Abstract
Carboxypeptidase M (CPM) is a zinc-dependent phospho-inositol-anchored protease that cleaves carboxy-terminal basic residues such as arginine or lysine from peptides. CPM is primarily membrane-bound, glycosylated, has a neutral pH optimum, and occurs in placental microvilli, seminal plasma, amniotic fluid, peripheral nerves, alveolar epithelial cells and macrophages. In this work, we examined whether CPM is expressed in various cells in the bone marrow (BM) including hematopoietic stem/progenitor cells (HSPC) and whether it plays a role in the stromal cell-derived factor (SDF)-1α-directed mobilization of HSPC from the BM) to peripheral blood (PB). SDF-1α produced by BM stromal cells retains HSPC in the BM and its proteolytic degradation results in mobilization. When exposed to serum, full-length SDF-1α (1–68) undergoes rapid cleavage of the C-terminal lysine yielding SDF-1α (1–67) which is then cleaved at the N-terminus by matrix metalloproteinases, CD26 and serine proteases or elastases, generating a truncated form of SDF-1α (3–67) with reduced chemoattractant activity. In this work, we present the first evidence that CPM can cleave the C-terminal lysine and furthermore reduces the ability of SDF-1α (1–67) to chemoattract HSPC. We found that CPM (i) is expressed by BM CD34+ cells strongly and weakly by PB CD34+ cells, mononuclear cells, neutrophils, mesenchymal stem cells and leukemic cell lines (THP-1 monocytic, KG-1 acute myeloid) by RT-PCR and flow cytometry; (ii) occurs on the cell surface of these cells and co-localizes with the SDF-1α receptor CXCR4 (by confocal microscopy); and (iii) is present on myeloid and megakaryocytic precursor cells, but not erythroid cells. G-CSF, the most commonly used agent for mobilization, slightly increased (1.2-fold) the expression of CPM in CD34+ cells at the gene level. Moreover, because in vivo SDF-1α (i.e., in serum) already lacks the C-terminal lysine, we used biologically active synthetic SDF-1α (1-67), and after treatment with CPM observed a significantly reduced chemoattraction for CD34+ cells. Furthermore, prolonged exposure of SDF-1α (over 24 h) to CPM completely obliterated its chemotactic activity but pre-incubating CPM with the peptidase inhibitor (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid) restored it. Because CPM is localized on the plasma membrane, it is ideally situated to modulate the activity of this chemokine. As it has been suggested that the C-terminal lysine of SDF-1α binds with heparin on the cell surface, preserving the activity of SDF-1α, we propose that CPM cleavage of lysine could release the SDF-1α from the cell surface and expose it to further proteolytic degradation, resulting in the mobilization of HSPC to the circulation. In conclusion, we present the first evidence that CPM cleaves the C-terminal lysine residue of SDF-1α, and that it is expressed by various cells in the BM microenvironment, which may facilitate HSPC mobilization; however, understanding the full biological functions of this enzyme requires further investigation.
Carboxypeptidase M Expressed by Human Bone Marrow Cells Cleaves the C-Terminal Lysine of Stromal Cell-Derived Factor-1α: Another Player in Hematopoietic Stem/Progenitor Cell Mobilization?