Mosaic expression of membrane peptidases by confluent cultures of Caco‐2 cells

Howell, Steven; Brewis, Ian Andrew; Hooper, Nigel M.; Kenny, A J; Turner, Anthony J.

doi:10.1016/0014-5793(93)81502-q

Cited by 23 publications

(15 citation statements)

References 8 publications

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“…Both enzymes have been localized by immunohistochemistry to polarized cell types in the kidney; the epithelia of proximal tubules and the endothelia of intrarenal vessels (3,30,49,50). The apical and basolateral distribution of ACE in MDCKII cells is in agreement with studies in humans (51,52).…”

Section: Discussionsupporting

confidence: 70%

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

Warner

Lew

Smith

et al. 2005

Journal of Biological Chemistry

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177

157

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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...

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Section: Discussionsupporting

confidence: 70%

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

Warner

Lew

Smith

et al. 2005

Journal of Biological Chemistry

Self Cite

177

157

View full text Add to dashboard Cite

show abstract

“…However, it has been demonstrated on tissue sections that ACE is present in the intracellular compartment on the perinuclear membrane and in the endoplasmic reticulum of renal vascular endothelial and proximal tubular cells (4). ACE, aminopeptidase N, and aminopeptidase W were detected both on the surface and in an intracellular pool in Caco-2 cells (15). Recently, the intracytoplasmic localization of ACE in the human gastric HGT-1 cell line has been found; however, most of the ACE within these ceils was immuuologically active, but enzymatically inactive (24).…”

Section: Discussionmentioning

confidence: 96%

Modulation of angiotensin-converting enzyme in cultured human vascular endothelial cells

Balyasnikova

Danilov

Muzykantov

et al. 1998

In Vitro Cell.Dev.Biol.-Animal

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Previous work has suggested that not all immunoreactive angiotensin-converting enzyme (ACE) in tissues or cells is in a biologically active state. We have explored this possibility in cultured human umbilical vein endothelial cells (HUVEC), one of the most widely studied in vitro endothelial cell systems. Our approach included characterization of the effect of increasing passage number on ACE activity and expression of immunoreactive ACE at the single cell level, the subcellular compartmentalization of active ACE, and the effect of phorbol ester (PMA) treatment. We found that both ACE activity and expression of ACE antigen were downregulated by cultivation (30% of ACE-positive cells at seventh passage vs. 90% in primary culture). ACE downregulation is specific (number of CD31-positive cells did not change with cultivation) and correlated with downregulation of factor VIII-antigen. The percentage of ACE-positive cells in permeabilized HUVEC at third passage was almost twice that in nonpermeabilized HUVEC (90% vs. 50%), indicating that HUVEC contain intracellular immunoreactive ACE. ACE activity, however, was similar when measured in intact cells and in cell lysates. Moreover, diazonium salt of sulfanilic acid (DASA), a membrane-impermeable ACE inhibitor, inhibited ACE activity in intact cells and in cell lysates at the same extent, thus implying that intracellular ACE is inactive. PMA (100 nM) treatment increased the percentage of ACE-positive cells at third passage from 57 to 96%. ACE activity was increased 3-fold in cell and 1.5-fold in the culture medium of PMA-treated cells. Analysis of ACE activity in intact monolayers and cell lysates of control and PMA-treated cells revealed that all enzymatically active ACE in PMA-treated cells is localized on the plasma membrane and acts as an ectoenzyme. We conclude that expression of ACE by HUVEC is downregulated by repeated passage in culture but can be restored by PMA treatment. In addition, ACE expression is heterogeneous between neighboring cells, and total immunoreactive ACE protein associated with HUVEC includes an inactive pool of the enzyme.

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“…In an effort to investigate whether the observed changes regarding tight junctions had influenced intestinal cell differentiation, we studied the localization pattern of aminopeptidase N (APN) (Howell et al, 1993; Mina-Osorio, 2008) in both WT and Ctsk −/− mice. We observed that cathepsin K deficiency induced no alterations in the distribution of this differentiation marker (data not shown).…”

Section: Discussionmentioning

confidence: 99%

Effects of cathepsin K deficiency on intercellular junction proteins, luminal mucus layers, and extracellular matrix constituents in the mouse colon

Arampatzidou

Schutte²,

Hansson³

et al. 2012

Biological Chemistry

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Cathepsin K has been shown to exhibit antimicrobial and anti-inflammatory activities in the mouse colon. To further elucidate its role, we used Ctsk−/− mice and demonstrated that the absence of cathepsin K was accompanied by elevated protein levels of related cysteine cathepsins (cathepsins B, L, and X) in the colon. In principle, such changes could result in altered subcellular localization; however, the trafficking of cysteine cathepsins was not affected in the colon of Ctsk−/− mice. However, cathepsin K deficiency affected the extracellular matrix constituents, as higher amounts of collagen IV and laminin were observed. Moreover, the localization pattern of the intercellular junction proteins E-cadherin and occludin was altered in the colon of Ctsk−/− mice, suggesting potential impairment of the barrier function. Thus, we used an ex vivo method for assessing the mucus layers and showed that the absence of cathepsin K had no influence on mucus organization and growth. The data of this study support the notion that cathepsin K contributes to intestinal homeostasis and tissue architecture, but the lack of cathepsin K activity is not expected to affect the mucus-depending barrier functions of the mouse colon. These results are important with regard to oral administration of cathepsin K inhibitors that are currently under investigation in clinical trials.

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Mosaic expression of membrane peptidases by confluent cultures of Caco‐2 cells

Cited by 23 publications

References 8 publications

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

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

Modulation of angiotensin-converting enzyme in cultured human vascular endothelial cells

Effects of cathepsin K deficiency on intercellular junction proteins, luminal mucus layers, and extracellular matrix constituents in the mouse colon

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