Altered storage of proteases in mast cells from mice lacking heparin:: A possible role for heparin carboxypeptidase A processing

Heparindeficient mice, generated by gene targeting of Ndeacetylase/Nsulfotransferase-2 (NDST-2), display severe mast cell defects, including an absence of stored mast cell proteases. However, the mechanism behind these observations is not clear. Here we show that NDST-2+/+ bone marrowderived mast cells cultured in the presence of IL-3 synthesise, in addition to highly sulphated chondroitin sulphate (CS), small amounts of equally highly sulphated heparinlike polysaccharide. The corresponding NDST-2/ cells produced highly sulphated CS only. Carboxypeptidase A (CPA) activity was detected in NDST+/+ cells but was almost absent in the NDST/ cells, whereas tryptase (mouse mast cell protease 6; mMCP-6) activity and antigen was detected in both cell types. Antigen for the chymase mMCP-5 was detected in NDST-2+/+ cells but not in the heparindeficient cells. Northern blot analysis revealed mRNA expression of CPA, mMCP-5 and mMCP-6 in both wildtype and NDST-2/ cells. A similar to36 kDa CPA band, corresponding to proteolytically processed active CPA, as well as a similar to50 kDa proCPA band was present in NDST-2+/+ cells. The NDST-2/ mast cells contained similar levels of proCPA as the wildtype mast cells, but the similar to36 kDa band was totally absent. This indicates that the processing of proCPA to its active form may require the presence of heparin and provides the first insight into a mechanism by which the absence of heparin may cause disturbed secretory granule organisation in mast cells.