Characterisation of Cystinyl aminopeptidase using newly developed angiotensine IV analogues.

Animal studies revealed that the hexapeptide angiotensin IV (Ang IV) improves memory and learning. High affinity Ang IV binding sites have recently been identified as the crystinyl aminopeptidase enzyme, a membrane-associated zinc-dependent exopeptidase of the M1 family. Its major substrates, oxytocin and vasopressin, are well known for their positive role in cognition. It has therefore been proposed that the physiological effects of Ang IV are related with its ability to inhibit the activity CAP. The large extracellular domain of CAP can be cleaved off proteolytically and the serum level of the resulting soluble enzyme increases with gestation. By decreasing the serum levels of oxytocin and vasopressin, this soluble enzyme may contribute to the maintenance of normal pregnancy. The same enzyme (but now denominated as insulin regulated aminopeptidase, IRAP) is also present in intracellular endosome-derived vesicles along with GLUT4, the major insulin-regulated trafficking to the cell membrane in type II diabetic patients.
Ang IV had been shown to trigger signalling pathways in several isolated cell systems and this raises questions about the underlying molecular mechanisms. In this respect, high affinity Ang IV binding has repeatedly been advanced to prove the implication of CAP in the biological activity of ANG IV. Recent research in the dept. MBFA revealed that binding of Ang IV to CAP only takes place in the presence of chelating agents and, hence, that binding occurs to the apo-enzyme. Nevertheless, ang IV and LVV-H7 (an Ang IV-related peptide which is present in the CNS) are able to inhibit the catalytic activity of CAP, albeit with lower potency than in binding experiments. This is partly due to the ability of CAP to degrade both natural peptides. Recently we demonstrated that Ang IV also inhibits the activity of aminopeptidase-N. In contrast to CAP, the AP-N apo-enzyme does not display high afiinity for Ang IV.
These intriguing findings prompt us to evaluate potential implication of CAP, AP-N (and other potential cellular targets) in Ang IV-mediated cell signalling. In addition, the radioligandbinding technique may offer an improved detection and quantification of CAP. Hence, we also wish to further develop/adapt this technique to explore the potential use of CAP as a bio-marker during pregnancy and type 2 diabetes. The development of stable Ang IV and LVV-H7 analogs and their fluorescent and/or radioactive labelling is essential for these approaches and it may also generate new tools in this research domain. Collaboration between the dept. MBFA and DSCH at the VUB will provide the necessary synergism to pursue such exploratory research to a good end.