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Characterisation of the structural motifs Involved in the cleavage and secretion of human angiotensin-converting enzyme
Includes bibliographical references.
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
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Division of Medical Biochemistry
2014
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| _version_ | 1867613184942145536 |
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| access_status_str | Open Access |
| author | Conrad, Nailah |
| author2 | Sturrock, Edward D |
| author_browse | Conrad, Nailah Sturrock, Edward D |
| author_facet | Sturrock, Edward D Conrad, Nailah |
| author_sort | Conrad, Nailah |
| collection | Thesis |
| description | Includes bibliographical references. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/8710 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:32:07.214Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2014 |
| publishDateRange | 2014 |
| publishDateSort | 2014 |
| publisher | Division of Medical Biochemistry |
| publisherStr | Division of Medical Biochemistry |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/8710 Characterisation of the structural motifs Involved in the cleavage and secretion of human angiotensin-converting enzyme Conrad, Nailah Sturrock, Edward D Schwager, Sylva L U Cell Biology Includes bibliographical references. Angiotensin converting enzyme is an ectoprotein prone to regulated proteolytic solubilisation by an as yet unknown protease or sheddase. Proteolytic cleavage of membrane proteins is an essential cellular process that controls their expression and function, and modulates cellular and physiological processes. Testis ACE (tACE) is shed at a higher rate than somatic ACE and it has been proposed that regions in its ectodomain direct its shedding. Discrete secondary structures on the surface of the distal ectodomain of tACE were replaced with their N-domain counterparts to determine their role in the ectodomain shedding of ACE. None of the regions investigated proved to be an absolute requirement for shedding, but the mutant ACE proteins were subject to variations in shedding compared to wild-type tACE. To investigate the role of the proximal ectodomain in shedding the residues H610-L614 were mutated to alanines, causing a decrease in shedding. An extension of this mutation on the N-terminal side to seven alanines resulted in a reduction in ACE activity and, more importantly, it affected the processing of the protein to the membrane, resulting in expression of an underglycosylated form of ACE. When E608-H614 was mutated to the homologous region of the N-domain, processing was normal and shedding only marginally reduced. These data suggest that this region is more crucial for the processing of ACE than is for regulating shedding. Construction of a P628L mutation in tACE showed an increase in shedding. Furthermore, MALDI analysis of a tryptic digest established that the putative glycosylation site N620WT became glycosylated. Further mutagenesis of the P628L mutant to remove the newly formed glycosylation site, resulted in an even greater increase in shedding. Soluble fluorogenic peptides mimicking the ACE stalk were used in a cell-based assay to characterise the contribution of the stalk to ACE shedding. Hydrolysis of the wild-type peptide Abz-NSARSEGPQ-EDDnp was not responsive to phorbol ester or the hydroxamate inhibitor (TAPI), however, it was inhibited by EDTA. The aminopeptidase inhibitor bestatin did not inhibit cleavage or alter the cleavage site. Therefore the protease involved in the cleavage of the ACE stalk peptides is likely different to the sheddase responsible for ACE shedding. Substitution of the P1 and P1' sites of the peptides did not significantly influence the rate of cleavage. All the peptides were cleaved at the E-G bond, which is C-terminal to the physiological R-S cleavage site. Removal of the fluorogenic capping groups resulted in no cleavage of the peptides and lengthening of the peptide did not result in cleavage. This confirms the need for the ACE sheddase and its substrate to be anchored in the membrane and suggests the use of soluble peptide substrates in a cell assay has limited application for investigating the ectodomain shedding of ACE. 2014-10-21T13:52:50Z 2014-10-21T13:52:50Z 2014 Doctoral Thesis Doctoral PhD http://hdl.handle.net/11427/8710 eng application/pdf Division of Medical Biochemistry Faculty of Health Sciences University of Cape Town |
| spellingShingle | Cell Biology Conrad, Nailah Characterisation of the structural motifs Involved in the cleavage and secretion of human angiotensin-converting enzyme |
| thesis_degree_str | Doctoral |
| title | Characterisation of the structural motifs Involved in the cleavage and secretion of human angiotensin-converting enzyme |
| title_full | Characterisation of the structural motifs Involved in the cleavage and secretion of human angiotensin-converting enzyme |
| title_fullStr | Characterisation of the structural motifs Involved in the cleavage and secretion of human angiotensin-converting enzyme |
| title_full_unstemmed | Characterisation of the structural motifs Involved in the cleavage and secretion of human angiotensin-converting enzyme |
| title_short | Characterisation of the structural motifs Involved in the cleavage and secretion of human angiotensin-converting enzyme |
| title_sort | characterisation of the structural motifs involved in the cleavage and secretion of human angiotensin converting enzyme |
| topic | Cell Biology |
| url | http://hdl.handle.net/11427/8710 |
| work_keys_str_mv | AT conradnailah characterisationofthestructuralmotifsinvolvedinthecleavageandsecretionofhumanangiotensinconvertingenzyme |