Purification and analysis of the core protein of the protease-resistant intracellular chondroitin sulfate E proteoglycan from the interleukin 3-dependent mouse mast cell.
Chondroitin sulfate E proteoglycan was extracted in the presence of protease inhibitors from 6 X 10(9) mouse bone marrow-derived, interleukin 3-dependent mast cells, of which 3 X 10(7) had been biosynthetically labeled with [35S]sulfate or [3H]glycine. Chondroitin sulfate E proteoglycan was purified to apparent homogeneity by density-gradient centrifugation, differential molecular weight dialysis, DEAE-52 ion exchange chromatography, and Sepharose CL-4B gel filtration chromatography. Chondroitin sulfate E proteoglycan, radiolabeled with [3H]glycine or [35S]sulfate, filtered as a single peak of radioactivity on Sepharose CL-4B with a Kav of 0.41. When purified [3H]glycine-labeled proteoglycan was digested with chondroitinase ABC and subjected to gel filtration, all of the radioactivity was shifted to a lower molecular weight. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr of the peptide core obtained by chondroitinase ABC treatment was approximately 10,000. The purified proteoglycan was resistant to degradation by collagenase, clostripain, trypsin, chymotrypsin, elastase, chymopapain, V8 protease, proteinase K, and Pronase, as assessed by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the core peptide of the intact proteoglycan revealed that glycine, serine, and glutamic acid/glutamine accounted for 70% of the total amino acids and were present in a molar ratio of 4.3/1.6/1.0. When analyzed for neutral hexose content by gas-liquid chromatography, the proteoglycan contained approximately 2% of its weight as mannose, fucose, galactose, and other sugars, indicating that oligosaccharides were linked to the peptide core. The mouse bone marrow-derived mast cell chondroitin sulfate E proteoglycan, like the rat serosal mast cell heparin proteoglycan, is markedly protease resistant, has highly sulfated glycosaminoglycans, and contains a peptide core that is rich in serine and glycine. These characteristics of the mast cell class of intracellular proteoglycans may contribute to their function in stimulus-induced granule secretion as well as in mediator storage, including retention of cationic neutral proteases.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001854:Bone Marrow Cells; D002499:Centrifugation, Density Gradient; D002808:Chondroitin Lyases / Q000378:metabolism; D011508:Chondroitin Sulfate Proteoglycans / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000378:metabolism; D007377:Interleukin-3; D008222:Lymphokines / Q000494:pharmacology*; D008407:Mast Cells / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis; D010447:Peptide Hydrolases / Q000378:metabolism; D011509:Proteoglycans / Q000302:isolation & purification*; D013431:Sulfates / Q000378:metabolism
D000596:Amino Acids; D011508:Chondroitin Sulfate Proteoglycans; D007377:Interleukin-3; D008222:Lymphokines; D009844:Oligosaccharides; D011509:Proteoglycans; D013431:Sulfates; D010447:Peptide Hydrolases; D002808:Chondroitin Lyases; D005998:Glycine