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, Lakshmi A. Devi

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)14704-14711
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number17
DOIs
StatePublished - Apr 25 2003

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Endothelins
Peptides
Enzymes
Processing
Endothelin-1
Peptide Biosynthesis
Proprotein Convertase 1
Peptide Hydrolases
Proprotein Convertases
Endothelin-Converting Enzymes
Subtilisin
Aromatic Amino Acids
Adrenal Medulla
Metalloproteases
Serine Proteases
Substrate Specificity
Neuropeptides
Proteolysis
Mass Spectrometry
Biosynthesis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of endothelin-converting enzyme-2 : Implication for a role in the nonclassical processing of regulatory peptides. / Mzhavia, Nino; Pan, Hui; Che, Fa Yun; Fricker, Lloyd D.; Devi, Lakshmi A.

In: Journal of Biological Chemistry, Vol. 278, No. 17, 25.04.2003, p. 14704-14711.

Research output: Contribution to journalArticle

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