotting analysis and immunocytochemical localization of the secretory polypeptide 7B2 in adrenal medulla.

The soluble proteins of bovine chromaffin granules were subjected to 2D-electrophoresis followed by immunoblotting with an antiserum against the pituitary peptide 7B2. One immunoreactive spot was visualized at a position corresponding to a molecular weight of 24,000 and to a pI of 5.2. Using peroxidase-antiperoxidase (PAP) pre-embedding immunocytochemical technique for electron microscopy, 7B2 has been localized within secretory granules with diameters of approximately 115 and 190 nm in noradrenergic and adrenergic cells respectively. These data establish that in chromaffin granules 7B2 represents a minor component of the acidic proteins which include the chromogranins A and B, secretogranin II and the enkephalin-containing peptides.

Adrenal Medulla↗