Biomedical subjects

G Boileau

Publications and source records attributed to G Boileau.

At least 37 records · Page 2Linked to original sources

PubMed1997

Patterns of compliance with once versus twice daily antihypertensive drug therapy in primary care: a randomized clinical trial using electronic monitoring.

OBJECTIVE: To evaluate patterns of compliance with once versus twice daily administration of antihypertensive therapy (primary-outcome measure) and relevance of partial compliance for blood pressure control (secondary outcome measure). DESIGN: Multicentre, nonblinded, parallel group randomized design. SETTING: Nonacademic primary care practices across Canada. STUDY POPULATION: Patients with mild essential hypertension (diastolic blood pressure 95 to 110 mmHg) of either sex (40% women), age 18 to 80 years (average 55 years). One hundred and ninety-eight patients were randomized to active treatment; 14 patients discontinued the study because of side effects. INTERVENTIONS: After a four-week placebo run-in period, patients were randomized to amlodipine 5 mg once-a-day or diltiazem slow release formulation (SR) 90 mg twice daily. Doses were increased to 10 mg and 180 mg to achieve sitting diastolic blood pressure of 90 mmHg or less. OUTCOME MEASURE: During 20 weeks on active treatment, compliance was assessed by pill counts and medication event monitoring system (MEMS), assessing percentage of prescribed doses taken, percentage days correct doses taken, percentage prescribed doses taken on time and blood pressure control as determined by office blood pressure measurement. RESULTS: The percentage prescribed doses taken (by either pill count of MEMS) showed a high degree of compliance, similar for the two treatments. However, other parameters of compliance were significantly better with once versus twice daily therapy. Partial compliance (less than 80% by pill count) led to less blood pressure control with the short acting diltiazem, but did not affect blood pressure control for the long acting amlodipine. Side effects profiles did not differ between the two treatments. CONCLUSIONS: Within the constraints of a clinical trial, hypertensive patients in primary care show a high degree of overall compliance with once or twice daily pill-taking, but patterns of pill-taking are more erratic with twice versus once daily medication, particularly in men. The results suggest that the negative consequences of partial compliance for blood pressure control can be offset by choosing agents with a duration of action well beyond the dosing interval.

Adolescent↗

PubMed1996

Identification of the cysteine residues implicated in the formation of alpha 2 and alpha/beta dimers of rat meprin.

Meprin (endopeptidase-24.18; EC 3.4.24.18) is a multisubunit zinc-metallopeptidase found in the brush-border membranes of rodent kidney and human intestine. The alpha and beta subunits of meprin are disulphide-linked to form either soluble alpha 2 homodimers or membrane-associated alpha/beta heterodimers. The aim of the present study was to identify the cysteine residue(s) implicated in the formation of alpha 2 and alpha/beta dimers and to investigate the effects of dimerization on intracellular transport and processing of the alpha subunit. Three cysteine residue candidates for the formation of disulphide bonds in the alpha subunit were selected by hydrophobic cluster analysis. These residues, located at positions 309, 560 and 562, were mutated to serine residues. When the resulting alpha subunit mutants were expressed alone in COS-1 cells, the alpha C560S and alpha C562S mutants were found to be secreted as alpha 2 homodimers whereas the alpha C309S mutant was found as monomers in the culture medium. In double-transfection experiments with the wild-type beta subunit, the alpha C560S and alpha C562S mutants behaved exactly as the wild-type alpha subunit and formed membrane-bound alpha/beta heterodimers. In contrast, the alpha C309S mutant was not retained at the cell surface but rather secreted as monomers in the culture medium, as was found in the simple transfection experiment. These results show that, despite the normal expression level and folding of the protein in a transport-competent from, the alpha C309S mutant is unable to form alpha 2 homodimers or alpha/beta heterodimers. This suggests that Cys309 is the unique residue of the alpha subunit implicated in the alpha 2 and alpha/beta dimerizations. Hydrophobic cluster analysis of the alpha and beta subunit sequences predicts that Cys309 is similar to Cys306 of the beta subunit. We mutated the latter residue to a serine and expressed the beta C306S mutant and the wild-type alpha subunit in the same COS-1 cells. No beta 2 or alpha/beta dimers were observed on immunoblotting, showing that Cys306 of the beta subunit is required for the formation of intermolecular disulphide bonds both in beta 2 homodimers and in alpha/beta heterodimers. Taken together, these results suggest that the alpha/beta heterodimeric form of meprin is held together by a single disulphide bond linking Cys309 in the alpha subunit to Cys306 in the beta subunit.

Amino Acid Sequence↗

PubMed1996

A highly selective assay for neutral endopeptidase based on the cleavage of a fluorogenic substrate related to Leu-enkephalin.

An intramolecularly quenched fluorogenic peptide structurally related to Leu-enkephalin, containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-Gly-Gly-D-Phe-Leu-Arg-Arg-Val-EDDnp (Abz-GGDFLRRV-EDDnp), was selectively hydrolyzed at the Arg-Val bond by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) with kinetic parameters (Km = 3 microM, kcat = 127 min-1 and kcatsolidusKm = 42 min-1 microM-1) similar to those of the Leu-enkephalin. The specificity of the NEP assay was demonstrated by incubating Abz-GGDFLRRV-EDDnp with a kidney homogenate or with crude membrane preparations of brain and lung: more than 95% of all products released were the complementary fragments Abz-GGDFLRR and V-EDDnp which were totally inhibited by 1 microM thiorphan, a highly specific NEP inhibitor. The blocked amino- and carboxyl-terminal amino acids protected this substrate against the action of aminopeptidases as well as of carboxypeptidases. Furthermore, D-Phe amino acid also ensured a very good protection of Abz-GGDFLRRV-EDDnp against the action of other tissue endopeptidases distinct from NEP. A continuous fluorometric assay for only 5 min was sufficient to quantify the NEP activity with a minimum sensitivity of 5 ng of purified enzyme or the equivalent enzymatic activity in crude tissue preparations. Therefore, amounts as little as 0.5 ng of enzyme could be quantified employing longer times of incubation.

Amino Acid Sequence↗

PubMed1996

Short-term effect of pudendal nerve electrical stimulation on detrusor hyperreflexia in spinal cord injury patients: importance of current strength.

Twenty patients with chronic suprasacral spinal cord injury presenting with detrusor hyperreflexia were examined. In a preliminary study in ten patients we investigated the reproducibility of bladder capacity through the repetition of three cystometries. The effect of electrical stimulation (ES) on detrusor hyperreflexia was then investigated in ten patients during three consecutive cystometries, the first one without ES (baseline) and the other two with continuous ES of the dorsal penile or clitoris nerve via surface electrodes. Parameters of stimulation were 5 Hz frequency, 0.50 msec pulse duration, and stimulation strength of 1 and 2 times the bulbocavernosus reflex threshold. No significant differences in bladder capacity were found between the three consecutive cystometries without ES (respectively 97.0 ml, 101.5 ml and 105.6 ml). A current at the bulbocavernosus threshold (mean 24.4 mA) failed to induce a significant increase in bladder capacity compared to baseline (173.0 ml vs 155.5 ml, P = 0.17) whereas a current of twice the bulbocavernosus threshold (mean 48.9 mA) was highly significant (318.5 ml vs 155.5 ml, P < 0.007). ES of twice the threshold resulted in perineal contraction in all of the patients, the threshold ES never did. Our results emphasise the decisive roles of perineal contraction and of current strength for achieving short-term bladder inhibition in spinal cord injury patients. The carry-over effect may also be dependent on the current strength. If so, maximal pudendal ES could represent an alternative procedure in the treatment of detrusor hyperreflexia in these patients.

Adolescent↗

PubMed1995

The nature of topogenic sequences determines the transport competence of topological mutants of neutral endopeptidase-24.11.

Type II integral membrane proteins are anchored by a signal-peptide/membrane-anchor domain (SA domain) located near their N-terminus, whereas type I membrane proteins are anchored by stop-transfer sequences usually located near the C-terminus. In this study we have attempted to transform neutral endopeptidase-24.11 (EC 3.4.24.11; NEP), a type II membrane protein, into a type I membrane protein. Three type I mutant proteins were constructed by fusion of topogenic sequences to the C-terminus of SecNEP, a soluble form of NEP. The first two type I mutants, SecNEP-TMC and SecNEP-TMIC, were constructed by fusing in frame the cytosolic and SA domains of NEP to the C-terminus of SecNEP. These two fusion proteins differ only in the orientation of the cytosolic tail. The third type I mutant, SecNEP-ACE, was constructed by fusing in frame the stop-transfer and cytosolic domains of angiotensin I-converting enzyme (EC 3.4.15.1; ACE) to the C-terminus of SecNEP. Our results suggest that: (1) the NEP ectodomain can be anchored with a type I topology in the endoplasmic reticulum (ER) membrane by both NEP and ACE topogenic sequences; (2) SecNEP-TMC and SecNEP-TMIC were transport-incompetent and needed proteolytic cleavage in the C-terminal region to leave the ER, whereas SecNEP-ACE was transported out of the ER as a type I membrane protein. Therefore we concluded that the nature of topogenic sequences determines the transport-competence of topological mutants of neutral endopeptidase-24.11.

Amino Acid Sequence↗

PubMed1995

Evidence that Asn542 of neprilysin (EC 3.4.24.11) is involved in binding of the P2' residue of substrates and inhibitors.

Neprilysin (EC 3.4.24.11) is a Zn2+ metallopeptidase involved in the degradation of biologically active peptides, e.g. enkephalins and atrial natriuretic peptide. The substrate specificity and catalytic activity of neprilysin resemble those of thermolysin, a crystallized bacterial Zn2+ metalloprotease. Despite little overall homology between the primary structures of thermolysin and neprilysin, many of the amino acid residues involved in catalysis, as well as Zn2+ and substrate binding, are highly conserved. Most of the active-site residues of neprilysin have their homologues in thermolysin and have been characterized by site-directed mutagenesis. Furthermore, hydrophobic cluster analysis has revealed some other analogies between the neprilysin and thermolysin sequences [Benchetrit, Bissery, Mornon, Devault, Crine and Roques (1988) Biochemistry 27, 592-596]. According to this analysis the role of Asn542 in the neprilysin active site is analogous to that of Asn112 of thermolysin, which is to bind the substrate. Site-directed mutagenesis was used to change Asn542 to Gly or Gln residues. The effect of these mutations on substrate catalysis and inhibitor binding was examined with a series of thiorphan-like compounds containing various degrees of methylation at the P2' residue. For both mutated enzymes, determination of kinetic parameters with [D-Ala2,Leu5]enkephalin as substrate showed that the large decrease in activity was attributable to an increase in Km (14-16-fold) whereas kcat values were only slightly affected (2-3-fold decrease). This is in agreement with Asn542 being involved in substrate binding rather than directly in catalysis. Finally, the IC50 values for thiorphan and substituted thiorphans strongly suggest that Asn542 of neprilysin binds the substrate on the amino side of the P2' residue by formation of a unique hydrogen bond.

Amino Acid Sequence↗

PubMed1995

Proteolytic processing of the alpha-subunit of rat endopeptidase-24.18 by furin.

Endopeptidase-24.18 (EC 3.4.24.18; meprin) is a multisubunit metallopeptidase of the astacin family. It is found in brush-border membranes of rodent kidney and human intestine. The membrane-bound enzyme is composed of alpha/beta dimers. Molecular cloning has shown that both subunits have a similar structural domain organization. Soluble alpha 2 dimers have also been observed in vivo and in transfected cells. The structures of all known alpha-subunits contain, upstream from the transmembrane domain, the sequence RXKR, which corresponds to the RXK/RR consensus sequence for specific cleavage by furin. In order to investigate the involvement of this putative cleavage site in the secretion process of endopeptidase-24,.18 alpha-subunit, we expressed in COS-1 cells rat alpha-subunits in which residues R655 or S656 (within the sequence R652PKRS656) were mutated to valine or leucine respectively. In contrast to the wild-type protein, the alpha R655V and alphaS656L mutants were not secreted in the culture medium. Moreover, when cells expressing the alpha-subunit were infected with a furin-encoding vaccinia virus, immunoblotting showed a shift of the major cell-associated form of endopeptidase-24.18 alpha-subunit from 98 kDa to 85 kDa and an increase in the amounts of secreted alpha-subunit. This shift in molecular mass was not observed with the mutant alpha-subunits. As observed for the 98 kDa species, the 85 kDa cell-associated protein was sensitive to endoglycosidase H treatment, suggesting that the proteolytic cleavage occurred in the endoplasmic reticulum or in an early Golgi compartment. Similar experiments using PACE4 and PC5 instead of furin showed that these enzymes were not able to generate the 85 kDa species. We conclude that furin is most probably the cellular enzyme involved in the proteolysis resulting in secretion of rat endopeptidase-24.18 alpha-subunit.

Amino Acid Sequence↗

PubMed1995

Comparative proteolytic processing of rat prosomatostatin by the convertases PC1, PC2, furin, PACE4 and PC5 in constitutive and regulated secretory pathways.

Recombinant vaccinia virus vectors were used to coexpress each of the candidate prohormone convertases PC1, PC2, furin, PACE4 and PC5 with rat prosomatostatin (rProSOM) in the constitutive secreting cell line LoVo and in the endocrine corticotroph cell line AtT-20, which exhibits regulated secretion. Mammalian ProSOM is cleaved at a dibasic Arg-Lys decreases site to produce somatostatin-14 (S-14) and at a monobasic Gln-Arg decreases site to yield somatostatin-28 (S-28). The analysis of processed products by gel-permeation high performance liquid chromatography shows that in LoVo cells PC1, furin and PACE4 generate S-14, S-28 and a mixture of S-14 and S-28, respectively, while PC2 is unable to process ProSOM in these constitutive cells. In contrast, PC2 can generate S-14 in AtT-20 cells. The convertase PC5 is unable to process ProSOM in either cell line. These data suggest that PC2, PC1 and PACE4 are candidate S-14 convertases, while PACE4 and furin are candidate S-28 convertases.

Animals↗

PubMed1995

Role of amino acid sequences flanking dibasic cleavage sites in precursor proteolytic processing. The importance of the first residue C-terminal of the cleavage site.

The amino acid sequences flanking 352 dibasic moieties contained in 83 prohormones and pro-proteins listed in a database were examined. Frequency calculations on the occurrence of given residues at positions P6 to P'4 allowed us to delineate a number of features which might be in part responsible for the in vivo discrimination between cleaved and uncleaved dibasic sites. These include the following: amino acids at these positions were characterized by a large variability in composition and properties; no major contribution of a given precursor subsite to endoprotease specificity was observed; some amino acid residues appeared to occupy preferentially certain precursor subsites (for instance, Met in P6 and P3, Asp and Ala in P'1, Pro in P6, Gly in P3 and P'2 etc.) whereas some others appeared to be excluded. Most amino acid residues occupying the P'1 position in these precursor cleavage sites were tolerated. But the beta-carbon branched side chain residues (Thr, Val, Leu, Ile) and Pro, Cys, Met and Trp were either totally excluded or poorly represented, suggesting that they might be unfavourable to cleavage. The biological relevance of these observations to the efficacy of dibasic cleavage by model propeptide convertases was in vitro tested using both pro-ocytocin convertase and Kex2 protease action on a series of pro-ocytocin related synthetic substrates reproducing the Pro7-->Leu15 sequence of the precursor in which the Ala13 residue (P'1 in the LysArg-Ala motif) was replaced by various amino acid residues. A good correlation was obtained on this model system indicating that P'1 residue of precursor dibasic processing sites is an important feature and may play the role of anchoring motif to S'1 convertase subsite. We tentatively propose that the present database, and the corresponding model, may be used for further investigation of dibasic endoproteolytic processing of propeptides and pro-proteins.

Amino Acid Sequence↗

PubMed1995

Direct targeting of neutral endopeptidase (EC 3.4.24.11) to the apical cell surface of transfected LLC-PK1 cells and unpolarized secretion of its soluble form.

LLC-PK1 cells were transfected with a cDNA encoding rabbit neutral endopeptidase (NEP; EC 3.4.24.11), an abundant enzyme of the kidney proximal brush border. Clones of cells expressing high levels of the protein were isolated. Selective biotinylation and radioimmunolabelling were used to determine that 85-95% of NEP was localized in the apical domain of filter-grown LLC-PK1 cells. Pulse-chase and selective biotinylation studies revealed that the majority (85%) of newly made NEP was directly targeted to the apical membrane. However, a soluble form of NEP was found to be secreted in approximately equal amounts from both sides of the monolayer when expressed in LLC-PK1 cells. Transfected pro-opiomelanocortin, a pituitary hormone precursor, was secreted almost exclusively into the basolateral medium, suggesting that the bulk flow is to the basolateral membrane. This behaviour contrasts with that observed in MDCK cells, where both the transmembrane and secreted forms of NEP are directly targeted to the apical membrane and where the secretion of pro-opiomelanocortin is unpolarized.

Animals↗

PubMed1995

Antihypertensive effects of amlodipine and hydrochlorothiazide in elderly patients with ambulatory hypertension.

Recent studies and authorities have advocated the use of low-dose thiazide diuretics as first-line treatment agents in elderly hypertensives. However, these recommendations were based solely on blood pressure (BP) measured in the clinic. The objective of the present 32-week double-blind study was to compare the effects of hydrochlorothiazide (HCTZ) and amlodipine (AML) in elderly patients with confirmed ambulatory hypertension. After a 4-week placebo washout period, 42 (25 men, 17 women) patients (mean age, 69 years) with clinic sitting diastolic BP of 95 to 114 mm Hg and daytime ambulatory diastolic BP of > or = 90 mm Hg were randomized double-blind to receive AML 5 to 10 mg (n = 21) or HCTZ 12.5 to 25 mg (n = 21) once daily. After 8 weeks of monotherapy, patients in whom clinic diastolic BP remained > or = 90 mm Hg were given combination therapy with the other agent. Amlodipine monotherapy induced significant reductions in clinic, mean 24-h, daytime and sleep systolic/diastolic BPs whereas only clinic BP decreased significantly in patients treated with HCTZ monotherapy. Moreover, 19/21 versus 8/21 patients on AML and HCTZ monotherapies achieved adequate BP control. At the end of the 32-week treatment period, combination therapy in the HCTZ group resulted in statistically significant reductions in clinic as well as in 24-h, daytime and sleep ambulatory BPs that were similar to those observed in the AML monotherapy group. In conclusion, the administration of AML monotherapy induced significant reductions in both clinic and ambulatory BPs in elderly patients whereas only clinic BP was significantly decreased by HCTZ monotherapy. Moreover, the addition of AML to HCTZ in patients inadequately controlled by monotherapy has permitted statistically significant decrements in clinic as well as in ambulatory BP. Consequently, the results of the present study suggest that the use of HCTZ in doses of up to 25 mg daily is inadequate for ambulatory BP control in the elderly despite official recommendations.

Aged↗

PubMed1995

A new fluorometric assay for neutral endopeptidase (EC 3.4.24.11).

An intramolecularly quenched fluorogenic peptide structurally related to Leu-enkephalin, Abz-GGDFLRRV-EDDnp, was selectively hydrolyzed at the R-V bond by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) with kinetic parameters (Km = 3 microM, kcat = 127/min and kcat/Km = 42/min microM) similar to those of Leu-enkephalin. The specificity of the assay for NEP was demonstrated by incubating Abz-GGDFLRRV-EDDnp with a kidney homogenate and with crude membrane preparations of brain and lung. For all three homogenates the complementary fragment Abz-GGDFLRR and V-EDDnp accounted for more than 95% of the products which are totally inhibited by 1 microM thiorphan, a highly specific NEP inhibitor. A continuous fluorometric assay for only 15 min was sufficient to quantify the NEP activity with a minimum sensitivity of 5 ng of purified NEP or the equivalent enzymatic activity in crude tissue preparations.

Animals↗

PubMed1994

Processing of human prosomatostatin in AtT-20 cells: S-28 and S-14 are generated in different secretory pathways.

Somatostatin-14 (S-14) and somatostatin-28 (S-28) are generated by differential processing of a single precursor at a dibasic (R-K) or monobasic (R) proteolytic cleavage site, respectively. To study the pathways of processing of prosomatostatin, we have expressed in AtT20 cells cDNA encoding human prosomatostatin and prosomatostatin mutated in one or the other processing site. Analysis of the peptides present in cell extracts or culture media before and after stimulation of the cells with 8-BrcAMP indicated that prosomatostatin can enter three distinct secretory pathways where it is differently processed: 1) prosomatostatin was secreted through the constitutive pathway; 2) the regulated secretory pathway generated S-14 which was released upon stimulation of the cells with 8-BrcAMP; 3) an alternative pathway, insensitive to 8-BrcAMP produced S-28 and S-14. Moreover, our results suggest that the R-K processing site used to produce S-14 is an important structural feature for targeting the precursor to the regulated secretory pathway.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1994

Role of glycosylation in transport and enzymic activity of neutral endopeptidase-24.11.

Neutral endopeptidase (NEP, EC 3.4.24.11) is a major ectoenzyme of the brush-border membrane. The ectodomain of NEP contains five putative N-glycosylation sites. In order to determine the role of the addition of sugar moieties on the activity and intracellular transport of NEP, we have used site-directed mutagenesis to remove all or some of the five potential sites of sugar addition in membrane-bound and secreted forms of the enzyme. Expression of NEP glycosylation mutants in COS-1 cells showed that all five sites are used for sugar addition. Immunoblotting of NEP in COS-1 cell extracts or culture media indicated that total expression of normal membrane-bound NEP was not affected by mutations at glycosylation sites, whereas this expression level appeared to be strictly dependent on the number of glycosylation sites retained on the soluble form. The transport to the cell surface was also reduced by decreased glycosylation, but again the phenomenon appeared more drastic in the case of the soluble form than for the membrane-bound enzyme. Enzyme activity was decreased by deglycosylation. However, the presence of either of two crucial sites (sites 1 and 5; numbered from the N-terminus of the protein) was sufficient to recover close-to-normal enzymic activities. Transport to the cell surface and enzyme activity of NEP are thus both dependent on sugar residues, probably through different conformational constraints. These constraints seem to be local for enzyme activity but more global for transport to the cell surface.

Animals↗

PubMed1994

A cleavable N-terminal signal peptide is not a prerequisite for the biosynthesis of glycosylphosphatidylinositol-anchored proteins.

During the biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins, an N-terminal signal peptide is used to direct biosynthesis to the endoplasmic reticulum. It was previously unknown whether or not this signal must be removed during the biosynthesis of GPI-anchored proteins. Using neutral endopeptidase (EC 3.4.24.11), a well characterized type II membrane protein that is attached to the membrane via an uncleaved N-terminal signal peptide, we extended its C terminus with 33 of the 37 amino acids of the GPI anchor signal sequence of decay-accelerating factor. When expressed in COS-1 and Chinese hamster fibroblast (CHW) cells, the protein was shown to possess both transmembrane and GPI anchors, indicating that a cleavable N-terminal signal peptide is not a prerequisite for the biosynthesis of GPI-anchored proteins.

Animals↗

PubMed1994

Expression of rat endopeptidase-24.18 in COS-1 cells: membrane topology and activity.

Endopeptidase-24.18 (E-24.18; EC 3.4.24.18) is a metallopeptidase of the astacin family and is highly expressed in kidney brush-border membranes of rodents. Rat E-24.18 consists of two disulphide-linked alpha/beta dimers [(alpha/beta)2]. In order to investigate the mechanisms of assembly and the importance of each subunit in the enzymic process, the cloned cDNAs for the rat alpha and beta subunits were transiently expressed either alone or together in COS-1 cells. Immunoblotting of cell extracts and spent culture media showed that, when expressed alone, the alpha subunit is secreted, whereas the beta subunit is membrane-bound. In alpha/beta-transfected cells, the alpha subunit remained membrane-bound, but could be released from the cell surface after papain treatment or after incubation with 10 mM dithiothreitol. Furthermore, mutants of the alpha subunit in which the putative C-terminal anchor domain was deleted could still form cell-associated alpha/beta dimers. These results are consistent with a topological model of E-24.18 in which the beta subunit is anchored in the plasma membrane and the alpha subunit is retained at the cell surface through disulphide bridge(s) with the beta subunit. Both the alpha and beta recombinant subunits expressed in COS-1 cells showed little azocasein-degrading activity. However, activity of either individual subunits of alpha/beta dimers was increased after mild trypsin digestion, suggesting that in COS-1 cells the enzymes are synthesized as zymogens. Finally, inactivation of the alpha subunit by site-directed mutagenesis of Glu-157, which is believed to play a role in catalysis, showed that both subunits participate in the enzymic activity of the heterodimer.

Amino Acid Sequence↗

PubMed1994

Expression of an enzymically active glycosylphosphatidylinositol-anchored form of neutral endopeptidase (EC 3.4.24.11) in Cos-1 cells.

Neutral endopeptidase (EC 3.4.24.11, NEP) is a type-II integral membrane protein found in a wide variety of cell types. We previously produced a secreted form of the enzyme by deletion of the cytoplasmic and transmembrane domains and in-frame fusion of the cleavable signal peptide of pro-opiomelanocortin [Lemay, Waksman, Roques, Crine and Boileau (1989) J. Biol. Chem. 264, 15620-15623]. Here we have used this secreted form of NEP and fused to it the glycosylphosphatidylinositol (GPI)-anchor attachment signal of decay-accelerating factor to produce a GPI-anchored form. Expression of this chimeric form in Cos-1 cells resulted in cell-surface activity. This activity could be released from the cell surface by phosphatidylinositol-specific phospholipase C and radiolabelling studies showed that the protein could incorporate [3H]ethanolamine, indicating that the enzyme was GPI-anchored. The Km value, using [D-Ala2,Leu5]enkephalin as substrate, of GPI-anchored NEP (62 +/- 5 microM) was comparable with that of wild-type NEP (70 +/- 4 microM), as were the sensitivities to the inhibitors phosphoramidon and thiorphan. However, pulse-chase studies showed that the biosynthesis and cell-surface delivery of GPI-anchored NEP was delayed compared with that of the wild-type transmembrane form of NEP. These results suggest a lower rate of biosynthesis and/or cellular transport for GPI-anchored NEP compared with its transmembrane counterpart.

Amino Acid Sequence↗

PubMed1994

Asp650 is crucial for catalytic activity of neutral endopeptidase 24-11.

Neutral endopeptidase (NEP) is a membrane-bound mammalian ectopeptidase that contains a catalytic zinc ion in its active site. Previous studies showed that the active site, and especially the zinc-binding site of NEP, have features in common with the prototypical bacterial zinc protease, thermolysin. Sequence comparison reveals that both enzymes have a conserved Asp residue (Asp650 in NEP and Asp170 in thermolysin) located four positions on the C-side of the third zinc ligand. In thermolysin, this residue is involved in a carboxylate-histidine-zinc interaction whose functional role has never been established [Christianson, D. W. & Alexander, R. S. (1990) Nature 346, 225]. To test the hypothesis that, in NEP, this residue is important for catalysis, we have changed Asp650 of NEP by site-directed mutagenesis and expressed the mutant enzymes in COS-1 cells. Substitution of Glu, Asn or Ala for Asp650 resulted in mutant enzymes exhibiting drastic decreases in specific activity. Binding experiments using the zinc-chelating inhibitor [3H]-N-[(2RS)-4-(hydroxyamino)-1,4-dioxo-2-(phenylmethyl)butyl]glycine suggested that the zinc ion is present in the active site of these mutant enzymes. These results strongly support the conclusion that Asp650 in NEP is crucial for hydrolytic activity.

Amino Acid Sequence↗