Abstract

Mast cells are highly granulated cells of the immune system that upon stimulation release a number of inflammatory mediators including heparin and/or chondroitin sulphate (CS) proteoglycan (PG) and various heparin-binding proteases such as tryptase, chymase and carboxypeptidase A (CPA). Mast cell CPA is a zinc-metalloexopeptidase, cleaving substrates with carboxyl-terminal aliphatic or aromatic amino acids. In this thesis, the storage and activation of CPA was investigated, using bone marrow derived mast cells (BMMCs) from mice lacking heparin, either due to loss of the gene coding for the heparin biosynthesis enzyme, NDST-2, or the gene coding for the PG core protein serglycin (SG). We found that BMMCs from NDST-2-/- mice that thus lack heparin, but produce CS, lack the chymase, mMCP-5 and mature CPA. Interestingly, the pro-form, but not the active form of CPA could be detected in the heparin-deficient cells, indicating a role for heparin in the processing of CPA. Furthermore, we have shown that the cysteine proteases, cathepsins C and S, are not involved in processing CPA, but rather that lack of cathepsin C or S cause increased levels of CPA as well as mMCP-5. In addition, neither cathepsins B nor L influence CPA processing at all, but instead, an aspartic protease, cathepsin E, plays a role in processing pro-CPA. Further, these studies led to the novel finding that cathepsin E is located inside the mast cell granules, where it is stored in complex with heparin. The activation of mast cells, which ultimately leads to degranulation, has been studied in detail; however, the process where mast cell granules are formed has not gained as much attention. We addressed this issue by the use of BMMCs from mice lacking SG. Here, we present evidence that secretory granules are formed independently of SG PG but that SG mediates selective condensation of certain granule constituents, while others are independent of SG. Mast cell proteases are correctly sorted into the granules but are subsequently degraded, exocytosed or remain unprocessed when SG is absent. These results indicate a model in which selected granule constituents are sorted into granules by SG-mediated retention.