The Plethora of Angiotensin-Converting Enzyme-Processed Peptides in Mouse Plasma

Abstract

Angiotensin-converting enzyme (ACE) converts angiotensin I into the potent vasoconstrictor angiotensin II, which regulates blood pressure. However, ACE activity is also essential for other physiological functions, presumably through processing of peptides unrelated to angiotensin. The goal of this study was to identify novel natural substrates and products of ACE through a series of mass-spectrometric experiments. This included comparing the ACE-treated and untreated plasma peptidomes of ACE-knockout (KO) mice, validation with select synthetic peptides, and a quantitative in vivo study of ACE substrates in mice with distinct genetic ACE backgrounds. In total, 244 natural peptides were identified ex vivo as possible substrates or products of ACE, demonstrating high promiscuity of the enzyme. ACE prefers to cleave substrates with Phe or Leu at the C-terminal P2' position and Gly in the P6 position. Pro in P1' and Iso in P1 are typical residues in peptides that ACE does not cleave. Several of the novel ACE substrates are known to have biological activities, including a fragment of complement C3, the spasmogenic C3f, which was processed by ACE ex vivo and in vitro. Analyses with N-domain-inactive (NKO) ACE allowed clarification of domain selectivity toward substrates. The in vivo ACE-substrate concentrations in WT, transgenic ACE-KO, NKO, and CKO mice correspond well with the in vitro observations in that higher levels of the ACE substrates were observed when the processing domain was knocked out. This study highlights the vast extent of ACE promiscuity and provides a valuable platform for further investigations of ACE functionality.

Figure 1.

Promiscuous peptidase specificity of ACE. (A,B) Probabilities of amino acid residues around the ACE cleavage site (P1|P1′) observed in 162 substrates (A) and in 680 nonsubstrates (B). (C) Differential residue probabilities of ACE substrates minus those of nonsubstrates. (D) Differential residue probabilities at the C termini of ACE nonsubstrates minus those of substrates. Coloring scheme: polar (STYQN), green; neutral (CG), black; basic (KRH), blue; acidic (DE), red; hydrophobic (AVLIPWFM), purple.

Figure 2.

Cleavage profiles of selected ACE substrates. (A) In vitro: synthetic ACE substrates treated with WT ACE, NKO ACE, or no enzyme, with the resulting peptides detected by MALDI-TOF or Orbitrap LC/MS. (B) In vivo: concentrations of endogenous ACE substrates in the blood plasma of mice with different genetic ACE backgrounds (WT, ACE KO, ACE NKO, or ACE CKO) as well as ramipril-treated WT mice (rtWT), as determined by triple-quadrupole MRM.