Molecular Identification and Characterization of Novel Membrane-bound Metalloprotease, the Soluble Secreted Form of Which Hydrolyzes a Variety of Vasoactive Peptides

One class of zinc metalloproteases, represented by neutral endopeptidase 24.11 and endothelin-converting enzyme, has been shown to be involved in proteolytic activation or inactivation of many regulatory peptides. Here, we report molecular cloning and characterization of a novel member of this type II membrane-bound metalloprotease family, termed soluble secreted endopeptidase (SEP). Alternative splicing results in the generation of another transcript, SEPΔ, which lacks a 69-base pair nucleotide segment following the transmembrane helix. Both SEP and SEPΔmRNA are detected in all mouse tissues examined. Transfection of an SEP cDNA expression construct resulted in the expression of the membrane-bound form of SEP in the early secretory pathway as well as the soluble secreted form of the enzyme in the culture medium. In contrast, transfection of the SEPΔ cDNA only results in the expression of the membrane-bound form. In vitroenzymological analysis of the recombinant soluble form of SEP demonstrated that it hydrolyzes a variety of vasoactive peptides, including endothelin-1, atrial natriuretic peptide, and angiotensin I. This activity of SEP was inhibited by phosphoramidon and the neutral endopeptidase 24.11 specific inhibitor thiorphan, but it was only partially inhibited by the endothelin-converting enzyme specific inhibitor FR901533. These findings suggest that SEP is a novel metalloprotease that possesses a broad substrate specificity and that it may be involved in the metabolism of biologically active peptides intracellulary as well as extracellularly. One class of zinc metalloproteases, represented by neutral endopeptidase 24.11 and endothelin-converting enzyme, has been shown to be involved in proteolytic activation or inactivation of many regulatory peptides. Here, we report molecular cloning and characterization of a novel member of this type II membrane-bound metalloprotease family, termed soluble secreted endopeptidase (SEP). Alternative splicing results in the generation of another transcript, SEPΔ, which lacks a 69-base pair nucleotide segment following the transmembrane helix. Both SEP and SEPΔmRNA are detected in all mouse tissues examined. Transfection of an SEP cDNA expression construct resulted in the expression of the membrane-bound form of SEP in the early secretory pathway as well as the soluble secreted form of the enzyme in the culture medium. In contrast, transfection of the SEPΔ cDNA only results in the expression of the membrane-bound form. In vitroenzymological analysis of the recombinant soluble form of SEP demonstrated that it hydrolyzes a variety of vasoactive peptides, including endothelin-1, atrial natriuretic peptide, and angiotensin I. This activity of SEP was inhibited by phosphoramidon and the neutral endopeptidase 24.11 specific inhibitor thiorphan, but it was only partially inhibited by the endothelin-converting enzyme specific inhibitor FR901533. These findings suggest that SEP is a novel metalloprotease that possesses a broad substrate specificity and that it may be involved in the metabolism of biologically active peptides intracellulary as well as extracellularly. neutral endopeptidase 24.11 endothelin-converting enzyme soluble secreted endopeptidase endothelin atrial natriuretic peptide Chinese hamster ovary reverse transcription polymerase chain reaction high pressure liquid chromatography phosphate-buffered saline angiotensin-converting enzyme endo-β-N-acetylglucosaminidase H peptide-N-glycosidase F A wide variety of biologically active peptide hormones, regulatory peptides, and neuropeptides have been shown to be proteolytically activated or inactivated by members of zinc metalloproteases (1Hooper N.M. FEBS Lett. 1994; 354: 1-6Crossref PubMed Scopus (672) Google Scholar). One such class of zinc metalloproteases, represented by neutral endopeptidase 24.11 (NEP)1and endothelin-converting enzyme (ECE), has recently been highlighted because of their implications in some disease states and should thus provide plausible therapeutic targets for certain diseases (2Yanagisawa M. Circulation. 1994; 89: 1320-1322Crossref PubMed Scopus (192) Google Scholar, 3Turner A.J. Murphy L.J. Biochem. Pharmacol. 1996; 51: 91-102Crossref PubMed Scopus (223) Google Scholar, 4Turner A.J. Tanzawa K. FASEB J. 1997; 11: 355-364Crossref PubMed Scopus (385) Google Scholar). In mammals, six members of this metalloprotease family have been identified: NEP; Kell blood group antigen (KELL); ECE-1 and ECE-2; PEX, which has been associated with X-linked hypophosphatemic rickets; and the recently identified “orphan” peptidase XCE. All these members are type II integral membrane proteins containing a highly conserved consensus sequence of a zinc-binding motif, HEXXH (whereX represents any amino acid), in their extracellular C-terminal domain. Despite the apparent structural similarity among the members of this family, a large diversity of physiological functions exists. NEP, which is especially abundant in kidney and brain, is also expressed in various tissues as an ectoenzyme that can degrade many circulating small peptide mediators, such as enkephalins, atrial natriuretic peptide (ANP), tachykinins, and endothelins (ETs) (5Roques B.P. Noble F. Dauge V. Fournie-Zaluski M.C. Beaumont A. Pharmacol. Rev. 1993; 45: 87-146PubMed Google Scholar). NEP is also known as the common acute lymphoblastic leukemia antigen, and its presence on leukemic cells has been associated with a better prognosis. Although the physiological substrates of NEP are still unknown, targeted disruption of the NEP gene in mice caused a dramatic sensitivity to endotoxin shock, suggesting that NEP may provide an unexpected protective role against endotoxin shock (6Lu B. Gerard N.P. Kolakowski Jr., L.F. Bozza M. Zurakowski D. Finco O. Carroll M.C. Gerard C. J. Exp. Med. 1995; 181: 2271-2275Crossref PubMed Scopus (143) Google Scholar). Moreover, in vivo pharmacological inhibition of NEP has led to a decrease in blood pressure, and NEP-deficient mice were noted to have lower mean blood pressure levels than wild-type littermates, thus indicating that NEP may also play an important role in blood pressure regulation (7Lu B. Figini M. Emanueli C. Geppetti P. Grady E.F. Gerard N.P. Ansell J. Payan D.G. Gerard C. Bunnett N. Nat. Med. 1997; 3: 904-907Crossref PubMed Scopus (140) Google Scholar). ECE, another well characterized member of this metalloprotease family, is involved in the regulation of vascular tone, as well as in the development of some sets of neural crest cells (3Turner A.J. Murphy L.J. Biochem. Pharmacol. 1996; 51: 91-102Crossref PubMed Scopus (223) Google Scholar, 4Turner A.J. Tanzawa K. FASEB J. 1997; 11: 355-364Crossref PubMed Scopus (385) Google Scholar). It converts the inactive ET precursors (big ETs) into biologically active ETs via a specific cleavage at Trp21-Val/Ile22 (8Yanagisawa M. Kurihara H. Kimura S. Tomobe Y. Kobayashi M. Mitsui Y. Yazaki Y. Goto K. Masaki T. Nature. 1988; 332: 411-415Crossref PubMed Scopus (10284) Google Scholar, 9Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (863) Google Scholar). ECE constitutes a potential regulatory site for the production of the active peptide. Two isozymes of ECE, ECE-1 and ECE-2, have been molecularly identified and make up a subfamily within this group of type II membrane-bound metalloproteases (9Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (863) Google Scholar, 10Emoto N. Yanagisawa M. J. Biol. Chem. 1995; 270: 15262-15268Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). Both enzymes have been shown to cleave big ET-1 to produce ET-1 with a similar overall profile of inhibitor sensitivity in vitro as well as in transfected cells. However, ECE-1 and ECE-2 exhibit the following striking differences: (i) ECE-1 cleaves big endothelins in neutral pH, whereas ECE-2 functions in an the sensitivity of ECE-1 to phosphoramidon is lower as with ECE-2, and ECE-1 is expressed in cells and known to produce whereas ECE-2 is detected in neural tissues including the the and the disruption of the ECE-1 gene in mice that ECE-1 is the enzyme to produce active ET-1 H. Yanagisawa M. D. Emoto N. PubMed Google Scholar). physiological of ECE-2 has been physiological substrates of the PEX, and are still expressed on cells and the for the Kell blood group antigen S. S. A. PubMed Scopus Google Scholar). Although the Kell blood group antigen is its activity has to be gene was identified by cloning as a gene for X-linked hypophosphatemic a characterized by in the kidney F. S. B. P. A. H. T. T. C. S. A. A. B. M. J. T. Nat. 1995; 11: PubMed Scopus Google Scholar). was recently by expressed sequence with the ECE-1 sequence O. A. PubMed Scopus Google Scholar). activity for has been its physiological is gene of ECE that ECE-1 is a for big ET-1 and big at specific H. Yanagisawa M. D. Emoto N. PubMed Google Scholar). However, the of ECE-1 resulted in and a of ET-1 peptide was still suggesting that can This led to for enzymes of this metalloprotease Here, we report the of a novel enzyme, termed soluble secreted endopeptidase by cDNA as cDNA sequence that SEP is a type II membrane-bound metalloprotease to NEP, and Transfection of the SEP cDNA into Chinese hamster ovary cells resulted in the presence of SEP only in the membrane of the cells but also in the suggesting that these cells soluble of the enzyme secretory analysis of the recombinant soluble SEP that SEP hydrolyzes a variety of peptides, which are known as substrates of NEP ECE, including big angiotensin and P. This suggest that SEP is a novel member of this metalloprotease family and may be involved in the metabolism of biologically active peptides. big ET-1 angiotensin and were thiorphan, and were was a were by Yanagisawa of A cDNA SEP was by reverse transcription against with on the highly conserved amino of ECE-2, NEP, and of of and of and of were at an of and were at an of were in a and an pair was the was into and A cDNA was by the against mouse the were with as of the cDNA was by of cDNA against and mouse cDNA was with reverse by a specific to amino of an was to the of the was as by the with a specific to amino of and a was to the by a specific to amino of of this was into and nucleotide of the cDNA were into the and by a Both of cDNA were at cDNA was with of mouse tissues and reverse II as by the a of a of of and of to amino of and to amino of were for of mouse were by against SEP was by with a peptide, to the C-terminal amino of mouse were with peptides in by analysis was with by the as by the SEP and the membrane and cells were with of endo-β-N-acetylglucosaminidase or peptide-N-glycosidase F in at for were in in the at for were to cells were as N. Y. H. J. T. Yanagisawa M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). transfection of and or SEPΔ was cells were with medium. was for an and was to a enzyme that big ET-1 and in vitro an SEP expression construct was transfected into cells. was for in II and was to a with and was and with the containing active was to a were and for was as N. Y. H. J. T. Yanagisawa M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). for cells were and in for at in phosphate-buffered saline containing was for at the was with containing against SEP C-terminal peptides. for at the cells were and in containing at the cells were were on with and cells were in containing for at in the cells were with the SEP and as were with with and of the of these resulted in for enzyme peptide, and an enzyme some the were at with various for to the of the peptide. were at for in enzyme reaction was by of was into a high pressure liquid chromatography that was with and was at a of with a of and by a for an were detected by at were on a was to the All were an of and a of A pair of highly was on conserved amino of known members of the membrane-bound metalloprotease ECE-2, NEP, and mouse cDNA of the these cDNA into and the nucleotide that the pair cDNA was a of cDNA mouse NEP, whereas the nucleotide sequence a sequence to members of this metalloprotease this novel metalloprotease the SEP as a we a mouse cDNA because SEP was expressed in and with the nucleotide of all these as nucleotide of the SEP a that was by an and by a amino sequence of SEP is shown in and amino sequence by the mouse SEP that the is by an for are the transmembrane domain. sequence that is in SEPΔ is zinc-binding is by a to a SEP we of cDNA on mouse brain, and mouse a of specific to the diversity of SEP This nucleotide sequence of was to the of the SEP cDNA by However, the results of that cDNA SEP but a 69-base pair nucleotide segment following the transmembrane to splicing and on these we termed this SEP cDNA sequence a novel which important structural with the NEP metalloprotease family (1Hooper N.M. FEBS Lett. 1994; 354: 1-6Crossref PubMed Scopus (672) Google A.J. Tanzawa K. FASEB J. 1997; 11: 355-364Crossref PubMed Scopus (385) Google Scholar). (i) cDNA a type II integral membrane with a a transmembrane and a large extracellular C-terminal extracellular of SEP constitutes the and a highly conserved consensus sequence of a zinc-binding motif, and an and a amino that is by many SEP has in the extracellular suggesting that SEP is a highly NEP and are in the extracellular that are conserved in all the proteins of this metalloprotease A the sequence a similarity of the SEP sequence to NEP, ECE-2, and sequence similarity is especially high within the C-terminal of the extracellular including the the zinc-binding this of the of mouse SEP with to mouse NEP, ECE-2, and are and analysis a variety of mouse tissues large of SEP in of SEP were also expressed in the was in including the brain, and the expression of SEP and SEPΔ in various mouse tissues by to of SEP Two to SEP and SEPΔ and were detected in all tissues as well as in Although is the suggest that SEP is the in whereas SEPΔ is expressed in the of SEP and SEPΔ, we and by expression by the N. Y. H. J. T. Yanagisawa M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). analysis with an C-terminal peptide that SEP and SEPΔ proteins are expressed as an in the membrane these cells In we detected of with an apparent molecular of in culture with suggesting that these cells soluble of SEP into the culture In contrast, we SEP in the of and cells. These that cells the membrane-bound form of SEP as well as the soluble form of SEP in the whereas cells only the membrane-bound form. cDNA cloning of SEP that it is a highly we that the in the apparent molecular on analysis was to the presence of the of we the sensitivity of the membrane-bound SEP and the soluble of SEP to endo-β-N-acetylglucosaminidase H and peptide-N-glycosidase F containing high in the early secretory including the and a of the are to H and F. In contrast, proteins of which the have been to which in the are to F but to H. of cells with H or F the apparent molecular to which to the molecular of These suggest that the in the membrane of the cells is the partially in the early secretory In contrast, F of the the to H on these indicating that the SEP in is to H. These suggest that the presence of the SEP in the is to its the these results suggest that cells membrane-bound SEP in the membrane of the the early secretory pathway in the and also a soluble form of in the culture secretory the of the membrane-bound SEP we and cells with that the common C-terminal of SEP cells indicating that these cells expressed SEP on the and cells A and These findings that the membrane-bound SEP expressed in cells to be the in the early secretory which is with its sensitivity to H. SEP can big ET-1 by a transfection (9Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (863) Google Scholar, 10Emoto N. Yanagisawa M. J. Biol. Chem. 1995; 270: 15262-15268Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, N. Y. H. J. T. Yanagisawa M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and cells were transfected with a with the of ET-1 secreted these cells into the by a enzyme shown in cells transfected with cDNA a of with the that cells have ECE activity (9Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (863) Google Scholar). the which transfected with the large of This is also with that the ECE-1 cDNA the to ET-1 to these cells (9Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (863) Google Scholar). and cells transfected with cDNA also of indicating that SEP has an to cleave big ET-1 to produce However, of ET-1 were by cells than by cells. These suggest that SEP may have activity than levels of ET-1 may also be to the that SEP may degrade the ET-1 the cleavage at the ECE-1 specific cleavage site in big we have shown that ECE-1 cleaves the of big to produce and the C-terminal of big of big ET-1 or ET-1 (9Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (863) Google Scholar). the cleavage of big ET-1 and ET-1 by recombinant SEP in vitro we partially the soluble form of SEP the of cells. large of big ET-1 and ET-1 with partially SEP for a of the cleavage by were and the peptides were identified by was to a by the soluble form of and at Two peptides and were with the of big and that were big and with an for of and These findings that the soluble form of SEP can produce ET-1 by at the specific site of big the it that ET-1 is by the soluble form of that ET-1 was to with by these indicating that the soluble form of SEP hydrolyzes ET-1 at These and ET-1 were with the and big ET-1 molecular peptide and was identified as peptide to be the which is by and and by cleavage at site and of J. Slaughter C. C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). A of the cells activity for big ET-1 and ET-1 These of the soluble form of SEP were inhibited by phosphoramidon These results suggest that big ET-1 is at the site by the soluble in the production of ET-1 and that the ET-1 may be by the soluble the of SEP by its ET-1 This activity of soluble form of SEP was vitro by the metalloprotease inhibitor and the specific NEP inhibitor Both phosphoramidon and inhibited SEP activity in a with apparent of enzyme was partially inhibited by the ECE specific inhibitor and was inhibited by the angiotensin-converting enzyme inhibitor Y. N. T. N. K. M. S. M. J. 1994; PubMed Scopus Google Scholar). A a neutral at with a These that soluble form of SEP represents a novel metalloprotease with a neutral and that it has an inhibitor sensitivity profile similar to that of inhibitor profile of SEP partially the of of SEP activity of SEP activity in a we the of peptides that have been characterized as substrates of ECE-1 NEP T. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was by and the results are in that angiotensin and were all to or by soluble whereas a of the cells These suggest that SEP possesses a broad substrate specificity that is similar to that of of biologically active of was by of and natriuretic of was by of and in a have the cloning and characterization of a novel soluble secreted metalloprotease that can a variety of vasoactive peptides. was to another endothelin-converting enzyme, and we have demonstrated that SEP can cleave big ET-1 to produce ET-1 in vitro as well as in transfected cells. However, we that SEP as a physiological ECE because it to ET-1 than it ET-1 big many to SEP structural and with NEP than with or members of this metalloprotease the sequence of SEP with to NEP is than of the In SEP and NEP are to in their of whereas only ECE-2, and in this the known to the substrate in NEP and in are conserved in SEP and in mouse A.J. Tanzawa K. FASEB J. 1997; 11: 355-364Crossref PubMed Scopus (385) Google Scholar). In contrast, only of the is conserved in ECE-1 in and ECE-2 in this has been shown to play an role in the substrate of ECE-1 K. M. A.J. Tanzawa K. Biochem. J. 1996; PubMed Scopus Google Scholar). the in known to be involved in the of is conserved in SEP or NEP K. M. A.J. Tanzawa K. Biochem. J. 1996; PubMed Scopus Google Scholar). SEP and NEP degrade big ET-1 and ET-1 at cleavage whereas ECE-1 cleaves at the of big ET-1 of big ET-1 or ET-1 (9Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (863) Google Scholar). the activity of SEP is inhibited by the specific NEP inhibitor but is inhibited by the specific ECE inhibitor Y. N. T. N. K. M. S. M. J. 1994; PubMed Scopus Google Scholar). SEP and NEP cleave many small peptides in a highly These findings suggest that SEP is a endothelin-converting enzyme and that SEP and NEP may a subfamily within this group of Although SEP important with known members of this metalloprotease family, it still striking Transfection of an expression construct of SEP results in the of a soluble form of the enzyme into the culture medium. This that the soluble form of SEP may as a circulating endopeptidase in These are in to the that NEP and ECE as membrane-bound and a soluble form A.J. Tanzawa K. FASEB J. 1997; 11: 355-364Crossref PubMed Scopus (385) Google Scholar). In we are of any member of this metalloprotease family that a soluble form of the proteolytic of an integral membrane-bound ectoenzyme is well for angiotensin-converting enzyme a member of another metalloprotease family, which a role in the of blood pressure in N.M. J. Biochem. PubMed Scopus Google Scholar). Although as a membrane-bound enzyme, a soluble form is in many including blood N.M. J. Biochem. PubMed Scopus Google Scholar, N.M. A.J. Biochem. J. 1997; PubMed Scopus Google Scholar). In mammals, as and are a gene by Transfection of the cDNA of the or results only in the expression of the membrane-bound form of on the but also in a secreted form as a of by some S. A. PubMed Scopus Google Scholar, I. H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, F. F. A. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. I. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). However, SEP the following (i) only of SEP a soluble form of the enzyme, whereas of produce soluble of the and membrane-bound is expressed on the as an whereas membrane-bound SEP to be expressed in the early secretory including and a of the metalloproteases are known to produce SEP is a novel the soluble form of SEP has in vivo activity to be SEP and SEPΔ the cDNA are for amino that are to the structural that the of the soluble form of SEP be within these amino a pair of are in the C-terminal of this SEP sequence and are the common of and precursors in Cell. Full Text PDF PubMed Scopus Google Scholar). Although we have the cleavage site of it is to that the biologically active soluble form of SEP is proteolytically the membrane-bound SEP by a SEP may be at by the membrane in a similar to that of some membrane such as the the or N.M. A.J. Biochem. J. 1997; PubMed Scopus Google Scholar). A as well as an amino sequence of the soluble form of is to this in vitro suggest that SEP and NEP may similar Both enzymes circulating vasoactive peptides, including angiotensin and in the of these peptides with a similar overall inhibitor However, the results in this we SEP can degrade these physiological substrates in are to the physiological of SEP in the of these vasoactive peptides. the physiological of it is that SEP was to be highly expressed in mouse which is known to a variety of metalloproteases including NEP, and (5Roques B.P. Noble F. Dauge V. Fournie-Zaluski M.C. Beaumont A. Pharmacol. Rev. 1993; 45: 87-146PubMed Google Scholar, P. C. J. PubMed Scopus Google Scholar, P. P. F. J. PubMed Scopus Google Scholar). Although the physiological of NEP and ECE-1 in the have been the high expression of NEP and as well as their peptide suggest that may have in the (5Roques B.P. Noble F. Dauge V. Fournie-Zaluski M.C. Beaumont A. Pharmacol. Rev. 1993; 45: 87-146PubMed Google Scholar, P. P. F. J. PubMed Scopus Google Scholar). In in mice demonstrated that the expression of in is for P. C. J. PubMed Scopus Google Scholar). SEP may have some in the by some known as unknown, In with an SEP and analysis SEP in should of the physiological of this have the membrane-bound form of which is to is the of partially we a of membrane-bound However, a transfection and SEPΔ resulted in the of of Moreover, ECE-1 has been shown to be active the of suggesting that is for ECE-1 enzyme activity K. Biochem. PubMed Scopus Google Scholar). These suggest that the membrane-bound SEP may as an of membrane-bound SEP in cells to be of the metalloprotease family results of the as well as findings of SEP sensitivity to suggest that a of membrane-bound SEP is in the early secretory In contrast, NEP is expressed on the and functions as an ectoenzyme A.J. Tanzawa K. FASEB J. 1997; 11: 355-364Crossref PubMed Scopus (385) Google whereas ECE-2 is to be in an on the findings that ECE-2 is active at an and is inactive at a neutral N. Yanagisawa M. J. Biol. Chem. 1995; 270: 15262-15268Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). of ECE-1 has been as it has been to be in as well as on the N. Y. H. J. T. Yanagisawa M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A.J. K. A. O. Pharmacol. Full Text Full Text PDF PubMed Scopus Google Scholar). we that ECE-1 which only in their exhibit and that the of novel that N. Y. H. J. T. Yanagisawa M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). SEP may novel that its should to the molecular of SEP including the generation and analysis of mice containing a targeted disruption of the SEP are to the physiological substrates of and the physiological functions of this the potential of NEP and ECE characterization and structural of SEP be in the of and of NEP and for of the