Altered storage of proteases in mast cells from mice lacking heparin: a possible role for heparin in carboxypeptidase A processing.
Heparin-deficient mice, generated by gene targeting of N-deacetylase/N-sulfotransferase-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 marrow-derived mast cells cultured in the presence of IL-3 synthesise, in addition to highly sulphated chondroitin sulphate (CS), small amounts of equally highly sulphated heparin-like 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 heparin-deficient cells. Northern blot analysis revealed mRNA expression of CPA, mMCP-5 and mMCP-6 in both wild-type and NDST-2-/- cells. A approximately 36 kDa CPA band, corresponding to proteolytically processed active CPA, as well as a approximately 50 kDa pro-CPA band was present in NDST-2+/+ cells. The NDST-2-/- mast cells contained similar levels of pro-CPA as the wild-type mast cells, but the approximately 36 kDa band was totally absent. This indicates that the processing of pro-CPA 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.
Henningsson
Frida
F
Swedish University of Agricultural Sciences, Department of Veterinary Medical Chemistry, The Biomedical Center, Uppsala.
Ledin
Johan
J
Lunderius
Carolina
C
Wilén
Maria
M
Hellman
Lars
L
Pejler
Gunnar
G
D000199:Actins / Q000378:metabolism; D000581:Amidohydrolases / Q000172:deficiency / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000378:metabolism* / Q000648:ultrastructure; D002268:Carboxypeptidases / Q000032:analysis / Q000378:metabolism*; D043422:Carboxypeptidases A; D002478:Cells, Cultured; D002809:Chondroitin Sulfates / Q000494:pharmacology; D006025:Glycosaminoglycans / Q000096:biosynthesis / Q000302:isolation & purification; D006493:Heparin / Q000378:metabolism*; D007377:Interleukin-3 / Q000494:pharmacology; D008407:Mast Cells / Q000201:enzymology* / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D012313:RNA / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D015238:Sulfotransferases / Q000172:deficiency / Q000378:metabolism; D053802:Tryptases
D000199:Actins; D006025:Glycosaminoglycans; D007377:Interleukin-3; C509190:Tpsb2 protein, mouse; D012313:RNA; D006493:Heparin; D002809:Chondroitin Sulfates; C491855:Ndst2 protein, mouse; D015238:Sulfotransferases; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D012697:Serine Endopeptidases; C509222:Tpsab1 protein, mouse; D053802:Tryptases; D000581:Amidohydrolases