Abstract
We have previously generated a mouse strain with a defect in its heparin biosynthesis by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2). The NDST-2(-/-) mice show reduced levels of various mast cell mediators such as histamine and various heparin-binding mast cell proteases, including chymases, tryptases, and carboxypeptidase A. In this work we have addressed the possible functional consequences of the lack of sulfated heparin. Peritoneal cells were harvested from normal and NDST-2(-/-) mice. After culturing the cells, conditioned media were collected and were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Several differences in the protein patterns were observed, including the presence of large amounts of a approximately 250-kDa protein in medium from NDST-2(-/-) mice that was absent in normal controls. Peptide microsequencing revealed identity of this protein with fibronectin. Western blot analysis showed the presence of fibronectin degradation products in cell cultures from normal mice, which were absent in cultures from NDST-2(-/-) animals. Further experiments showed that the degradation of fibronectin observed in cell cultures from NDST-2(+/+) mice was catalyzed by mast cell chymase in a strongly heparin-dependent manner. This report thus indicates a biological function for chymase/heparin proteoglycan complexes in fibronectin turnover.
Highlights
We have previously generated a mouse strain with a defect in its heparin biosynthesis by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2)
Further experiments showed that the degradation of fibronectin observed in cell cultures from NDST-2؉/؉ mice was catalyzed by mast cell chymase in a strongly heparin-dependent manner
Rat chymase and heparin proteoglycan were purified from peritoneal mast cells as described previously [17]. ␣1Antichymotrypsin and ␣1-antitrypsin were purchased from Calbiochem (La Jolla, CA), and donkey anti-rabbit Ig conjugated to horseradish peroxidase was from Amersham Pharmacia Biotech
Summary
We have previously generated a mouse strain with a defect in its heparin biosynthesis by targeting the gene for N-deacetylase/N-sulfotransferase-2 (NDST-2). The NDST-2؊/؊ mice show reduced levels of various mast cell mediators such as histamine and various heparin-binding mast cell proteases, including chymases, tryptases, and carboxypeptidase A. Western blot analysis showed the presence of fibronectin degradation products in cell cultures from normal mice, which were absent in cultures from NDST-2؊/؊ animals. Further experiments showed that the degradation of fibronectin observed in cell cultures from NDST-2؉/؉ mice was catalyzed by mast cell chymase in a strongly heparin-dependent manner. The connective tissue type of mast cells express the tryptases mouse mast cell protease 6 (mMCP-6) and mMCP-7 as well as the chymases mMCP-4 and mMCP-5 [7,8,9,10,11,12] Both the chymases and tryptases bind strongly to heparin proteoglycan within the secretory granules.
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