Carboxypeptidase M (CPM) is a cell-surface bound carboxypeptidase which removes C-terminal basic aminoacids from bioactive peptides. CPM has been reported to be distributed in many organs and fluids. CPM is also present on immunological and inflammatory cells. However, a physiological role of CPM in these tissues has not yet been clearly defined. Commonly, it is accepted that CPM can be a player in the processing or inactivation of peptide hormones, growth factors, inflammatory important peptides, et cetera.
As there is very little knowledge of the molecular properties of CPM, we aim to systematically characterise the catalytic mechanisme and substrate specificity of CPM. These investigations must allow to differentiate between the various carboxypeptidases, specifically concerning the substrate specificity. Carboxypeptidase U (CPU) inhibitors are currently under clinical investigation as adjuvans in the fibrinolytic therapy. The importance of studying the selectivity of the CPU inhibitors with regard to other carboxypeptidases is self-evident. In this context, CPM is particulary important because it is constitutionally expressed in many tissues.
In the first phase of our project, CPM will be cloned, expressed and purified. A comparative study of the recombinant and natural enzyme will be performed. We wish to study the different elements of the catalytic proces using enzymological methods. The substrate specificity of CPM will be studied using site-directed mutagenesis.
In the second phase, we aim to identify naturally occuring substrates of CPM in tissue extracts (proteomics analysis). Extracted polypeptides will be fractionated and analysed for the presence of substrate (LC-MS). The enzyme kinetics of these substrates will be studied in vitro.
A last aim is to set up a database of CPM values in normal and clinical samples.
The results of our study can be used in the development of new CPU inhibitors. New therapeutic strategies can be proposed if the involvement of CPM in inflammation is confirmed. The possibility of administering therpeutic peptides as aerosol is under research. The presence of CPM on type I alveolar cells can influence the absorption and pharmacokinetics of these drugs.