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G Boileau

Publications and source records attributed to G Boileau.

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

Molecular cloning of the alpha-subunit of rat endopeptidase-24.18 (endopeptidase-2) and co-localization with endopeptidase-24.11 in rat kidney by in situ hybridization.

Endopeptidase-24.18 (endopeptidase-2, EC 3.4.24.18, E-24.18) is a Zn-ectoenzyme of rat renal and intestinal microvillar membranes exhibiting an oligomeric structure, alpha 2-beta 2. The primary structure of the alpha-subunit of E-24.18 has been defined by molecular cloning and its expression mapped in rat kidney by in situ hybridization. A 2.9-kb cDNA coding for the alpha-subunit was isolated and sequenced. It had an open reading frame of 2,244 base pairs coding for a type I membrane protein of 748 amino acids. The deduced amino acid sequence showed 87% identity with that of meprin A, a mouse metallo-endopeptidase, sharing common properties with the rat enzyme, and 85% identity with the human intestinal enzyme, 'PABA-peptide hydrolase'. Northern blot analysis revealed the alpha-subunit to be encoded by a single mRNA species of 3.2-kb. In situ hybridization performed on rat kidney showed a co-localization of E-24.18 with endopeptidase-24.11 in proximal tubules of juxtamedullary nephrons, suggesting that the two enzymes have similar or complementary physiological functions in kidney.

Amino Acid Sequence↗
PubMed1992

Secretion of a functional soluble form of neutral endopeptidase-24.11 from a baculovirus-infected insect cell line.

Neutral endopeptidase (NEP; EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types. A secreted form of NEP (sec-NEP) was recently obtained by transfection of COS-1 cells with a recombinant expression vector consisting of the cDNA encoding the signal peptide of pro-opiomelanocortin fused in-frame to the cDNA sequence of the complete ectodomain of rabbit NEP [Lemay, Waksman, Roques, Crine & Boileau (1989) J. Biol. Chem. 264, 15620-15623]. In order to produce large quantities of this enzyme for structural studies we have expressed this recombinant soluble form of NEP at high yields using a baculovirus/insect-cell system. A recombinant Autographa californica nuclear polyhedrosis-virus genome containing the sec-NEP sequence was used to infect host Spodoptera frugiperda Sf9 cells. Infected cells secreted an N-glycosylated soluble form of neutral endopeptidase which was enzymically active. The yield was about 80 nmol of enzyme/litre of culture. The soluble form of the recombinant enzyme purified by immunoaffinity showed the same catalytic properties as the wild-type enzyme extracted from the kidney brush-border membranes. Treatment of the recombinant enzyme with endo-beta-N-acetylglucosaminidase H showed, however, that invertebrate cells did not glycosylate the enzyme to the same extent as did mammalian cells. Our findings demonstrate that insect cells can be used as hosts for the production of the soluble form of neutral endopeptidase. We also conclude that neither a full complement of carbohydrate side chains nor the membrane anchor appear to be essential for the production and targeting to the cell surface of a fully functional enzyme in this expression system.

Amino Acid Sequence↗
PubMed1992

The pro-region of the Kex2 endoprotease of Saccharomyces cerevisiae is removed by self-processing.

We have produced in the baculovirus/insect cells expression system a soluble secreted form of the Saccharomyces cerevisiae Kex2 endoprotease. This secreted enzyme was purified and its NH2-terminal sequence determined. The NH2-terminal sequence started at residue Leu109 of the sequence deduced from the KEX2 gene nucleotide sequence, showing that the Kex2 enzyme is produced as a proenzyme. Residue Leu109 is preceded by a pair of basic amino acid residues (Lys107-Arg108) which is a potential processing site for the Kex2 endopeptidase. Furthermore, expression of an inactive form of this truncated enzyme resulted in the production of a protein with a higher molecular weight. These observations suggest that the pro-region of Kex2 endoprotease is removed by a self-processing event.

Amino Acid Sequence↗
PubMed1992

Expression of the Saccharomyces cerevisiae Kex2p endoprotease in inset cells. Evidence for a carboxy-terminal autoprocessing event.

The pheromone-processing Kex2p endoprotease of Saccharomyces cerevisiae has been difficult to characterize due to its low level of expression in yeast cells. To overcome this problem, we have overexpressed Kex2p using the baculovirus/insect cell expression system. Spodoptera frugiperda Sf9 insect cells infected with a recombinant baculovirus, containing the complete KEX2 gene which encodes the Kex2p protease (814 amino acids), accumulate an 120-kDa functional form of the enzyme. The inhibition profile of the insect-cell-derived endoprotease is similar to that of the yeast enzyme. The recombinant infected insect cells also secrete into the medium about half of the total Kex2p activity produced. Deleting the carboxyl-terminal tail and the transmembrane domain of Kex2p (Kex2 delta p, 666 amino acids) does not measurably interfere with the enzyme characteristics and results in the secretion of up to 90% of the total enzyme activity. The truncated form, Kex2 delta p, of the endoprotease accumulates in the cell supernatant to 6.7 x 10(5) U/l. The molecular mass of the secreted forms for both the wild-type Kex2p and Kex2 delta p is the same (70 kDa) and is 50-kDa lower than the intracellular form. This result implicates a processing event which gives rise to shorter extracellular forms of both the wild-type Kex2p and Kex2 delta p and which trims their carboxy termini upsteam of amino acid 666. This processing event requires the integrity of the Ser385 of the Kex2p active site.

Animals↗
PubMed1992

Charge polarity reversal inverses the specificity of neutral endopeptidase-24.11.

Attempts to change enzyme specificity by charge polarity reversal have so far met with little success, probably due to a destabilization of the resulting ion pair in an environment naturally optimized for the inverted pair. In the zinc metallopeptidase neutral endopeptidase-24.11 (EC 3.4.24.11), Arg102, involved in substrate binding, is probably located at the edge of the active site (Bateman, R.C., Jr., Kim, Y.-A., Slaughter, C., and Hersh, L.B. (1990) J. Biol. Chem. 265, 8365-8368; Beaumont, A., Le Moual, H., Boileau, G., Crine, P., and Roques, B.P. (1991) J. Biol. Chem. 266, 214-220). This environment may be favorable for polarity reversal, as in water the energies of reverse ion pairs would be identical. We show here that, while mutating Arg102 to Glu reduces the specificity of a C-terminally negatively charged substrate 16-fold, it increases that of a substrate with an optimally positioned positive charge 29-fold. The concept of charge polarity reversal can be extended to other zinc metallopeptidases, and the mutated enzyme could also have applications in the enantiomeric separation of unnatural amino acids.

Binding Sites↗
PubMed1992

Expression and polarized apical secretion in Madin-Darby canine kidney cells of a recombinant soluble form of neutral endopeptidase lacking the cytosolic and transmembrane domains.

Neutral endopeptidase-24.11 (NEP; EC 3.4.24.11) is an abundant metalloendopeptidase of the brush border membrane of kidney proximal tubules. We have recently shown that NEP is delivered directly to the apical domain of the plasma membrane when expressed in polarized Madin-Darby canine kidney (MDCK) cells in culture (Jalal, F., Lemay, G., Zollinger, M., Berteloot, A., Boileau, G., and Crine, P. (1991) J. Biol. Chem. 266, 19826-19832). Here, a soluble form of NEP consisting of the signal peptide of pro-opiomelanocortin fused in-frame with the ectodomain of NEP has been expressed in MDCK cells. Enzymatic assays performed on apical and basolateral culture media of MDCK cells grown on semi-permeable supports indicated that the recombinant enzyme was predominantly released at the apical surface. In contrast, when the chimeric protein was expressed in NIH 3T3 cells or when pro-opiomelanocortin was expressed in MDCK cells, non-polarized secretion was observed into both the apical and basolateral compartments of the culture chamber. Our results suggest that the ectodomain of NEP is sufficient for directing the targeting of this protein to the apical membrane of polarized MDCK epithelial cells.

3T3 Cells↗
PubMed1991

Neutral endopeptidase, a major brush border protein of the kidney proximal nephron, is directly targeted to the apical domain when expressed in Madin-Darby canine kidney cells.

We have used a retroviral vector containing both the cDNA for rabbit neutral endopeptidase (EC 3.4.24.11; NEP) and the neomycin resistance gene to promote the expression of NEP in a polarized Madin-Darby canine kidney (MDCK) cell line. Cells resistant to G418 (a neomycin synthetic analog) were analyzed with a fluorescence-activated cell sorter to isolate a homogeneous population of cells which stably expressed NEP at their surface. When cells grown in Petri dishes were labeled with an antibody to NEP coupled to colloidal gold and examined under the electron microscope, a strong labeling of microvilli was observed, whereas very few particles were present on the basolateral domain, suggesting that the polarized distribution of this enzyme typical of proximal tubule cells is maintained in this MDCK cell population. To study more accurately the mechanism by which MDCK cells target NEP to the apical surface, cultures were grown to confluence on Costar Transwell chambers and used for pulse-chase experiments with [35S]methionine. Immunoprecipitation of recombinant NEP was then performed by adding an anti-NEP polyclonal antibody to the apical or basolateral surface of intact monolayers and by analyzing immunoprecipitates by gel electrophoresis and fluorography. Our results suggest that NEP is delivered directly to the apical domain and does not transit through the basolateral domain of the plasma membrane. This NEP-expressing MDCK cell line therefore constitutes a new model for investigating the molecular basis of apical membrane targeting in polarized epithelial cells.

Animals↗
PubMed1991

Identification of glutamic acid 646 as a zinc-coordinating residue in endopeptidase-24.11.

Neutral endopeptidase (EC 3.424.11, NEP) is a membrane-bound zinc-metallopeptidase. The substrate specificity and catalytic activity of NEP resemble those of thermolysin, a bacterial zinc-metalloprotease. Comparison of the primary structure of both enzymes suggests that several amino acids present in the active site of thermolysin are also found in NEP. Using site-directed mutagenesis of the cDNA encoding the NEP sequence, we have already shown that His residues 583 and 587 are two of the three zinc ligands. In order to identify the third zinc ligand, we have substituted Val or Asp for Glu616 or Glu646. Val616 NEP showed the same kinetic parameters as the non-mutated NEP. In contrast, the mutant Val646 NEP was almost completely devoid of catalytic activity and unable to bind the tritiated inhibitor [3H]N-[2(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxypropyl]gl ycine, the binding of which is dependent on the presence of the zinc ion. Replacing Glu for Asp at position 646 conserved the negative charge, and the mutant enzyme exhibited the same Km value as the non-mutated enzyme, but kCat was decreased to less than 3% of the value of the non-mutated enzyme. When compared to the non-mutated enzyme Asp646 NEP showed a higher susceptibility to chelating agents, but bound the tritiated inhibitor with the same affinity. Taken together, these observations strongly suggest that Glu646 of NEP is the third zinc-coordinating residue and is equivalent to Glu166 in thermolysin.

Binding Sites↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

Evidence that both arginine 102 and arginine 747 are involved in substrate binding to neutral endopeptidase (EC 3.4.24.11).

Neutral endopeptidase (EC 3.4.24.11, NEP) is a Zn-metallopeptidase involved in the degradation of biologically active peptides, notably the enkephalins and atrial natriuretic peptide. Recently, the structure of the active site of this enzyme has been probed by site-directed mutagenesis, and 4 amino acid residues have been identified, namely 2 histidines (His583 and His587), which act as zinc-binding ligands, a glutamate (Glu584) involved in catalysis, and an arginine residue (Arg102), suggested to participate in substrate binding. Site-directed mutagenesis has now been used to investigate the role of 4 other arginine residues (Arg408, Arg409, Arg659, and Arg747) that have been proposed as possible active site residues and to further analyze the role of Arg102. In each case, the arginine was replaced with a methionine, and both enzymatic activity and the IC50 values of several NEP inhibitors were measured for the mutated enzymes and compared to wild-type enzyme. The results suggest that 2 arginines, Arg102 and Arg747, could both be important for substrate and inhibitor binding. Arg747 seems to be positioned to interact with the carbonyl amide group of the P'1 residue and can be modified when the enzyme is treated with the arginine-specific reagents phenylglyoxal and butanedione. Arg102 could be positioned to interact with the free carboxyl group of a P'2 residue in some substrates and inhibitors and can be modified by phenylglyoxal but not by butanedione. The results could explain the dual dipeptidylcarboxypeptidase and endopeptidase nature of NEP.

Amino Acid Sequence↗
PubMed1991

Investigation of a possible role of the amino-terminal pro-region of proopiomelanocortin in its processing and targeting to secretory granules.

Proopiomelanocortin (POMC) is a polyprotein which is targeted to the regulated secretory pathway of neuroendocrine cells where it undergoes tissue-specific proteolysis to yield peptides such as adrenocorticotropic hormone, beta-lipotropin and beta-endorphin. The pro-region of POMC is 49 amino acid long with two disulfide bonds between cysteine residues 2 and 24 and 8 and 20. These cysteine residues are conserved across the species. The pro-region contains no known hormonal sequence. Sorting to the regulated secretory pathway is thought to involve targeting signals encoded in the structure of secretory proteins. In the present study, we have examined the possibility that the disulfide bridges located in the NH2-terminal portion of the pro-region of POMC are essential for maintaining a determinant involved in the sorting of POMC to the regulated secretory pathway. Using site-directed and deletion mutagenesis of the porcine POMC cDNA, we created mutants in which one or both disulfide bridges were disrupted or in which the first 26 amino acid residues of the pro-region were deleted. Recombinant retroviruses carrying the mutated POMC cDNAs were used to infect Neuro2A cells. Immunofluorescence and immunoelectron microscopy studies performed on infected cells revealed that the unmutated and mutated POMC-immunoreactive peptides were localized in dense-core vesicles at the tips of cellular extensions. Analysis of the POMC-immunoreactive peptides extracted from the infected Neuro2A cells indicated that the mutated precursors in which one disulfide bridge was disrupted (POMC-S2 or POMC-S8) were stored and processed as efficiently as the unmutated POMC. By contrast, the mutated precursor in which both disulfide bridges were disrupted (POMC-S2,8) did not accumulate in intracellular compartments to the same extent as unmutated POMC. Moreover, this mutant was very inefficiently processed and no release could be observed upon stimulation of the cells with K+/Ca2+. These results suggest that POMC-S2,8 entered the regulated secretory pathway less efficiently than the unmutated precursor. However, when both disulfide bridges were removed from the precursor from the precursor by deletion of the first 26 amino acid residues of POMC, the truncated precursor (POMC delta 1-26) behaved as the unmutated POMC. Taken together our results indicate that the NH2-terminal portion of the pro-region including both disulfide bridges can be deleted without affecting the targeting of the molecule to secretory granules. However, when the entire POMC sequence is expressed in Neuro2A cells, the proper folding of the NH2-terminal region might be important for efficient processing and targeting.

Amino Acid Sequence↗
PubMed1991

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.

Animals↗
PubMed1991

Prohormone processing by yeast proteases.

Investigations of the precursors of alpha-pheromone and killer toxin in the yeast Saccharomyces cerevisiae have defined the genes coding (KEX1 and KEX2) for the proteases which are responsible for their processing. In addition to processing at pairs of basic residues it is evident that yeast can also process at monobasic sites. We present data on the Kex1p and Kex2p enzymes, their cellular localization, and their post-translational modification. In addition initial characterisation of the monobasic specific protease and the isolation of mutants defective in this activity are presented. The use of the yeast system as a model for the processing of mammalian prohormones is discussed.

Amino Acid Sequence↗
PubMed1991

[Correlation and validation of different methods of evaluation of results after surgery of the rotator cuff. Plea for a standardized method].

Several charts elaborated for the assessment of the results of surgical treatment of cuff rotators tears were critically analysed. Twenty-one patients were reviewed and results assessed with three different charts, the Patte, Kenesi and Constant ones. The correlation of the parameters was studied and was found to be satisfactory.

Evaluation Studies as Topic↗
PubMed1990

Effect of mRNA secondary structure on the efficiency of translational initiation by eukaryotic ribosomes.

We have used site-directed mutagenesis to determine whether the structural context surrounding the AUG triplet influences its ability to be selected as an initiation codon by the eukaryotic preinitiation complex. AUG triplets were introduced in a loop and stem structure naturally occurring at the midpoint of the 129-nucleotides-long 5'-untranslated region of the porcine proopiomelanocortin mRNA; one AUG triplet was inserted in the loop while another was inserted in the stem of the hairpin structure. The proopiomelanocortin cDNA and the mutant cDNAs were inserted downstream from the early promoter of an expression vector derived from simian virus 40 (SV40) and transfected into monkey kidney COS-1 cells. Analysis of the proopiomelanocortin-related peptides present in the culture medium 72 h after transfection revealed that both mutant cDNAs direct the synthesis of more proopiomelanocortin than the non-mutant cDNA. The increased translational efficiency observed with both mutants is probably due to the decreased secondary structures of the shortened 5'-untranslated region. In addition, comparison of the two mutants indicates that the mutant mRNA with the AUG triplet inserted in the loop region of the hairpin structure directs the synthesis of approximately 75% more proopiomelanocortin than the mutant mRNA with the AUG triplet inserted in the stem region of the same hairpin structure, supporting a role for the structural context in the efficiency of translational initiation.

Animals↗