Browsing by Subject "ACE"

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    Deletion of the cytoplasmic domain increases basal shedding of angiotensin-converting enzyme
    (Elsevier, 2004) Chubb, Anthony J; Schwager, Sylva L U; van der Merwe, Elizabeth; Ehlers, Mario R W; Sturrock, Edward D
    Ectodomain shedding generates soluble isoforms of cell-surface proteins, including angiotensin-converting enzyme (ACE). Increasing evidence suggests that the juxtamembrane stalk of ACE, where proteolytic cleavage-release occurs, is not the major site of sheddase recognition. The role of the cytoplasmic domain has not been completely defined. We deleted the cytoplasmic domain of human testis ACE and found that this truncation mutant (ACE-ΔCYT) was shed constitutively from the surface of transfected CHO-K1 cells. Phorbol ester treatment produced only a slight increase in shedding of ACE-ΔCYT, unlike the marked stimulation seen with wild-type ACE. However, for both wild-type ACE and ACE-ΔCYT, shedding was inhibited by the peptide hydroxamate TAPI and the major cleavage site was identical, indicating the involvement of similar or identical sheddases. Cytochalasin D markedly increased the basal shedding of wild-type ACE but had little effect on the shedding of ACE-ΔCYT. These data suggest that the cytoplasmic domain of ACE interacts with the actin cytoskeleton and that this interaction is a negative regulator of ectodomain shedding.
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    Investigation of subcellular localization of Angiotensin-converting enzyme
    (University of Cape Town, 2026) Tuwani, Lutendo; Sturrock, Edward; Savulescu, Anca
    Angiotensin-converting enzyme (ACE) is a highly glycosylated peptidyl-dipeptidase ectoenzyme that is involved in the breakdown of biologically active peptides. It is most known for its regulation of blood pressure through the renin angiotensin system, and it occurs in two isoforms: somatic ACE (sACE) and testis ACE (tACE). Furthermore, ACE is found in a soluble secreted form and a membrane-bound form. Historically, research on ACE has focused on its presence on the cell surface, with less emphasis on its distribution within the cell; however, ACE moves to the cell membrane from the endoplasmic reticulum (ER) making it highly likely that it will be localised in the ER, perinuclear region, and cytoplasm. Given that protein subcellular localisation is typically correlated to protein function, characterisation of ACE's subcellular distribution is essential for uncovering its intracellular functions. This study aimed to explore the role of glycosylation in ACE expression, processing, and transport to the cell membrane in Chinese hamster ovary cells (CHO), as well as the effect of site occupancy and the composition of the glycan on subcellular localisation. To achieve this, the expression of ACE in CHO cells, along with various ACE and glycosylation mutants (tACEg3, g13, g1234, and sACE-Y465D), was quantified using a sensitive fluorometric assay and western blotting. Additionally, confocal laser scanning microscopy was employed to determine the subcellular localisation of ACE in CHO cells. The effects of glycosylation on ACE localisation and processing were further examined using the glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) and ACE mutants. We found that N-linked glycans play a crucial role in the localisation, shedding and processing of ACE. The western blot, immunofluorescence and shedding quantifications revealed that the absence of these glycans (glycans 2, 4, 5 and 6) led to reduced ACE expression and, in some instances, caused ACE to be retained within the cell rather than reaching the cell membrane. Additionally, the lack of N-linked glycans increased the rate of ACE shedding, as observed in tACE-g13 and tACE-g3 glycosylation mutants. Taken together, these findings advance our knowledge of the role of glycosylation in ACE localisation, provide new insights into its effect on ACE shedding and highlight its importance in the overall processing of ACE.