Characterization of Endothelin-converting Enzyme-2
IMPLICATION FOR A ROLE IN THE NONCLASSICAL PROCESSING OF
REGULATORY PEPTIDES*
Nino
Mzhavia
,
Hui
Pan,
Fa-Yun
Che§,
Lloyd D.
Fricker§, and
Lakshmi A.
Devi¶
From the Department of Pharmacology and Biological
Chemistry, Mount Sinai School of Medicine, New York, New York 10029 and
the § Department of Molecular Pharmacology, Albert Einstein
College of Medicine, Bronx, New York 10461
Most neuroendocrine peptides are
generated by proteolysis of the precursors at basic residue cleavage
sites. Prohormone convertases belonging to the subtilisin family of
serine proteases are primarily responsible for processing at these
"classical sites." In addition to the classical cleavages, a
subset of bioactive peptides is generated by processing at
"nonclassical" sites. The proteases responsible for these cleavages
have not been well explored. Members of several metalloprotease
families have been proposed to be involved in nonclassical processing.
Among them, endothelin-converting enzyme-2 (ECE-2) is a good candidate
because it exhibits a neuroendocrine distribution and an acidic pH
optimum. To examine the involvement of this protease in neuropeptide
processing, we purified the recombinant enzyme and characterized its
catalytic activity. Purified ECE-2 efficiently processes big
endothelin-1 to endothelin-1 by cleavage between
Trp21 and Val22 at acidic pH. To
characterize the substrate specificity of ECE-2, we used mass
spectrometry with a panel of 42 peptides as substrates to identify the
products. Only 10 of these 42 peptides were processed by ECE-2. A
comparison of residues around the cleavage site revealed that ECE-2
exhibits a unique cleavage site selectivity that is related to but
distinct from that of ECE-1. ECE-2 tolerates a wide range of amino
acids in the P1-position and prefers aliphatic/aromatic residues in the
P1'-position. However, only a small fraction of the aliphatic/aromatic
amino acid-containing sites were cleaved, indicating that there are
additional constraints beyond the P1- and P1'-positions. The enzyme is
able to generate a number of biologically active peptides from peptide
intermediates, suggesting an important role for this enzyme in the
biosynthesis of regulatory peptides. Also, ECE-2 processes
proenkephalin-derived bovine adrenal medulla peptides, and this
processing leads to peptide products known to have differential
receptor selectivity. Finally, ECE-2 processes PEN-LEN, an
endogenous inhibitor of prohormone convertase 1, into products that do
not inhibit the enzyme. Taken together, these results are consistent
with an important role for ECE-2 in the processing of regulatory
peptides at nonclassical sites.
*
This work was supported by National Institutes of Health
Grants NS 26880 and DA00458 (to L. A. D.) and DA04494 (to
L. D. F.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Pharmacology and Biological Chemistry, Box 1603, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Tel.: 212-241-8345; Fax: 212-996-7214; E-mail: Lakshmi.Devi@mssm.edu.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
