Characterization of endothelin-converting enzyme-2. Implication for a role in the nonclassical processing of regulatory peptides

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 Trp(21) and Val(22) 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.

Fig. 1. Purification of ECE-2 activity from the media of Sf9 cells overexpressing ECE-2

A, fractions containing ECE-2 activity were subjected to purification by ion exchange and metal ion chromatography as described under “Experimental Procedures.” The resulting fractions were subjected to electrophoresis under denaturing conditions on an 8% polyacrylamide gel. B, Western blot analysis of ECE-2 using a polyclonal antiserum directed against the C-terminal region of ECE-2. The positions of protein standards (in kDa) are indicated.

Fig. 2. Characterization of purified ECE-2

A, pH optimum of purified ECE-2 was determined with 10 μM McaBk2 in sodium acetate (circles), Tris acetate (squares), and sodium citrate (triangles) at the indicated pH values. The data represent means of quadruplicate determinations. Error bars show the standard error of the mean. B, the effect of metal ions on purified ECE-2 was determined with 10 μM McaBk2 in 0.2 M sodium acetate buffer, pH 5.5. The reaction was carried out in the presence of the indicated concentration of metal ion. “Relative units” represent fluorescence units generated per min, and the data represent means of triplicate determinations.

Fig. 3. Cleavage site determination using MALDI-TOF mass spectrometry

One nanomole of peptide was incubated in the presence or absence of purified ECE-2 (5 ng) in 0.2 M sodium acetate buffer, pH 5.5, for 100 min at 37 °C. The reaction was terminated by quick freezing, and the samples were subjected to MALDI-TOF mass spectrometry as described (16, 28).

Fig. 4. Kinetic analysis of ECE-2 hydrolysis of two representative bioactive peptides

Purified ECE-2 (5 ng) was assayed using the indicated concentrations of the various peptides. The reaction mixtures were subjected to high pressure liquid chromatography analysis, and peptides were detected by absorbance at 215 nm. The initial rate of substrate hydrolysis was determined by measuring the appearance of product under initial rate conditions (less than 10% substrate hydrolysis). Representative figures from the analysis of peptide E and bradykinin hydrolysis are shown.