Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.6.4 (chondroitinase)
2,039 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The synthesis and secretion of mucin-like high-molecular glycoprotein was studied in 2 human colon cancer cell lines that spontaneously differentiate in culture (Caco-2 and T84) and in 2 cell lines that do not spontaneously differentiate (LS174T and HT29). Mucin, quantitated by 3H-glucosamine labelling and chromatography on Sepharose CL-4B was found to be produced by all 4 cell lines. The mucinous nature of the labelled high-molecular glycoprotein was verified by enzymatic degradation treatments (heparinase, hyaluronidase, chondroitinase ABC, and N-glycanase), alkaline-borohydride treatment, inhibition of labelling by the glycosylation inhibitor benzyl-alpha-GalNAc, and by CsCl-density-gradient centrifugation. In all 4 cell lines, an inverse correlation of mucin synthesis with cell density was demonstrated. In Caco-2 cells, the spontaneous post-confluent enterocytic differentiation with increased brush-border enzyme expression was associated with a decrease in mucin synthesis and in the activities of polypeptidyl GalNAc transferase and beta 1,3-galactosyltransferase activity. Using cDNA probes for 2 distinct human intestinal mucins (MUC2 and MUC3), we found that all 4 colon cancer cell lines expressed mucin message, but the types of mucin mRNA expressed differed. These data indicate that mucin-like glycoproteins can be synthesized by cell lines derived from non-mucinous colon cancer, whether or not they undergo spontaneous differentiation in culture. These cell lines may serve as in vitro models for studying apomucin heterogeneity and control of mucin gene expression.
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PMID:Mucin synthesis and secretion in relation to spontaneous differentiation of colon cancer cells in vitro. 172 5

Small proteoglycans were dissociatively extracted from normal human thoracic aorta with 4 M guanidine hydrochloride containing protease inhibitors, and purified by Sepharose CL-4B chromatography, dissociative cesium chloride density gradient centrifugation, and diethylaminoethyl cellulose chromatography. The intact proteoglycans migrated in the 270,000-340,000 range on 4-20% sodium dodecyl sulfate polyacrylamide gradient gels. Core proteins prepared following digestion of the intact proteoglycan monomer with chondroitinase ABC consisted of a major Coomassie blue-staining protein band of 50,000 along with a minor band of 44,000. Subsequent studies using endoglycosidases H, F, and N-glycanase demonstrated that mainly complex type N-linked glycans were present on the 50,000 cores while the 44,000 cores appeared to be devoid of N-linked glycans. Western blotting demonstrated that both of these cores were recognized by the monoclonal antibody 2-B-6, indicating the presence of the terminal 4-sulfated unsaturated disaccharide (delta Di-4S) remaining on the linkage region following chondroitinase ABC digestion. In contrast, a diffuse pattern of delta Di-4S epitopes ranging from 50,000 to approximately 60,000 was observed following chondroitinase AC II digestion of the dermatan sulfate proteoglycan, suggesting the presence of iduronate residues in close proximity to the glycosaminoglycan-linkage region. Conversely, the large chondroitin sulfate-proteoglycan core proteins from aorta (Mr 200,000-400,000) did not react with either monoclonal antibody 3-B-3 (recognizing the terminal delta DI-6S) or 2-B-6 following chondroitinase AC II digestion, although both delta DI-4S and delta DI-6S were present on these cores following chondroitinase ABC digestion. Additional studies using antisera against synthetic peptides derived from sequences of the core proteins of human bone small PG I and PG II indicated the presence of both gene products in PG isolated from human thoracic aorta. The Mr approximately 44,000 and 50,000 core proteins represent small PG I type cores while a closely spaced doublet (Mr 49,000 and 51,000) represented small PG II type cores. The results demonstrate that the core proteins of dermatan sulfate proteoglycan from human aorta are heterogeneous in primary structure and in the content of N-linked glycans.
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PMID:Heterogeneity in glycosylation of dermatan sulfate proteoglycan core proteins isolated from human aorta. 212 40

The low molecular weight proteoglycan fraction extracted from articular discs with 4 M guanidinium chloride was found to consist predominantly of an iduronate-rich dermatan sulphate proteoglycan, together with chondroitin sulphate-containing material. The dermatan sulphate proteoglycan was purified by ion-exchange and gel-filtration chromatography and its core protein isolated after digestion with chondroitinase ABC. The amino acid composition and pattern of cyanogen bromide peptides obtained from this core were closely similar to those of the protein core of bovine skin proteodermatan sulphate. Four monoclonal antibodies raised against bovine skin proteodermatan sulphate also reacted with the disc protein core and its cyanogen bromide peptides. Results of digestion with glycopeptidase F demonstrated the presence of three N-linked oligosaccharides. The combined size of these oligosaccharides appeared to be somewhat less than the size of those on skin proteodermatan sulphate. The glycosaminoglycan chain released by digestion with cathepsin C had a higher molecular weight than that from skin. These differences in glycosylated structures may be responsible for the different effects on collagen fibrillogenesis in vitro; whereas skin proteodermatan sulphate only reduced the rate of fibril growth, disc dermatan sulphate proteoglycan also increased the length of the lag-phase and the final opacity.
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PMID:Proteoglycans of the articular disc of the bovine temporomandibular joint. II. Low molecular weight dermatan sulphate proteoglycan. 279 47

Dermatan sulphate proteoglycans (DSPGs) synthesized in the presence of 35SO4 were characterized in culture media of fibroblast lines obtained from skin, synovium, and gingiva. The molecular mass of DSPG varied from 95-130 kDa as estimated by SDS/polyacrylamide-gel electrophoresis. Gingival fibroblasts constantly produced larger DSPGs than skin fibroblasts. This was due to the larger dermatan sulphate (DS) chains, which also showed tissue-related heterogeneity in the distribution of 4- and 6-sulphated disaccharide units. The N-glycosylated cores (44 and 47 kDa) obtained following chondroitinase ABC treatment were of identical size in all tissues. The cores from the different tissues were also of the same size (38 kDa) when addition of the N-linked oligosaccharides was inhibited by tunicamycin or when they were removed by N-glycanase treatment. No evidence for low-molecular-mass sulphated oligosaccharides was found. All tissues contained two mRNA species (1.6 and 1.9 kb) for the DSPG core protein. These data suggest that the pattern of transferase activities involved in the construction of DS chains differs from one tissue to another. This variation may modulate the functions of DSPG in the extracellular matrix.
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PMID:The small dermatan sulphate proteoglycans synthesized by fibroblasts derived from skin, synovium and gingiva show tissue-related heterogeneity. 322 8

Chick-embryo cartilage contains a unique set of proteoglycans. Type H proteoglycan (PG-H) is the most abundant, constituting over 90% of the total cartilage hexuronate. We previously showed that treatment of PG-H with chondroitinase ACII and keratanase yields a protein-enriched core molecule [PG(-CS,KS)] with enzymically modified linkage oligosaccharides of the chondroitin sulphate and keratan sulphate chains. We report here that further treatment of PG(-CS,KS) with pepsin and N-oligosaccharide glycopeptidase (almond glycopeptidase) released four distinct types of mannose-containing oligosaccharide. Two of them were shown to be: (Formula: see text). Of the mannose-containing glycopeptides formed by pepsin digestion, about 40% (as mannose) were resistant to N-oligosaccharide glycopeptidase. Since the resistant fraction was enriched in keratan sulphate remnants, it is suggest that the mannose-containing oligosaccharides in this fraction represent those located in a keratan sulphate-enriched region of PG-H.
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PMID:The core molecule from type H proteoglycan. Release of mannose-containing oligosaccharides by digestion with N-oligosaccharide glycopeptidase. 405 10

A large brain-specific chondroitin sulfate proteoglycan, identified with monoclonal antibody 6B4 (6B4 proteoglycan/phosphacan), was isolated from rat brain. Soluble proteoglycans in the phosphate-buffered saline extract from 20-day-old rat whole brain were fractionated by anion exchange chromatography and CsCl density gradient centrifugation. 6B4 proteoglycan was further purified by gel filtration and additional ion exchange chromatography. The molecular mass of 6B4 proteoglycan shifted from 800 to 300 x 10(3) mol. wt after chondroitinase ABC digestion. The core protein was substituted with chondroitin sulfate chains with an average molecular weight of 21,000, keratan sulfate and HNK-1 carbohydrates. Glycosidase digestion of 6B4 proteoglycan with O-glycanase, N-glycanase, endo-beta-galactosidase, or keratanase did not remove the HNK-1 epitopes. The expression of 6B4 proteoglycan was developmentally regulated in the rat cerebral cortex; appearing first at embryonic day 14, peaking at postnatal day 0, and persisting throughout adulthood at a lower level. Immunohistochemical analysis indicated that 6B4 proteoglycan was distributed along the radial glial fibers and on the migrating neurons in the embryonal rar cerebrum. The radial glial fibers were stained intensely all along their length, but the neurons in the cortical plate were not stained in contrast to the moderate staining of the migrating neurons in the intermediate zone and the subplate. From postnatal day 5 to postnatal day 20, 6B4 proteoglycan was present throughout the cortex. After postnatal day 30, staining of the neuropil was weakened, and the expression of 6B4 proteoglycan was restricted around subsets of neurons. The positive neurons were mostly non-pyramidal cells (> 95%) and were relatively concentrated in layers IV and VI of the primary somatosensory cortex. Immunohistochemical analysis of the dissociated cortical neurons indicated that 6B4 proteoglycan was distributed on the cell bodies and neurites. 6B4 proteoglycan strikingly promoted neurite extension of cortical neurons from embryonic day-16 rat embryos when coated on coverslips as a substrate. 6B4 proteoglycan is a brain-specific chondroitin sulfate proteoglycan which carries keratan sulfate and HNK-1 carbohydrates. The spatiotemporal expression profile and effects on the dissociated cerebral neurons suggest that 6B4 proteoglycan plays important roles in the migration and differentiation of neurons in the immature cortex, and also in the maintenance of subsets of neurons in the mature cortex.
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PMID:Purification, characterization and developmental expression of a brain-specific chondroitin sulfate proteoglycan, 6B4 proteoglycan/phosphacan. 747 3

Immunological and biochemical characteristics of a 100,000 MW biglycan-like haemopoietic factor, purified from thymic myoid cells 871207B, were studied to distinguish them from macrophage colony-stimulating factor (M-CSF), which they resemble in activity and biochemical properties. Rabbit antibody raised against a synthetic peptide fragment (J-1) designed from amino acid sequences specific to the 100,000 MW factor responded to 871207B cells, the conditioned medium of 871207B, and capillary-like structures in the thymus, but not to M-CSF producer L-929 cells or the conditioned medium of L-929 cells. In contrast, M-CSF epitope was detected in L-929 cells and the conditioned medium cells but not in 871207B cells or the conditioned medium, even after enzymatic digestion of glycosaminoglycan chains. Treatment of the 100,000 MW factor with chondroitinase ABC and AC produced a 50,000 MW component. Digestion of this product with N-glycanase resulted in a 40,000 MW protein component. These results suggest that the 100,000 MW factor is a proteoglycan consisting of a core protein with an apparent molecular mass of 40,000 MW, a 50,000 MW chondroitin sulphate chain and 10,000 MW N-linked oligosaccharide chains. A small amount of a 40,000 MW monocytic cell growth activity was also found in the 871207B cell-conditioned medium. An enzymatically obtained 40,000 MW factor, the conditioned medium 40,000 MW factor, and the 100,000 MW factor were specifically eluated from an anti-J-1 IgG-immobilized affinity column with monocytic cell growth activity, suggesting that the biological activity resides in the 40,000 MW core protein. The 100,000 MW factor induced the proliferation and differentiation of monocytic lineage cells from a variety of sources, such as bone marrow cells, peritoneal exudated cells and brain microglia cells.
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PMID:Immunological and biochemical characterization of biglycan-like haemopoietic factor. 754 45

The deduced amino acid sequence of an estrogen-dependent sheep oviductal glycoprotein (M(r) 90,000-116,000) revealed the presence of several potential sites for glycan substitution on a protein backbone of M(r) approximately 66,500, and identity with chitinases. In order to further define the nature of the secreted glycoprotein, the objectives of the present study were 1) to devise a method to significantly enrich for the glycoprotein from oviductal secretions, 2) to biochemically characterize the glycoprotein by use of lectin blotting and enzymatic and chemical digestion, and 3) to determine whether unfractionated and enriched fractions containing the glycoprotein have chitinase activity. Oviducts were obtained from ovariectomized ewes treated with estradiol for 6 days and explant-cultured for 24 h. The oviductal glycoprotein was enriched approximately 80-85% from explant culture media by Maackia amurensis agglutinin (MAA) lectin affinity chromatography. Enriched fractions containing the oviductal protein were separated on SDS gels, transferred to polyvinyl difluoride, and probed with digoxigenin-labeled lectins. Lectin blotting revealed that the glycoprotein contained the carbohydrate moieties N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, and sialic acid both in alpha(2,3) and alpha(2,6) linkages, typical of sialomucins. Enzymatic digestion with neuraminidase and N-glycanase indicated that approximately 20% and approximately 6% of the molecular weight of the oviductal glycoprotein can be accounted for by sialic acid and N-linked glycans, respectively. The oviductal glycoprotein was resistant to digestion with O-glycanase alone and chondroitinase ABC, with the latter indicating that it was not a proteoglycan. Treatment with trifluoromethanesulfonic acid resulted in a deglycosylated product of M(r) approximately 66,000 immunoreactive with antibodies to the oviductal glycoprotein. No chitinase activity could be detected for unfractionated culture medium proteins or enriched fractions containing the M(r) 90,000-116,000 oviductal glycoprotein when the substrate methylumbelliferyl chitotriose was used. These data show that 1) MAA lectin chromatography can significantly enrich for the M(r) 90,000-116,000 glycoprotein from oviductal secretions, 2) the secreted glycoprotein contains saccharide residues typical of sialomucins, and 3) despite primary amino acid sequence identity, the oviductal glycoprotein does not share an enzymatic relationship with chitinases.
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PMID:An estrogen-dependent sheep oviductal glycoprotein has glycan linkages typical of sialomucins and does not contain chitinase activity. 856 10

Synchrotron x-ray diffraction patterns from macular corneal dystrophy (MCD) corneas contain an unusual reflection that arises because of an undefined ultrastructure with a periodic repeat in the region of 4.6 A. In this study, we compared with wide-angle x-ray diffraction patterns obtained from four normal human corneas and four MCD corneas. Moreover, portions of two of the MCD corneas were pretreated with a specific glycosidase to shed light on the origin of the 4.6 A reflection. None of the normal corneas produced an x-ray reflection in the region of 4.6 A, whereas all four of the MCD corneas did (MCD type I at 4.65 A and 4.63 A, MCD type II at 4.63 A and 4.67 A). This reflection was diminished after incubation of the MCD tissues with either chondroitinase ABC or N-glycanase. The findings indicate that glycosaminoglycans or proteoglycans contribute to the unusual MCD x-ray reflection and hence most likely contain a periodic 4.6 A ultrastructure. Furthermore, the results imply that periodic 4.6 A MCD ultrastructures reside in either intact, unsulfated lumican molecules and regions of the CS/DS-containing molecules or in a region of a hybrid macromolecular aggregate formed by the interaction of the two molecules.
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PMID:Proteoglycans contain a 4.6 A repeat in muscular dystrophy corneas: x-ray diffraction evidence. 878 55

A material of Mr 24,000 has been isolated from a cachexia-inducing mouse tumor (MAC16) and shown to initiate protein degradation in isolated gastrocnemius muscle. Biological activity was destroyed by preincubation with peptide N-glycosidase F (PNGase F) and endo-alpha-N-acetylgalactosaminidase (O-glycosidase) but not by neuraminidase or trypsin. Antibody reactivity was destroyed by treatment with periodate, indicating carbohydrate moieties to be the antigenic determinants. Antigenic activity was also reduced by treatment with PNGase F and O-glycosidase and was completely destroyed by treatment with chondroitinase ABC but was unaffected by treatment with either trypsin or chymotrypsin, confirming that the N- and O-linked sulfated oligosaccharide chains were both the antigenic and biological determinants. Biosynthetic labeling of MAC16 cells using a combination of [35S]sulfate and [6-3H]GlcN gave a single component of Mr 24,000 containing both radiolabels. Similar material could not be isolated from a cell line (MAC13) originating from a tumor that does not cause cachexia in vivo. Digestion of 3H/35S material with PNGase F produced two fragments of Mr 14,000 and 10,000 containing both radiolabels, and digestion with O-glycosidase produced three fragments of Mr 14,000, 6,000, and 4, 000, the first two contained both radiolabels and the third contained only 3H. Digestion of the fragment of Mr 14,000 released by PNGase F with O-glycosidase also gave fragments of Mr 6,000 and 4, 000. The products from both digestions were acidic as determined by anion exchange chromatography on DEAE-cellulose. The negative charge on the fragment of Mr 4,000 was removed by treatment with alkaline phosphatase. This suggests that the charge originated from phosphate residues, and this has been confirmed by biosynthetic labeling of MAC16 cells with [32P]orthophosphate, where radiolabel was incorporated into material of Mr 24,000 and into the fragment of Mr 4,000 after treatment with O-glycosidase. To determine the size of the polypeptide core MAC16 cells were biosynthetically labeled with L-[2,5-3H]His which after chemical deglycosylation produced a major component of Mr 4,000. These results suggest a model for the Mr 24, 000 material consisting of a central polypeptide chain of Mr 4,000 and with phosphate residues that may be attached to the polypeptide or a short oligosaccharide chain containing GlcN, one O-linked sulfated oligosaccharide chain containing GlcN, and of Mr 6,000 and one N-linked sulfated oligosaccharide chain of Mr 10,000 also containing GlcN. Neither chain was cleaved into disaccharides with chondroitinase ABC, suggesting that the material is a sulfated glycoprotein.
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PMID:Structural analysis of a tumor-produced sulfated glycoprotein capable of initiating muscle protein degradation. 913 70


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