Abstract
Murine mastocytoma cells were incubated in vitro with inorganic [35S]sulfate, in the absence or presence of 2.5 mM n-butyrate, and labeled heparin was isolated. The polysaccharide produced in the presence of butyrate showed a lower charge density on anion exchange chromatography than did the control material and a 3-fold increased proportion (54 versus 17% for the control) of components with high affinity for antithrombin. Structural analysis of heparin labeled with [3H] glucosamine in the presence of butyrate showed that approximately 35% of the glucosamine units were N-acetylated, as compared to approximately 10% in the control material; the nonacetylated glucosamine residues were N-sulfated. The presence of butyrate thus leads to an inhibition of the N-deacetylation/N-sulfation process in heparin biosynthesis, along with an augmented formation of molecules with high affinity for antithrombin. Preincubation of the mastocytoma cells with butyrate was required for manifestation of either effect; when the preincubation period was reduced from 24 to 10 h the effects of butyrate were no longer observed. Assays for microsomal N-acetylheparosan deacetylase activity failed to show any significant inhibition of the enzyme at butyrate concentrations well above those found to affect heparin biosynthesis in intact mastocytoma cells. Moreover, a polysaccharide formed on incubating mastocytoma microsomal fraction with UDP-[3H]glucuronic acid, UDP-N-acetylglucosamine, and 3'-phosphoadenylylsulfate in the presence of 5 mM butyrate showed the same N-acetyl/N-sulfate ratio as did the corresponding control polysaccharide, produced in the absence of butyrate. These findings suggest that the effect of butyrate on heparin biosynthesis depends on the integrity of the cell.
Highlights
Murine mastocytoma cells were incubated in vitro ment with N-sulfate groups, followed by epimerization of a with inorganic [3SS]sulfate,in theabsence or presence large proportion of the D-glucuronic acid residues into Lof 2.5 mM n-butyrate, andlabeled heparin waissolated. iduronic acid units, and, by O-sulfation in three dif
The antithrombin-binding region has required for manifestation of either effect; when the been identified as a pentasaccharide sequence in which at preincubation period was reduced from24 to 10 h the least four differently located sulfate groups are required for effects of butyrate wereno longer observed
In the present report we show that the addition of butyrate to mouse mastocytoma cells in uitro leads to increased N-acetylation of the newly synthesized heparin, and at the same time, to an augmented formation of heparin molecules with high affinity for antithrombin
Summary
The biosynthesis of heparin occurs by formation of a nonsulfated proteoglycan intermediate, the polysaccharide chains of which are composed of alternating D-glucuronic acid and N-acetyl-D-glucosamine residues. Inorganic [35S]sulfate(carrier free) and D-[6-3H]glucosamine (20-40 Ci/mmol) were purchased from the Radiochemical Center, Amersham, Bucks, United Kingdom. ~-[l-~H]Gluco(se5Ci/ mmol) was purchased from New England Nuclear (Dreieich, West Germany) and was converted enzymatically into UDP-~-[~H]glucuronic acid as described [21]. Unlabeled UDP-N-acetylglucosamine, Pronase, and chrondroitin lyase ABCwere purchased from Sigma; DEAE-cellulose (DE52) from Whatman, Maidstone, Kent, United Kingdom; and Sephadex G-25 and Sepharose 4B-C1 from Pharmacia. Assays for microsomal N-aCetyl-D-glUCOSaminylN-deacetylase activity were performed as described [28],using a N-[3H]acetyl-labeled capsular polysaccharide from Escherichia coli K5 as substrate
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