Endothelin-converting Enzyme-2 Is a Membrane-bound, Phosphoramidon-sensitive Metalloprotease with Acidic pH Optimum (*)
- From the (1)Howard Hughes Medical Institute and Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050
- ¶ Associate Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Inst., University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9050. Tel.: 214-648-5082; Fax: 214-648-5068.
Abstract
Endothelins (ET) are a family of potent vasoactive peptides that are produced from biologically inactive intermediates, termed
big endothelins, via a proteolytic processing at Trp
-Val/Ile
. We recently cloned and characterized a membrane-bound metalloprotease that catalyzes this proteolytic activation, endothelin-converting
enzyme-1 (ECE-1) (Xu, D., Emoto, N., Giaid, A., Slaughter, C., Kaw, S., deWit, D., and Yanagisawa, M.(1994) Cell 78, 473-485). This enzyme was shown to function in the secretory pathway as well as on the cell surface. Here we report molecular
cloning of another novel enzyme, ECE-2, that produces mature ET-1 from big ET-1 both in vitro and in transfected cells. The cDNA sequence predicts that bovine ECE-2 is a metalloprotease structurally related to ECE-1,
neutral endopeptidase 24.11, and human Kell blood group protein. The deduced amino acid sequence of ECE-2 is most similar
to ECE-1, with an overall identity of 59%. ECE-2 resembles ECE-1 in that it is inhibited in vitro by phosphoramidon and FR901533 but not by thiorphan or captopril, and it converts big ET-1 more efficiently than big ET-2
or big ET-3. However, ECE-2 also exhibits the following striking differences from ECE-1. (i) The sensitivity of ECE-2 to phosphoramidon
is 250-fold higher as compared with ECE-1, while FR901533 inhibits both enzymes at similar concentrations. (ii) ECE-2 has
an acidic pH optimum at pH 5.5, which is in sharp contrast to the neutral pH optimum of ECE-1. ECE-2 has a narrow pH profile
and is virtually inactive at neutral pH. Chinese hamster ovary (CHO) cells, which lack detectable levels of endogenous ECE
activity, secrete mature ET-1 into the medium when doubly transfected with ECE-2 and prepro-ET-1 cDNAs. However, ECE-2-transfected
CHO cells do not efficiently produce mature ET-1 when present with an exogenous source of big ET-1 through coculture with
prepro-ET-1-transfected CHO cells. These findings suggest that ECE-2 acts as an intracellular enzyme responsible for the conversion
of endogenously synthesized big ET-1 at the trans-Golgi network, where the vesicular fluid is acidified.
Footnotes
-
↵§ Associate of the Howard Hughes Medical Institute.
-
↵* This study is supported in part by research grants from the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank
/EMBL Data Bank with accession number(s) U27341.
-
↵1 The abbreviations used are:
- ET
-
endothelin
- ECE
-
endothelin-converting enzyme
- pCMS
-
p-chloromercuriphenylsulfonic acid
- MES
-
2-(N-morpholino)ethanesulfonic acid
- EIA
-
enzyme immunoassay
- RT-PCR
-
reverse transcription-polymerase chain reactions
- RACE
-
rapid amplification of cDNA ends
- kb
-
kilobase(s)
- CHO
-
Chinese hamster ovary.
