Angiotensin-converting Enzyme 2 (ACE2), But Not ACE, Is Preferentially Localized to the Apical Surface of Polarized Kidney Cells

Anthony J. Turner,Nigel M. Hooper,Daniel W. Lambert,A. Ian Smith,Rebecca A. Lew,Fiona J. Warner

doi:10.1074/jbc.m508914200

Anthony J. Turner, Nigel M. Hooper + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m508914200

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Abstract

Angiotensin-converting enzyme-2 (ACE2) is a homologue of angiotensin-I converting enzyme (ACE), the central enzyme of the renin-angiotensin system (RAS). ACE2 is abundant in human kidney and heart and has been implicated in renal and cardiac function through its ability to hydrolyze Angiotensin II. Although ACE2 and ACE are both type I integral membrane proteins and share 61% protein sequence similarity, they display distinct modes of enzyme action and tissue distribution. This study characterized ACE2 at the plasma membrane of non-polarized Chinese hamster ovary (CHO) cells and polarized Madin-Darby canine kidney (MDCKII) epithelial cells and compared its cellular localization to its related enzyme, ACE, using indirect immunofluorescence, cell-surface biotinylation, Western analysis, and enzyme activity assays. This study shows ACE2 and ACE are both cell-surface proteins distributed evenly to detergent-soluble regions of the plasma membrane in CHO cells. However, in polarized MDCKII cells under steady-state conditions the two enzymes are differentially expressed. ACE2 is localized predominantly to the apical surface ( approximately 92%) where it is proteolytically cleaved within its ectodomain to release a soluble form. Comparatively, ACE is present on both the apical ( approximately 55%) and basolateral membranes ( approximately 45%) where it is also secreted but differentially; the ectodomain cleavage of ACE is 2.5-fold greater from the apical surface than the basolateral surface. These studies suggest that both ACE2 and ACE are ectoenzymes that have distinct localization and secretion patterns that determine their role on the cell surface in kidney epithelium and in urine.

Highlights

Ment for hypertension, have been shown to have the broadest impact of any drug in cardiovascular medicine [1]
For nearly 60 years, angiotensin-I converting enzyme (ACE) has been considered the central enzyme in the renin-angiotensin system (RAS), converting angiotensin I (Ang I) to the potent vasoconstrictor, angiotensin II (Ang II)
Northern blot analysis and real-time PCR studies have shown angiotensin-converting enzyme-2 (ACE2) gene expression to be highest in heart, kidney, testes, and the gastrointestinal tract [2, 3, 18]

Summary

Characteristics of primary antibodies used in this study

Lium and, present an applicable cell line in which to study both ACE2 and ACE. The present study has investigated the cellular localization and cell fate of ACE2 in comparison with ACE, an analogous protein to ACE2 both in sequence and membrane topology, in non-polarized Chinese Hamster Ovary (CHO) cells and polarized MDCKII cells. Establishing an in vitro model of ACE2 and ACE expression in stably transfected polarized MDCKII cells provides a useful model to study the possible mechanisms regulating the cell fate and steady-state distribution of ACE2 and ACE in kidney epithelium

EXPERIMENTAL PROCEDURES

TABLE TWO

Primary cultures RPTECs HUVECs HCAECs CF SVSMCs

Findings

DISCUSSION