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Characterization of angiotensin-converting enzyme 2 ectodomain shedding from mouse proximal tubular cells.

ABSTRACT: Angiotensin-converting enzyme 2 (ACE2) is highly expressed in the kidney proximal tubule, where it cleaves angiotensin (Ang) II to Ang-(1-7). Urinary ACE2 levels increase in diabetes, suggesting that ACE2 may be shed from tubular cells. The aim of this study was to determine if ACE2 is shed from proximal tubular cells, to characterize ACE2 fragments, and to study pathways for shedding. Studies involved primary cultures of mouse proximal tubular cells, with ACE2 activity measured using a synthetic substrate, and analysis of ACE2 fragments by immunoblots and mass spectrometry. The culture media from mouse proximal tubular cells demonstrated a time-dependent increase in ACE2 activity, suggesting constitutive ACE2 shedding. ACE2 was detected in media as two bands at ∼ 90 kDa and ∼ 70 kDa on immunoblots. By contrast, full-length ACE2 appeared at ∼ 100 kDa in cell lysates or mouse kidney cortex. Mass spectrometry of the two deglycosylated fragments identified peptides matching mouse ACE2 at positions 18-706 and 18-577, respectively. The C-terminus of the 18-706 peptide fragment contained a non-tryptic site, suggesting that Met(706) is a candidate ACE2 cleavage site. Incubation of cells in high D-glucose (25 mM) (and to a lesser extent Ang II) for 48-72 h increased ACE2 activity in the media (p<0.001), an effect blocked by inhibition of a disintegrin and metalloproteinase (ADAM)17. High D-glucose increased ADAM17 activity in cell lysates (p<0.05). These data indicate that two glycosylated ACE2 fragments are constitutively shed from mouse proximal tubular cells. ACE2 shedding is stimulated by high D-glucose, at least partly via an ADAM17-mediated pathway. The results suggest that proximal tubular shedding of ACE2 may increase in diabetes, which could enhance degradation of Ang II in the tubular lumen, and increase levels of Ang-(1-7).

SUBMITTER: Xiao F

PROVIDER: S-EPMC3893316 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Angiotensin converting enzyme (ACE) is known for converting inactive angiotensin I into the potent vasoconstrictor angiotensin II, playing a critical role in blood pressure regulation. However, there is evidence that the dicarboxypeptidase activity of ACE is also essential for other physiological processes likely through processing of various peptide substrates. This study used mass spectrometry for the comprehensive detection and identification of natural substrates and products of ACE within the mouse plasma peptidome. The plasma peptidome of ACE KO mice was obtained through a multi-step purification process which included organic TCA precipitation as well as size exclusion and reversed phase chromatography. The obtained complex mixture of endogenous peptides was then subjected to in vitro cleavage by ACE. ACE-treated and untreated samples were then analyzed by LC/MS on an Orbitrap mass spectrometer, followed by alignment of MS1 data by Progenesis QI software. The MS1 signals that gained or lost intensity after treatment with ACE, were considered as possible products and substrates of ACE, respectively, and were selected for a targeted MS/MS analysis, and subsequently identified with PEAKS 8.5 and Proteome Discoverer software. Results. Close to 250 natural peptides were identified as possible substrates and products of ACE, demonstrating the high promiscuity of the enzyme. The use of internal standards as well as detection of some expected endogenous peptides, such as angiotensin II and bradykinin, supported the validity of the approach. Some of the newly identified substrates of ACE are known for their biological activities. For example, a fragment of complement C3, the 17-amino acid peptide C3f, exhibits spasminogenic activity and was processed by ACE. ACE cleavage of select peptides was further confirmed in vitro. Also, concentrations of ACE substrates in plasma from mice with variant genetic ACE domain backgrounds were determined by LC/MS using multiple reaction monitoring on a triple quadrupole mass spectrometer. The in vivo results were consistent with the in vitro results, in the sense that higher levels of the ACE substrates were observed when the respective processing domain was knocked out. The use of transgenic mice as well as ACE with single active domain allowed clarifying the ACE domain selectivity towards individual peptide substrates. This study resulted in creation of a library of substrates and products of ACE that can be further tested for their biological function and can help to elucidate the link between ACE and the numerous physiological effects attributed to its activity.

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Characterization of angiotensin-converting enzyme 2 ectodomain shedding from mouse proximal tubular cells.

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