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

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 (ϳ92%) where it is proteolytically cleaved within its ectodomain to release a soluble form. Comparatively, ACE is present on both the apical (ϳ55%) and basolateral membranes (ϳ45%) 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. Angiotensin-I converting enzyme (ACE)3 is a zinc metallopeptidase, which functions as a fundamental regulator of the renin-angiotensin system (RAS) by converting angiotensin I (Ang I) to the potent vasoconstrictor, angiotensin II (Ang II). ACE is an important target for the control of blood pressure and ACE inhibitors, being the primary treatment for hypertension, have been shown to have the broadest impact of any drug in cardiovascular medicine (1).In 2000, a human homologue of ACE, angiotensin-converting enzyme-2 (ACE2), was independently identified by our group (2) and by others (3). ACE2 is an 805-amino acid protein that shares ϳ42% sequence identity to the N-and C-domains of somatic ACE (2). The predicted topology of ACE2 is that of a type I integral membrane protein consisting of a short C-terminal cytoplasmic domain (22 amino acids), a transmembrane domain anchoring ACE2 to the plasma membrane, and a large N-terminal ectodomain of 740 amino acids in which the catalytic zinc binding motif, HEXXH, is located. ACE2 functions exclusively as a carboxypeptidase (4, 5) and, therefore, differs in specificity from ACE, which can behave either as a peptidyl dipeptidase or an endopeptidase (6). ACE2 most efficiently cleaves apelin-13, dynorphin A (1-13), and des-Arg 9 bradykinin (4, 5). However, it is the ability of ACE2 to cleave the vasoconstrictor peptide Ang II to a putative cardi...

Assigning functions to distinct regions of the N-terminus of the prion protein that are involved in its copper-stimulated, clathrin-dependent endocytosis

“…This latter observation in particular would not be expected if Cu 2+ was required to promote the interaction of PrP C with a protein that engages the clathrin endocytic machinery. It should be noted that the transmembrane and cytosolic domains from angiotensin-converting enzyme used in the PrP-CTM construct (Walmsley et al, 2001) lack recognisable endocytosis signals and that angiotensin-converting enzyme itself does not undergo constitutive endocytosis from the cell surface in the time scale measured here (Warner et al, 2005). Together, these data indicate that binding of Cu 2+ to the octapeptide repeats is required to promote the dissociation of PrP C from rafts (summarised in Fig.…”

Intrarenal Renin Angiotensin System Revisited

“…Megalin and an intact endosomal apparatus may be necessary for ACE surface expression, whereas altered retrieval from the membrane may be specifically relevant for ACE2 as demonstrated previously for NaPi-2 (36). Although divergent regulation for ACE and ACE2 has been described (52), their membrane surface expression was considered to be similar, because their spontaneous internalization from the membrane was shown to be rather modest compared with other integral membrane proteins (27). The cause for the obvious difference in BBM representation and likely the concomitant surface expression upon the defects in endocytosis and membrane recycling caused by megalin deficiency in Cre(ϩ) mice is unclear but may be related to distinct interactions of ACE versus ACE2 with cytosolic proteins, which may be differently affected by the drop in megalin-induced signaling, endocytosis of other proteins, or recycling steps (29,36,53).…”

“…Whatever the source of conversion, proximal tubule Ang II levels were approximately 100-fold higher than those in plasma, confirming that the proximal tubule produces and secretes Ang II at levels greater than can be accounted for by glomerular filtration (1,11,20). The role of ACE2, an ACE homologue that is highly expressed in the proximal tubule, has received recent interest because ACE2 may counterbalance classical RAS effects in part by metabolizing Ang II to the heptapeptide, Ang-(1-7), which activates the receptor Mas, thus antagonizing the effects of local Ang II (26,27).…”

Functional and molecular evidence for expression of the renin angiotensin system and ADAM17-mediated ACE2 shedding in COS7 cells

“…Therefore, ACE2 was proposed as a potential early biomarker of kidney disease. Indeed, increased ACE2 shedding into urine has been described for diabetic mice (6, 38, 52) and for patients with chronic kidney disease, in diabetic renal transplant patients, and in patients with diabetic nephropathy (29,48,53).ACE2 colocalizes in the kidney with a disintegrin and metalloproteinase (ADAM17) (6, 38), a metalloprotease capable of cleaving the ectodomain of ACE2 but not that of ACE (24,25,49), suggesting that ADAM17 may be directly involved in the ectodomain shedding of ACE2. Evidence for ACE2 shedding via an ADAM17-mediated pathway has been provided in human proximal tubular HK-2 kidney cells showing that release of ACE2 into the media was blocked by inhibition with a specific ADAM17 inhibitor (38).…”

“…ACE2 colocalizes in the kidney with a disintegrin and metalloproteinase (ADAM17) (6, 38), a metalloprotease capable of cleaving the ectodomain of ACE2 but not that of ACE (24,25,49), suggesting that ADAM17 may be directly involved in the ectodomain shedding of ACE2. Evidence for ACE2 shedding via an ADAM17-mediated pathway has been provided in human proximal tubular HK-2 kidney cells showing that release of ACE2 into the media was blocked by inhibition with a specific ADAM17 inhibitor (38).…”