Mouse heparin proteoglycan. Synthesis by mast cell-fibroblast monolayers during lymphocyte-dependent mast cell proliferation.

Abstract

I5Mouse mast cells in primary mouse embryonic fibroblast monolayers were induced to proliferate under the influence of mouse lymphocytes.Resulting mast cellrich cultures incorporated [35S]sulfate into intracellular proteoglycans which had an average molecular weight estimated at approximately 750,000.The [36S]proteoglycans were identified by use of selective polysaccharidases as a mixture of approximately 60 to 70% heparin and 30 to 40% chondroitin sulfate and dermatan sulfate.Incubation of the [35S]proteoglycans with chrondroitin ABC lyase to remove chondroitin sulfate and dermatan sulfate did not change the molecular size of the remaining heparin proteoglycan.This heparin proteoglycan was insensitive to pronase, papain, trypsin, and chymotrypsin.When C3H]serine was included in the culture media, 3H-labeled material was found to chromatograph in the same fractions as 35S-labeled compounds.Incubation of the [35S]proteoglycans for 14 h at 22 "C in 0.5 16 NaOH degraded the radiolabeled proteoglycan to their [36S]glyc~samin~glycan side chains which had average molecular weights of approximately 40,000 to 60,000.DEAE-Sephacel chromatography of the [35S]glycosaminoglycans revealed two separate peaks of activity.The first peak appeared before an internal commercial heparin marker and was degradable by chondroitin ABC lyase, while the second peak appeared after the heparin marker and was resistant to chondroitin ABC lyase, consistent with its identity as mouse heparin glycosaminoglycan.Thus, mouse mast cell-rich fibroblast monolayers synthesized a protease-resistant heparin proteoglycan similar to heparin proteoglycan previously isolated from rat skin and rat peritoneal mast cells.These findings indicate that protease-resistant heparin proteoglycan occurs in nature more widely than previously appreciated, and is not limited to rat heparin.Heparin from most sources, including beef lung, human lung, porcine intestinal mucosa, and mouse mastocytoma has been isolated as single glycosaminoglycan chains.These chains may be products of an endoglycosidase which has been