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G Boileau

Publications and source records attributed to G Boileau.

At least 55 records · Page 3Linked to original sources
PubMed1994

Insertion of hydrophilic amino acid residues in the signal peptide/membrane anchor domain of neprilysin (neutral endopeptidase-24.11) results in its cleavage: role of the position of insertion.

We have expressed in COS-1 cells mutants of neprilysin (neutral endopeptidase-24.11; NEP) in which the hydrophilic sequence S-Q-N-S was either substituted for V42-T-M-I or inserted after T38 in the signal peptide/membrane anchor (SA) domain. These mutations were introduced in full-length NEP (mutants NEP(H1) and NEP(H2), respectively) and a form of NEP lacking its cytosolic tail (mutants NEP delta cyto(H1) and NEP delta cyto(H2), respectively). Immunoblotting showed that NEP(H1) was membrane-bound while NEP delta cyto(H1), NEP(H2), and NEP delta cyto(H2) were secreted. Furthermore, carbonate treatment of isolated intracellular membranes suggested that cleavage of the SA domain was performed in the endoplasmic reticulum, presumably by signal peptidase. Sequencing of the secreted proteins indicated that cleavage of the SA domain mostly occurred at the carboxy side of Ala46 but also at the carboxy side of Ala41 in NEP(H2) and NEP delta cyto(H2). We conclude that the position of the S-Q-N-S sequence influences the accessibility of the cleavage site and, in the case of NEP(H1) and NEP(H2), the efficiency of cleavage of the SA domain.

Amino Acid Sequence↗
PubMed1993

Rat endopeptidase-24.18 alpha subunit is secreted into the culture medium as a zymogen when expressed by COS-1 cells.

Endopeptidase-24.18 (EC 3.4.24.18, E-24.18) is an oligomeric Zn-ectoenzyme. The alpha and beta subunits have been cloned from both rat and mouse kidneys. The primary structure of these subunits revealed that they both contain the consensus Zn binding site and that they are members of the astacin family. Analysis of the hydropathy plot also suggested that they are anchored by a C-terminal hydrophobic domain. In order to verify the mode of anchoring of the rat E-24.18 alpha subunit and to test the functionality of the astacin-like domain in the alpha subunit when expressed alone, COS-1 cells were transfected with a cloned cDNA for rat alpha subunit. Despite the presence of its putative transmembrane domain, the alpha subunit was not anchored in the plasma membrane but rather secreted as a dimer into the culture medium. When the enzymatic activity of the secreted recombinant protein was tested in the azocasein degradation assay, the alpha subunit was found to be inactive. Activity could, however, be revealed after mild trypsin digestion. This activity was abolished by replacing the Glu-157 in the active site by Val. Taken together our results suggest that the alpha subunit of Endopeptidase-24.18 contains a latent astacin-like Zn metallopeptidase activity which could be secreted as a soluble enzyme by kidney and intestine.

Animals↗
PubMed1993

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.

Amino Acid Sequence↗
PubMed1993

Substitution of potential metal-coordinating amino acid residues in the zinc-binding site of endopeptidase-24.11.

Neutral endopeptidase (EC 3.4.24.11; NEP) is a membrane-bound zinc-metallopeptidase. The catalytic zinc ion is coordinated to three amino acid residues (His538, His587 and Glu646) and a water molecule. Here, we have systematically substituted potential metal-coordinating amino acid residues (His, Glu, Asp, Cys, Tyr, Ser) for each of the three zinc ligands of NEP using a recombinant polymerase chain reaction procedure. NEP mutants at positions 583 and 587 were devoid of catalytic activity. However, Glu587 NEP and Cys583 NEP were able to bind partially a tritiated inhibitor, the binding of which is dependent on the presence of the zinc atom. At position 646, the aspartate and cysteine mutants exhibited activity. For both mutants Km values were unaltered but kcat values were decreased by about 20-fold. Both mutants bound the tritiated inhibitor with Kd values similar to that of the wild-type enzyme. Our data suggest that neither histidine-583 nor -587 can be replaced by any other ligands. On the other hand, the glutamic acid at position 646 can be converted to an aspartic acid or a cysteine indicating the importance of a negative charge at this position.

Binding Sites↗
PubMed1993

Processing of Kex2 pro-region at two interchangeable cleavage sites.

In Saccharomyces cerevisiae, the Kex2 endoprotease (Kex2p) is required for the proteolytic maturation of alpha-pheromone and also for the removal of its own pro-region. Kex2p is specific for pairs of basic amino acid residues. Two putative processing sites are present in the pro-region of Kex2p. We have expressed processing site mutants of Kex2p and assayed the production of active Kex2p. Mutations affecting either putative cleavage site do not alter the activity. However, mutations affecting both sites led to a reduction in both Kex2 activity and the amount of protein. These results suggest that removal of Kex2p pro-peptide is required for the production of a stable enzyme and can occur at either processing site.

Enzyme Stability↗
PubMed1993

Kinetic evidence that His-711 of neutral endopeptidase 24.11 is involved in stabilization of the transition state.

Neutral endopeptidase 24.11 (EC 3.4.24.11; NEP) is a membrane-bound Zn-metalloendopeptidase with a catalytic activity and a specificity very similar to that of thermolysin, a bacterial zinc-endoprotease. NEP can be inactivated by reaction with diethylpyrocarbonate, due to the modification of a histidine residue present in the active site of the enzyme. This histidine residue was proposed to be analogous to His231 in thermolysin, which is involved in the stabilization of the tetrahedral intermediate during the transition state. Using site-directed mutagenesis of the cDNA encoding rabbit NEP, we have created two mutants of NEP where His711 was replaced by either Gln or Phe (NEP-Gln711 and NEP-Phe711). Determination of kinetic parameters showed that both mutants had Km values very similar to that of the non-mutated enzyme but that their kcat values were 25-fold lower. The calculated difference in free energy needed to form the transition state complex was increased by 2.2 kcal/mol for both mutants. These observations strongly suggest that His711 is involved in the stabilization of the transition state by forming an hydrogen bond with the oxyanion of the tetrahedral intermediate.

Animals↗
PubMed1993

Localization of neprilysin (EC 3.4.24.11) mRNA in rat brain by in situ hybridization.

The distribution of the messenger RNA (mRNA) coding for neprilysin (EC 3.4.24.11) has been studied by in situ hybridization in the adult rat brain. A markedly heterogenous distribution among various brain regions was found. A strong signal was observed in the glomerular layer of the olfactory bulb, the olfactory tubercle, the caudate putamen, the habenular, anterior pretectal, interpeduncular, red, dorso tegmental, pontine, and vestibular nuclei, the mammillary bodies, the Purkinje cells, and the choroid plexus of the fourth ventricle. A large number of areas such as the cortex, the dentate gyrus, the hippocampus, the medial terminal nucleus of the accessory tract, the accumbens and the arcuate nuclei, the superior and inferior colliculi, and a few regions in the thalamus at the mesencephalic level exhibited a moderate or low signal of hybridization. The majority of these regions are also known to contain the neprilysin protein. On the other hand, the globus pallidus, the substantia nigra, and the central gray matter, which show a high or a moderate amount of neprilysin, did not contain any neprilysin mRNA. Comparison of the regional distribution of neprilysin mRNA with that of its translation product provides insight into neprilysin neuronal pathways in the central nervous system.

Animals↗
PubMed1993

Transformation of the signal peptide/membrane anchor domain of a type II transmembrane protein into a cleavable signal peptide.

Rabbit neutral endopeptidase-24.11 is a type II transmembrane protein with a 27-amino acid residue positively charged NH2-terminal cytoplasmic domain, a 23-amino acid residue hydrophobic signal peptide/membrane anchor domain, and a large catalytic COOH-terminal domain exposed on the exoplasmic side of the membrane. In order to study the mechanism of membrane anchoring of neutral endopeptidase-24.11, we created mutants in which the cytoplasmic tail was deleted. Expression of these mutants in COS-1 cells resulted in the secretion of approximately 10-20% of the protein into the culture medium, due possibly to the cleavage of part or all of the signal peptide/membrane anchor domain by the rough endoplasmic reticulum signal peptidase. In a second set of mutants, a hydrophilic sequence (GSQNS) was inserted midway in the signal peptide/membrane anchor domain of neutral endopeptidase-24.11. When this hydrophilic sequence was introduced into the full-length neutral endopeptidase-24.11, approximately 20% of the enzyme activity was recovered in the culture medium. This proportion increased to 93% when the cytosolic tail was deleted. Sequencing of the [3H]tyrosine- or [3H]isoleucine-labeled secreted protein indicated that proteolysis, possibly by signal peptidase, occurred on the COOH-terminal side of the signal peptide/membrane anchor domain. We conclude that the efficient cleavage of the signal peptide/membrane anchor domain and secretion of the protein require both the deletion of the cytosolic domain and the presence of a hydrophilic sequence.

Amino Acid Sequence↗
PubMed1993

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)

Adrenocorticotropic Hormone↗
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↗