PubMed Health⌕ Search

Biomedical subjects

N G Seidah

Publications and source records attributed to N G Seidah.

At least 19 recordsLinked to original sources
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

Affinity purification of proteinases by a combination of immobilized peptidyl aldehyde and semicarbazone.

D-Phe-Phe-argininal semicarbazone and Tyr-Gly-Gly-Phe-Leu-Arg-argininal semicarbazone were prepared using the solution phase synthesis method and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The tripeptide and heptapeptide semicarbazones were individually immobilized on affi-Gel 15 resulting in two affinity columns called S3 and S7, respectively. A third affinity column was obtained by hydrolysing the semicarbazone moiety in column S3 to aldehyde (column A3). Serine proteinases such as trypsin or rat plasma kallikrein almost quantitatively bind to either S3 or A3 affinity columns. Under optimized conditions, more than 97% of trypsin bound to both columns S3 and A3. At a lower ionic strength and higher pH, 80-85% of rat plasma kallikrein bound to the same columns. Elution of both enzymes was achieved using mild conditions at near neutral pH and in the presence of a small amount of denaturant. Both proteinases were identified and characterized by high-performance liquid chromatography, sodium dodecylsulphate polyacrylamide gel electrophoresis and by their substrate specificity and inhibition profiles. A single purification (six-to seven-fold) step using either column S3 or A3 allowed the preparation of pure trypsin from commercial sources. Starting from rat plasma partially purified by a phenyl boronate column, fractionation on the S3 column allowed approximately an 87-fold purification of rat plasma kallikrein. However, serial purification of rat plasma kallikrein on column S7 followed by column A3 resulted in a purification factor of about 455.

Aldehydes↗
PubMed1992

Dynamics of 7B2 and galanin expression in solitary magnocellular hypothalamic vasopressin neurons of the homozygous Brattleboro rat.

The homozygous Brattleboro rat (di/di), displaying a hypothalamic form of diabetes insipidus, synthesizes a vasopressin (VP) precursor with an abnormal C-terminus. The phenotypic expression of coexisting peptides in mutant magnocellular VP cells shows a differential pattern. 7B2 is one of the peptides which is not detectable, whereas there is a clear galanin expression. During postnatal life a small but increasing number of solitary post-mitotic VP neurons of the di/di rat undergoes a switch to a heterozygous phenotype. Here we report the presence of 7B2 and galanin in these heterozygous cells, which suggests that for the expression of 7B2, but not for that of galanin, the relative amount of mutant VP precursor must be diminished. Possible underlying mechanisms for this differential phenotypic expression of coexisting peptides are compartmentalization of precursor synthesis within the RER or a competition for sites involved in the translocation of the functionally reduced RER.

Animals↗
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↗
PubMed1992

The cDNA sequence of the human pro-hormone and pro-protein convertase PC1.

Using a probe consisting of the full-length cDNA sequence of the mouse pro-hormone convertase PC1 (mPC1), we isolated from a lambda gt10 human pituitary cDNA library a number of contiguous clones, of which composite sequence of 3.3-kb defined the complete coding sequence of human PC1 (hPC1). The cDNA sequence of hPC1 encodes a protein containing 753 amino acids and potentially two N-glycosylation sites, one carboxy-terminal amidation site, a cAMP-dependent protein kinase Ser phosphorylation site, a tyrosine kinase phosphorylation site, and an ArgGlyAsp (RGD) sequence. Like mPC1, the carboxy-terminal sequence of hPC1 exhibits an amphipathic domain potentially involved in membrane association. The coding region of hPC1 exhibits an overall 92.6% protein sequence identity to the mouse mPC1 sequence, with the highest homology (98%) found in the catalytic segment of the molecule (residues 84-399). Whereas Northern blot analysis of tissues obtained from mouse, rat and porcine demonstrated the presence of two mRNAs of 3 kb and 5 kb, Northern blots of human tissues and cells demonstrated the presence of a dominant transcript of 6.2 kb and the presence of smaller transcripts in some tissues. The major site of production of hPC1 seems to be the pituitary and brain, although detection was also possible in pancreas and heart.

Amino Acid Sequence↗
PubMed1992

Testicular expression of PC4 in the rat: molecular diversity of a novel germ cell-specific Kex2/subtilisin-like proprotein convertase.

The rat cDNA sequence of PC4 (rPC4), representing a new member of the Kex2/subtilisin-like proprotein convertases, demonstrated the presence of at least three rPC4 mRNAs resulting in the production of rPC4-A (654 amino acids), rPC4-B (619 amino acids), and rPC4-C (609 amino acids) with different C-terminal sequences. Analogous to rat PC4, three cDNAs were also found for the mouse PC4. The observed molecular diversity of PC4 mRNA possibly results from the differential splicing and/or exon skipping of the parent gene. PC4 mRNA, with a major form at 2.8 kilobases, was highly abundant in the rat testis but could not be detected by Northern analysis in any other tissues including the central nervous system and peripheral tissues. Testicular cell separation studies combined with Northern analysis indicate the high expression levels of PC4 in germ cells but not in Leydig, Sertoli, or peritubular cells. In situ hybridization histochemistry confirms the site of PC4 gene expression as the pachytene spermatocytes and the round spermatids but not in the elongating spermatids. We also demonstrate the colocalization of PC4 with proenkephalin in testicular germ cells by in situ hybridization. A study of the ontogeny of PC4 indicated that PC4 mRNA was first expressed postnatally between days 19 and 22, coinciding with the first stages of spermiogenesis. The stage-specific expression of PC4 in testis indicates its potential role in the developmental maturation of germ cells and that this convertase may play a specific physiological function in reproduction.

Amino Acid Sequence↗
PubMed1992

Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary.

PC1 and PC2 are enzymes involved in the activation of prohormones via the cleavage of pairs of basic amino acids. The expression levels of each of these enzymes were evaluated in the rat anterior and neurointermediate pituitary lobes by in situ hybridization and Northern gel analysis and after various pharmacological manipulations. All intermediate lobe melanotrophs expressed high levels of PC2 mRNA and lower levels of PC1 mRNA. PC1 mRNA was highly expressed throughout the anterior lobe; however, appreciable PC2 mRNA levels were also found. Based on colocalization studies, anterior lobe corticotrophs were found to express PC1 mRNA, but very little PC2 mRNA. Neurointermediate lobe levels of PC1, PC2, and POMC mRNA increased 2- to 6-fold in rats treated with haloperidol, while they decreased to 10-25% of their control values after bromocriptine treatment. These results indicate that in the intermediate lobe, dopamine is involved in the regulation of PC1 and PC2. In the anterior lobe, haloperidol had a strong effect on PC2 mRNA, increasing its levels by 8- to 12-fold compared to the control value, while PC1 mRNA was unaffected. Both PC1 and PC2 mRNA levels were increased 5- to 9-fold in animals made hypothyroid by treatment with 6-n-propyl-2-thiouracil. Adrenalectomy had no significant effect on anterior lobe PC1 mRNA levels. However, both PC1 and PC2 mRNA levels were responsive to dexamethasone treatment in the AtT-20 cell lines. Our results indicate that dopamine, thyroid hormones, and corticosteroids are involved in PC1 and/or PC2 gene expression. These data are also consistent with the role of PC1 and PC2 as prohormone-processing enzymes.

Amino Acid Sequence↗
PubMed1991

Immunological identification and sequence characterization of a peptide derived from the processing of neuroendocrine protein 7B2.

A newly raised antiserum against the C-terminal region of neuroendocrine protein 7B2 was used to purify a novel peptide from the culture media of the mouse corticotroph cell line AtT-20. Based on partial sequencing, this peptide, which we call Cter-7B2, begins at Ser156 and appears to result from the cleavage of pro7B2 after a five-basic-residue sequence. Thus, 7B2 processing may contribute to the diversity of peptides found in neuronal and endocrine cells.

Amino Acid Sequence↗
PubMed1991

N-linked glycosylation affects the processing of mouse submaxillary gland prorenin in transfected AtT20 cells.

Most mouse inbred strains carry two renin genes, Ren-1 and Ren-2, Renin-2, the product of the Ren-2 gene, is highly expressed in the submaxillary gland. It is a renin isoenzyme 96% similar to kidney renin-1, but unglycosylated. In order to investigate if glycosylation of prorenin affects its processing and/or secretion we have introduced two potential N-linked glycosylation sites into preprorenin-2 cDNA using site-directed mutagenesis. Expression plasmids were derived from wild-type and mutant renin-2 cDNA and were transfected into AtT20 cells. Both transfected cells, expressing glycosylated or unglycosylated forms, secreted prorenin and renin by the constitutive and regulated pathways, respectively. Prorenin was correctly processed to active renin but the second maturation site was not cleaved in AtT20 cells. The comparison of glycosylated and unglycosylated renin expression showed a diminished secretion of glycosylated active renin. Prevention of glycosylation with tunicamycin resulted in an improved secretion of active renin. Moreover, the efficiency of the trypsin activation in vitro was reduced for glycosylated prorenin and it was restored when the activation was performed on mutant renin secreted from tunicamycin-treated cells. It is proposed that the bulky carbohydrates attached to prorenin constitute a steric hindrance to proteolysis by maturation enzymes.

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

Gene structure and chromosomal localization of plasma kallikrein.

Plasma kallikrein (Fletcher factor) is a hepatic serine proteinase that participated in the early phase of blood coagulation. From two genomic libraries, we succeeded to isolate four overlapping clones representing the entire rat plasma kallikrein gene. Using selective DNA sequencing, polymerase chain reactions, and restriction mapping, we demonstrated that the gene for rat plasma kallikrein was 22 kb in length. Similar to human factor XI [Asakai et al. (1987) Biochemistry 26, 7221-7228], we also found that the plasma kallikrein gene is composed of 15 exons and 14 introns. A potential transcription initiation step was determined by a novel application of the polymerase chain reaction technique. Computer analysis of the 5'-promoter region of this gene revealed some putative control elements that might regulate the rat plasma kallikrein gene expression. These data and the results of chromosomal localization reported in the present study for mouse (chromosome 8) and human (chromosome 4) plasma kallikrein genes strongly corroborate a genic duplication event from a common ancestor to both plasma kallikren and factor XI.

Amino Acid Sequence↗
PubMed1991

The production by alternate splicing of two mRNAs differing by one codon could be an intrinsic property of neuroendocrine protein 7B2 gene expression in man.

Two types of mRNAs for neuroendocrine protein 7B2 were deduced from the sequence of cDNAs clones isolated from a human pituitary cDNA library. One type lacks an Ala100 codon present in the other. The difference is located at an intron site within the human 7B2 gene and can be explained by the transcriptional utilization of two alternate acceptor splice sites, three nucleotides apart. Heteroduplex analysis of DNA fragments amplified by the polymerase chain reaction indicated that this 7B2 mRNA dimorphism occurs in several human endocrine tissues as well as in other species, suggesting that the alternate processing of 7B2 gene transcripts may be an intrinsic mechanism of its expression and could underlie some yet unknown biological functions.

Animals↗
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

Expression and sorting of rat plasma kallikrein in POMC-producing AtT-20 cells.

A vaccinia virus (VV) vector was used to express rat plasma kallikrein (rPK) in the constitutively secreting cells, BSC-40, and in the endocrine regulated cells, AtT-20. Using a specific rPK antibody and a fluorogenic substrate, Phe-Phe-Arg-AMC, we demonstrated that in both cell lines VV infections resulted in the synthesis of an immunoreactive enzyme predominantly present as a zymogen which can be activated with trypsin. Stimulation of VV:rPK-infected AtT-20 cells with either 5mM 8-bromo-cAMP or 56 mM KCl resulted in a different pattern of rPK and ACTH secretion, strongly suggesting that rPK follows the constitutive secretory pathway. Finally, the 10% rPK activity found within AtT-20 cell extracts had no effect on pro-opiomelanocortin (POMC) processing either intracellularly or extracellularly. The above data show that the biosynthetic machinery of both cell lines analyzed does not allow the efficient activation of plasma prekallikrein. Finally, despite the PK's demonstrated ability to cleave various hormone precursors in vitro at pairs of basic residues, in vivo, we did not obtain evidence that this hepatic enzyme can also act as an intracellular pro-protein processing enzyme.

Adrenocorticotropic Hormone↗
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↗