Investigation of subcellular localization of Angiotensin-converting enzyme

Tuwani, Lutendo

Investigation of subcellular localization of Angiotensin-converting enzyme

Thesis / Dissertation

2026

Publisher

University of Cape Town

Publisher

University of Cape Town

Department

Department of Integrative Biomedical Sciences (IBMS)

Faculty

Faculty of Health Sciences

Abstract

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.