ECE-1 and ECE-2 also differ in their sensitivity to the inhibitor phosphoramidon. Both forms of ECE exist as type I1 integral membrane proteins and are related to ...
Biochemical Society Transactions ( 1 996) 24
Mutagenesis and modelling of endothelin converting enzyme V. M. HOANG, C. E. SANSOM and A. J. TURNER Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K. Hndothelin converting enzyme (ECE) catalyses the final key step in the proteolytic processing of the endothelin (ET) precursor. Two forms of ECE have currently been cloned, ECE-1 with a pH optimum in the neutral range and ECE-2 with an acidic pH optimum [see 1 for review]. ECE-1 and ECE-2 also differ in their sensitivity to the inhibitor phosphoramidon. Both forms of ECE exist as type I1 integral membrane proteins and are related to neutral endopeptidase-24.11 (NEP; neprilysin), thermolysin and Kell blood group antigen [2, 31. Unlike NEP or thermolysin, ECE-I exists as a disulphidelinked dimer, as shown by SDS-PAGE under reducing and nonreducing conditions and by cross-linking experiments [4]. Alignment of the sequences of human ECE with NEP and thermolysin indicates two non-conserved cysteine residues (C395 and C416) as candidates for disulphide-bond formation. We have therefore carried out site-directed mutagenesis to identify the residue involved in dimerisation. Site-directed mutagenesis was performed according to the method of Kunkel [5], changing Cys416 to Gly. Mutations were verified by DNA sequencing. The wild-type and C416G expression vectors pcDL-SRa296hECE were used to transfect Cos-l cells. For transient expression, Cos-l cells were plated in 24-well plates at 33% confluence or in 150 cm2 flasks at approx 2 x lo6 cells. After 24 h of growth, the cells were washed twice with Opti-Mem and transfected (0.2 pcg DNNwell or 5 pg DNNflask) by using Lipofect-Amine as ca3onic lipid (DNA:lipid, 1:lO). The cells were incubated for 2 h /and Dulbecco's modified Eagle medium containing 10% foetal calf serum was added. After 24 h, the medium was replaced with fresh and the cells were incubated for another 48 h before membrane preparation [6]. Reducing and non-reducing SDS gels were run in a 7.5% acrylamide minigel system. Approx. 10 pg detergentsolubilized membrane preparation was loaded onto each gel track. After electrophoresis, immunoblotting was performed using the anti-ECE monoclonal antibody AEC23-326 [2] and protein detected by the enhanced chemiluminescence method (Amersham, U.K.). Under reducing conditions, both wild-type and C416G mutant migrate as polypeptides of M, 130 000, indicating similar degrees of glycosylation. However, under non-reducing conditions, the M, of the wild-type is 250 000300 000, but that of the mutant remains at 130 000. Thus, C416 in human ECE-I appears to be responsible for dimer formation. This is consistent with the demonstration that the equivalent residue (C412) in rat ECE-I is involved in dimerisation [7]. Since the equivalent residue in ECE-2 is also a cysteine, it too is likely to occur as a dimer under native conditions. Both the wild-type and mutant ECE-1 were shown to be expressed at the cell-surface at comparable levels by immunocytochemistry . ECE activity was assayed by a novel HPLC assay involving the synthetic peptide substrate: DII WFNTPEHVVPYGLGamide in the presence and absence of 100 pM phosphoramidon. Assays were carried out in 100 mM Tris/HCI buffer, pH 7.0, with 200 pM substrate and 10-20 pg membrane protein,
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containing a protease inhibitor cocktail (amastatin, pepstatin A, E-64, thiorphan, all at 10 pM, and 3,4-dichloroisocoumarin and di-isopropylfluorophosphateat 100 pM) to prevent non-specific degradation of substrate or products. Cleavage occurred exclusively at the W-F bond and the products were resolved by HPLC and quantified by use of the appropriate standards. Hydrolysis of substrate was fully inhibited by phosphoramidon and non-transfected Cos-l cells showed no significant hydrolysis of the substrate. The assay is of sufficient sensitivity for assay of ECE in cell membrane preparations, is convenient for kinetic studies and much more rapid than previous assays of ECE involving radioimmunoassay of the ET product. Kinetic comparisons of expressed wild-type and mutant ECE-I suggest that dimerisation favours conversion of big ET-I into ET-I . Sequence alignments of ECE- I , NEP and thermolysin coupled with molecular modelling based on the structure of thermolysin have allowed prediction of a number of other residues that may be important in the structure and mechanism of ECE-I [8]. The availability of a suitable expression and mutagenesis system, coupled with a sensitive assay for enzyme activity, should allow the validation of such structural models for ECE.
VMH is in receipt of a M.R.C. studentship. We thank Dr K. Tanzawa for the gift of human ECE-I cDNA and anti-ECE monoclonal antibody. 1. Turner, A. J. and Murphy, L. J. (1996) Biochem.
Pharmacol. 51, 91-102. 2. Shimada, K., Takahashi, M. and Tanzawa, K. (1994) J. Biol. Chem. 269, 18275-1 8278. 3. Emoto, N. and Yanagisawa, M. (1995) J. Biol. Chem. 270, 15262- 15268. 4. Takahashi, M., Fukuda, K., Shimada, K., Barnes, K., Turner, A. J., Ikeda, M., Koike, H., Yamamoto, Y. and Tanzawa, K. (1995) Biochem. J. 311,657-665. 5 . Kunkel, T. A., Roberts, J. D. and Zarkour, R. A. (1 987) Meth. Enzymol. 154, 367-383. 6. Keynan, S., Hooper, N. M. and Turner, A. J. (1994) FEBS Lett. 349, 50-54. 7. Shimada, K., Takahashi, M., Turner, A. J. and Tanzawa, K. (1996) Biochem. J., in press. 8. Sansom, C. E., Hoang, V. M. and Turner, A. J. (1 995) J. Cardiovasc. Pharmacol. 26 (Suppl. 3), S75-S77.
