Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a procedure for the use of minislab gels to electrophoretically separate proteoglycans (PGs), large macromolecules with molecular masses greater than 2.5 million Da. Our procedure is a modification of the method of C.A. McDevitt and H. Muir (Anal. Biochem. 44, 612-622, 1971) for agarose/polyacrylamide, composite tube gels. These 1% agarose/1.2% acrylamide minigels are run at 35 mA for 75 min; bands are visualized by toluidine blue staining. The subtle size differences between the large aggregating PGs isolated from rat chondrosarcoma, bovine nasal septal cartilage, and adult bovine articular cartilage (which consists of two subpopulations) can be distinguished by their migration on these large pore gels. Chondroitin sulfate chains, added to all wells as a marker of constant mobility, ran immediately behind the dye front. The distance of migration into the gel of PGs incubated overnight with cathepsin B, carboxypeptidase A, papain, plasmin, elastase, or cathepsin G varied with the size of the cleavage products. We propose the use of this procedure for a convenient assessment of cartilage PGs and a rapid, reproducible assay for proteoglycanase activity.
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PMID:Agarose/polyacrylamide minislab gel electrophoresis of intact cartilage proteoglycans and their proteolytic degradation products. 178 94

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.
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PMID:Purification and analysis of the core protein of the protease-resistant intracellular chondroitin sulfate E proteoglycan from the interleukin 3-dependent mouse mast cell. 393 50

1. Mast cells derive from the bone marrow and are responsible for the development of allergic and possibly inflammatory reactions. Mast cells are stimulated by immunoglobulin E (IgE) and specific antigen, but also by a number of neuropeptides such as neurotensin (NT), somatostatin or substance P (SP), to secrete numerous pro-inflammatory molecules that include histamine, cytokines and proteolytic enzymes. 2. Chondroitin sulphate, a major constituent of connective tissues and of mast cell secretory granules, had a dose-dependent inhibitory effect on rat peritoneal mast cell release of histamine induced by the mast cell secretagogue compound 48/80 (48/80). This inhibition was stronger than that of the clinically available mast cell 'stabilizer' disodium cromoglycate (cromolyn). Inhibition by chondroitin sulphate increased with the length of preincubation and persisted after the drug was washed off, while the effect of cromolyn was limited by rapid tachyphylaxis. 3. Immunologic stimulation of histamine secretion from rat connective tissue mast cells (CTMC) was also inhibited, but this effect was weaker in umbilical cord-derived human mast cells and was absent in rat basophilic leukemia (RBL) cells which are considered homologous to mucosal mast cells (MMC). Oligo- and monosaccharides were not as effective as the polysaccharides. 4. Inhibition, documented by light and electron microscopy, involved a decrease of intracellular calcium ion levels shown by confocal microscopy and image analysis. Autoradiography at the ultrastructural level showed that chondroitin sulphate was mostly associated with plasma and perigranular membranes. 5. Chondroitin sulphate appears to be a potent mast cell inhibitor of allergic and nonimmune stimulation with potential clinical implications.
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PMID:Chondroitin sulphate inhibits connective tissue mast cells. 1108 9

Chondroitin sulfate E (CS-E), a chondroitin sulfate isomer containing GlcAbeta1-3GalNAc(4,6-SO(4)) repeating unit, was found in various mammalian cells in addition to squid cartilage and is predicted to have several physiological functions in various mammalian systems such as mast cell maturation, regulation of procoagulant activity of monocytes, and binding to midkine or chemokines. To clarify the physiological functions of GalNAc(4,6-SO(4)) repeating unit, preparation of CS-E with a defined content of GalNAc(4,6-SO(4)) residues is important. We report here the in vitro synthesis of CS-E from chondrotin sulfate A (CS-A) by the purified squid N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) which catalyzed transfer of sulfate from 3(')-phosphoadenosine-5(')-phosphosulfate to position 6 of GalNAc(4SO(4)) residues of CS-A and dermatan sulfate (DS). When CS-A was used as an acceptor, about half of GalNAc(4SO(4)) residues, on average, were converted to GalNAc(4,6-SO(4)) residues. Anion exchange chromatography of the CS-E synthesized in vitro showed marked heterogeneity in negative charge; the proportion of GalNAc(4,6-SO(4)) in the most negative fraction exceeded 70% of the total sulfated repeating units. GalNAc4S-6ST also catalyzed the synthesis of oversulfated DS with GalNAc(4,6-SO(4)) residues from DS. Squid GalNAc4S-6ST thus should provide a useful tool for preparing CS-E and oversulfated DS with a defined proportion of GalNAc(4,6-SO(4)) residues.
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PMID:Enzymatic synthesis of chondroitin sulfate E by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid cartilage. 1242 30

Chondroitin sulfate (CS) and dermatan sulfate (DS) containing N-acetylgalactosamine 4,6-bissulfate (GalNAc(4,6-SO(4))) show various physiological activities through interacting with numerous functional proteins. N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate in CS or DS to yield GalNAc(4,6-SO(4)) residues. We here report generation of transgenic mice that lack GalNAc4S-6ST. GalNAc4S-6ST-null mice were born normally and fertile. In GalNAc4S-6ST-null mice, GalNAc(4,6-SO(4)) residues in CS and DS disappeared completely, indicating that GalNAc4S-6ST should be a sole enzyme responsible for the synthesis of GalNAc(4,6-SO(4)) residues in both CS and DS. IdoA-GalNAc(4,6-SO(4)) units that account for approximately 40% of total disaccharide units of DS in the liver of the wild-type mice disappeared in the liver DS of GalNAc4S-6ST-null mice without reduction of IdoA content. Bone marrow-derived mast cells (BMMCs) derived from GalNAc4S-6ST-null mice contained CS without GlcA-GalNAc(4,6-SO(4)) units. Tryptase and carboxypeptidase A activities of BMMCs derived from GalNAc4S-6ST-null mice were lower than those activities of BMMCs derived from wild-type mice, although mRNA expression of these mast cell proteases was not altered. Disaccharide compositions of heparan sulfate/heparin contained in the mast cells derived from BMMCs in the presence of stem cell factor were much different from those of heparan sulfate/heparin in BMMCs but did not differ significantly between wild-type mice and GalNAc4S-6ST-null mice. These observations suggest that CS containing GalNAc(4,6-SO(4)) residues in BMMCs may contribute to retain the active proteases in the granules of BMMCs but not for the maturation of BMMCs into connective tissue-type mast cells.
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PMID:Mice deficient in N-acetylgalactosamine 4-sulfate 6-o-sulfotransferase are unable to synthesize chondroitin/dermatan sulfate containing N-acetylgalactosamine 4,6-bissulfate residues and exhibit decreased protease activity in bone marrow-derived mast cells. 2043 88