Ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia

Abstract

verting enzyme 2 (ACE2) is a terminal carboxypeptidase and the receptor for the SARS and NL63 coronaviruses (CoV). Loss of ACE2 function is implicated in severe acute respiratory syndrome (SARS) pathogenesis, but little is known about ACE2 biogenesis and activity in the airways. We report that ACE2 is shed from human airway epithelia, a site of SARS-CoV infection. The regulation of ACE2 release was investigated in polarized human airway epithelia. Constitutive generation of soluble ACE2 was inhibited by DPC 333, implicating a disintegrin and metalloprotease 17 (ADAM17). Phorbol ester, ionomycin, endotoxin, and IL-1␤ and TNF␣ acutely induced ACE2 release, further supporting that ADAM17 and ADAM10 regulate ACE2 cleavage. Soluble ACE2 was enzymatically active and partially inhibited virus entry into target cells. We determined that the ACE2 cleavage site resides between amino acid 716 and the putative transmembrane domain starting at amino acid 741. To reveal structural determinants underlying ACE2 release, several mutant and chimeric ACE2 proteins were engineered. Neither the juxtamembrane stalk region, transmembrane domain, nor the cytosolic domain was needed for constitutive ACE2 release. Interestingly, a point mutation in the ACE2 ectodomain, L584A, markedly attenuated shedding. The resultant ACE2-L584A mutant trafficked to the cell membrane and facilitated SARS-CoV entry into target cells, suggesting that the ACE2 ectodomain regulates its release and that residue L584 might be part of a putative sheddase "recognition motif." Thus ACE2 must be cell associated to serve as a CoV receptor and soluble ACE2 might play a role in modifying inflammatory processes at the airway mucosal surface. a disintegrin and metalloprotease 17; severe acute respiratory syndrome; coronavirus SEVERE ACUTE RESPIRATORY SYNDROME (SARS) emerged as a regional and global health threat in 2002-2003 resulting in ϳ8,000 cases and 800 deaths. The causative agent was identified as a novel human coronavirus Refs. 8,33,51). Studies (13,47) of patients with SARS demonstrated that the respiratory tract is a major site of SARS-CoV infection and disease-associated morbidity. In 2003, angiotensin-converting enzyme 2 (ACE2) was discovered as a receptor for the virus (39). ACE2 is expressed in vascular endothelia, lung, renal and cardiovascular tissues, testes, and epithelia of the small intestine (9,17,18). It is a terminal carboxypeptidase that cleaves a single residue from ANG II, generating ANG-(1-7) (10, 32, 60). In addition, ACE2 cleaves the terminal residues from several other bioactive peptides, including neurotensin, dynorphin A (1-13), apelin-13, and des-Arg bradykinin (9, 60). As a type I transmembrane protein, ACE2 is comprised of a short cytoplasmic domain, a transmembrane domain, and a large ectodomain (57). A region of the ACE2 ectodomain that includes the first ␣-helix and lysine 353 and proximal residues of the N-terminus of ␤-sheet 5 interacts with high affinity with the receptor-binding domain of the SARS-CoV S glycoprotein (40).Previ...

Differential regulation of circulating and renal ACE2 and ACE in hypertensive mRen2.Lewis rats with early-onset diabetes

“…Regression analysis of urinary angiotensinogen (uAogen; ng/mg Cr) and proteinuria (mg/mg Cr). vascular surface through metallosecretases such as ADAM 10 and ADAM 17, previously shown to release the enzyme from renal and pulmonary membranes (1,18). Despite the increased ACE2 activity and reduced levels of angiotensinogen in males, circulating ANG II was significantly higher in both diabetic groups.…”

Paricalcitol modulates ACE2 shedding and renal ADAM17 in NOD mice beyond proteinuria

“…Interestingly, compared with nontreated diabetic animals, in diabetic animals, paricalcitol at both doses resulted in significantly decreased circulating ACE2 activity. Although the source for circulating ACE2 is not currently known, different studies suggest that ACE2 may be actively shed from the cell surface through metallosecretases such as ADAM10 and ADAM17 (17). Recent studies have explored the relationship between ACE2 and ADAM17 in experimental models of diabetic nephropathy (8,42).…”

“…In addition, this increase can be prevented by chronic administration of insulin (33). Although the source for circulating ACE2 is not currently known, different studies suggest that ACE2 may be actively shed from the vascular surface through metallosecretases such as a disintegrin and metalloproteinase domain-10 and -17 (ADAM10 and ADAM17, respectively) (17). Recent studies have explored the relationship between ACE2 and ADAM17 in experimental models of diabetic nephropathy (8,42).…”

Cell cycle dependence of ACE-2 explains downregulation in idiopathic pulmonary fibrosis

“…In the human lung, airway epithelial cells are one of the first sites of contact by the SARS coronavirus during lung infection; moreover, ACE-2 has been shown to be the site to which the SARS virus binds to initiate tissue infection [28]. In studies of cultured human airway epithelial cells, shedding of the ACE-2 ectodomain is believed to be an important determinant of the extent and outcome of SARS infection [29]. Related in vitro investigations have shown that shedding of the ACE-2 ectodomain is upregulated by phorbol esters and, furthermore, is dependent on the binding of calmodulin to a specific binding domain in the cytoplasmic tail of ACE-2 [30].…”