Synthesis of chondroitin sulfate D and heparin proteoglycans in murine lymph node-derived mast cells: The dependence on fibroblasts

Proteoglycans synthesized in cultured mast cells derived from horse serum-immunized lymph node cells were analyzed. Treatment of the ³⁵S-proteoglycans extracted from these cells with either chondroitinase ABC or AC resulted in 95% ± 7% and 84% ± 7%, respectively (mean ± S.E., n = 3), of the radioactivity associated with disaccharides eluting in the included volume of PD-10. The ³⁵S-proteoglycans were not hydrolyzed by nitrous acid elimination treatment. The chondroitinase ABC-generated disaccharides were analyzed by aminocyano high performance liquid chromatography. ³⁵S-Disaccharides eluted in a major peak at a retention time of 8.1 min, corresponding to the disaccharide of chondroitin 4-sulfate proteoglycan (Δ Di-4S), and a second peak at 12 min, corresponding to the disaccharide of chondroitin sulfate D proteoglycan (ΔDi-diS_D.) Further treatment with chondro-4-sulfatase did not affect the retention time of the disaccharide corresponding to ΔDi-diS_D whereas this peak disappeared after the digested proteoglycan was treated either by chondro-6-sulfatase or by both sulfatases. Therefore, this disaccharide was identified as chondroitin sulfate D. Quantification of the radiolabeled disaccharides showed that ΔDi-diS_D contributed 20% ± 2% (n = 3) of the total sulfated disaccharides of the chondroitin sulfate of these cultured cells. The role of fibroblasts in inducing the shift of chondroitin sulfate D into heparin proteoglycan in these mast cells was also investigated by using three types of monolayers: mouse embryonic skin fibroblasts (MESF), rat embryonic skin fibroblasts (RESF), and 3T3 fibroblasts. ³⁵S-Proteoglycans that were extracted from the lymph node-derived mast cells cultured for 30 days on MESF and on 3T3 fibroblast monolayers were 93% ± 4% and 30% ± 7% (n = 3) susceptible to nitrous acid elimination, respectively. No degradation by nitrous acid was observed in ³⁵S-proteoglycans extracted from cells cultured on RESF monolayer. Since the MESF was found to be the most potent monolayer in the induction of heparin synthesis, the kinetics of changes in the synthesis of proteoglycan types were determined in lymph node-derived mast cells cultured on MESF for up to 30 days. It was found that the synthesis of chondroitin sulfate gradually declined whereas that of heparin starting between 4 and 7 days after plating gradually increased. From the 17th day on, only the synthesis of heparin was detected.