The characterisation of ectodomain shedding of angiotensin-converting enzyme 2 (ACE2)
2017-05-26T07:52:08Z (GMT) by
Angiotensin-converting enzyme 2 (ACE2) is the only mammalian homologue of the well-characterised angiotensin-I converting enzyme (ACE). ACE2 shares 40% identity and 60% similarity in overall protein sequence with ACE. Like ACE, ACE2 is a type I integral membrane protein and a zinc dependent metalloprotease. ACE2 has recently been shown to undergo a proteolytic cleavage event, releasing an active soluble ectodomain. This cleavage-event is also commonly known as ectodomain shedding. The studies outlined in this thesis were designed to characterise the regulation of ACE2 ectodomain shedding, specifically looking at the interaction of this carboxypeptidase with calmodulin, potential site of cleavage mediated by tumour necrosis factor-α converting enzyme (also known as TACE or ADAM17), as well as the mechanism(s) responsible for the activation of this cleavage event. Calmodulin gel shift assays have showed that this calcium regulatory protein is able to bind to both peptides that mimic the cytoplasmic tail of ACE2. Furthermore, we showed that GST-calmodulin fusion proteins were successfully co-immunoprecipitated along with full-length ACE2, in vitro. By using increasing concentrations of the calmodulin specific inhibitor, trifluoroperazine and calmidazolium, we have observed increase in the shedding activity of ACE2 endogenously expressed in Huh-7 cells (n≥3, one-way ANOVA, P < 0.05). We also provided evidence that calmodulin-induced shedding of ACE2 in the Huh-7 cells were non-responsive to protein kinase C inhibitor, suggesting that the activation pathway for this shedding mechanism differs from that mediated by phorbol esters. To characterise the cleavage-secretion site for ACE2 ectodomain release, peptide mimetics of ACE2 juxtamembrane region were incubated with purified recombinant TACE for analysis by MALDI-TOF/TOF. Peptide fragment analysis showed that ADAM17 cleaves peptide mimetics between Arg708 – Ser709 bond. Shedding profiles of an ACE2 mutant in CHO-K1 and Huh-7 cells were carried out, indicating that Arg708 → Glu708 and double mutant Arg708/Arg710 → Glu708/Glu710 displayed increased shedding activity when stimulated by phorbol ester. Lastly, to explore the possibility that the phorbol ester (PMA) stimulated shedding could be a consequence of a G-protein coupled receptor activation, Huh-7 cells and HEK 293 cells expressing the AT1R receptor were transfected with ACE2 and treated with angiotensin peptides (angiotensin I and angiotensin II) in the presence of different angiotensin receptor antagonists. At various time points of angiotensin treatment, significant increases were observed in ACE2 shedding in cells treated with either angiotensin I or angiotensin II peptides. However when Huh-7 cells were incubated with the metabolically stable AT1 receptor agonist, L-162,313, we observed significant changes in cell morphology, suggesting the likeliness of cell death, and were also reflected in the gradual decrease in ACE2 activity detected in the medium. In summary these studies have provided strong evidence suggesting the involvement of calmodulin in regulating the ectodomain shedding of ACE2, an event which is independent of protein kinase C signaling pathway. We also propose that ADAM17 is able to cleave ACE2 between Arg708 – Ser709, and that AT1R receptor activation may lead to stimulation of ectodomain shedding in ACE2. Given that ACE2 has recently been shown to be the functional receptor for the severe-acute respiratory syndrome (SARS) coronavirus (SARS-CoV) and also its implications in cardiovascular, renal and respiratory regulations, these studies add to our knowledge of how the ectodomain shedding of this important membrane protein may be actually regulated.