scholarly journals The control of chondroitin sulphate biosynthesis and its influence on the structure of cartilage proteoglycans

1982 ◽  
Vol 202 (2) ◽  
pp. 387-395 ◽  
Author(s):  
D Mitchell ◽  
T Hardingham
Keyword(s):  
Actinomycin D ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Chain Elongation ◽  
Chondrosarcoma Cell ◽  
Acceptor Concentration ◽  
The Core ◽  
Chain Synthesis ◽  
Cartilage Proteoglycans ◽  
Elongation Activity

Chondroitin sulphate synthesis on proteoglycans was decreased in rat chondrosarcoma cell cultures in the presence of cycloheximide (0.1-1.0 muM) or p-nitrophenyl beta-D-xyloside (50 microM). In the presence of cycloheximide the proteoglycan monomer was of larger size, the chondroitin sulphate chains were increased in length, but a similar number of chains was attached to each proteoglycan and the size of the core protein was unaltered. In the presence of p-nitrophenyl beta-D-xyloside (50 microM), chondroitin sulphate synthesis was increased (by 60-80%), but the incorporation into proteoglycans was decreased (by 70%). The chondroitin sulphate chains were of shorter length than in control cultured and the number of chains attached to each proteoglycan was decreased. In cultures with cycloheximide or actinomycin D the synthesis of chondroitin sulphate was less inhibited on beta-xyloside than on endogenous proteoglycan. When the rate of chondroitin sulphate synthesis was decreased by lowering the temperature of cultures, the chains synthesized at 22 and 4 degrees C were much longer than at 37 degrees C, but in the presence of p-nitrophenyl beta-D-xyloside the chains were of the same length at all three temperatures. A model of chain elongation is thus proposed in which the rate of chain synthesis is determined by the concentration of xylosyl acceptor and the length of the chains is determined by the ratio of elongation activity to xylosyl-acceptor concentration.

scholarly journals The relation of RNA synthesis to chondroitin sulphate biosynthesis in cultured bovine cartilage

1986 ◽  
Vol 235 (2) ◽  
pp. 499-505 ◽  
Author(s):  
D J McQuillan ◽  
C J Handley ◽  
H C Robinson ◽  
K Ng ◽  
C Tzaicos
Keyword(s):  
Half Life ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Glycosaminoglycan Synthesis ◽  
First Order ◽  
The Core ◽  
First Order Kinetics ◽  
Bovine Cartilage

Addition of actinomycin D (or cordycepin, an alternative inhibitor of RNA synthesis) to cartilage cultures resulted in a first-order decrease in the rate of incorporation of [35S]sulphate into proteoglycan (half-life = 7.5 +/- 1.1 h). Addition of 1.0 mM-benzyl beta-D-xyloside relieved the initial inhibition of glycosaminoglycan synthesis induced by actinomycin D; however, after a lag of about 10 h the rate of xyloside-initiated glycosaminoglycan synthesis also decreased with apparent first-order kinetics (half-life = 7.1 +/- 1.8 h), which paralleled the decrease in the rate of core-protein-initiated glycosaminoglycan synthesis. The hydrodynamic size of the proteoglycans formed in the presence of actinomycin D remained essentially constant (Kav. 0.21-0.23), whereas the constituent glycosaminoglycan chains were larger than those formed by control cultures, which suggested that the core protein was substituted with fewer but larger glycosaminoglycan chains. Proteoglycans formed in the presence of beta-D-xyloside were significantly smaller (Kav. approximately 0.33) than those synthesized by control cultures, and were further diminished in size after exposure of cultures to actinomycin D. Glycosaminoglycan chains synthesized by these same cultures on to both core-protein and xyloside acceptors were also smaller than those of control cultures. The decrease in synthesis observed after exposure to actinomycin D was not reflected by any significant decrease in the activities of several glycosyltransferases involved in chondroitin sulphate synthesis (galactosyltransferase-I, galactosyltransferase-II, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II).

scholarly journals The relation of protein synthesis to chondroitin sulphate biosynthesis in cultured bovine cartilage

1984 ◽  
Vol 224 (3) ◽  
pp. 977-988 ◽  
Author(s):  
D J McQuillan ◽  
C J Handley ◽  
H C Robinson ◽  
K Ng ◽  
C Tzaicos ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Culture Medium ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Bovine Articular Cartilage ◽  
The Core ◽  
Elevated Rate ◽  
Chain Synthesis ◽  
Chondroitin Sulphate Chain ◽  
Bovine Cartilage

The effect of cycloheximide on chondroitin sulphate biosynthesis was studied in bovine articular cartilage maintained in culture. Addition of 0.4 mM-cycloheximide to the culture medium was followed, over the next 4h, by a first-order decrease in the rate of incorporation of [35S]sulphate into glycosaminoglycan (half-life, t 1/2 = 32 min), which is consistent with the depletion of a pool of proteoglycan core protein. Addition of 1.0 mM-benzyl beta-D-xyloside increased the rate of incorporation of [35S]sulphate and [3H]acetate into glycosaminoglycan, but this elevated rate was also diminished by cycloheximide. It was concluded that cycloheximide exerted two effects on the tissue; not only did it inhibit the synthesis of the core protein, but it also lowered the tissue's capacity for chondroitin sulphate chain synthesis. Similar results were obtained with chick chondrocytes grown in high-density cultures. Although the exact mechanism of this secondary effect of cycloheximide is not known, it was shown that there was no detectable change in cellular ATP concentration or in the amount of three glycosyltransferases (galactosyltransferase-I, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II) involved in chondroitin sulphate chain synthesis. The sizes of the glycosaminoglycan chains formed in the presence of cycloheximide were larger than those formed in control cultures, whereas those synthesized in the presence of benzyl beta-D-xyloside were consistently smaller, irrespective of the presence of cycloheximide. These results suggest that beta-D-xylosides must be used with caution to study chondroitin sulphate biosynthesis as an event entirely independent of proteoglycan core-protein synthesis, and they also indicate a possible involvement of the core protein in the activation of the enzymes of chondroitin sulphate synthesis.

The intermediates of aggrecanase-dependent cleavage of aggrecan in rat chondrosarcoma cells treated with interleukin-1

2000 ◽  
Vol 351 (1) ◽  
pp. 161-166 ◽  
Author(s):  
John D. SANDY ◽  
Vivian THOMPSON ◽  
Kurt DOEGE ◽  
Christie VERSCHAREN
Keyword(s):  
Core Protein ◽  
Degradation Products ◽  
Chondroitin Sulphate ◽  
Interleukin 1 ◽  
Rich Region ◽  
Chondrosarcoma Cell ◽  
Enzyme Binding ◽  
The Core ◽  
Attachment Region ◽  
The Individual

We have examined the abundance and structure of intermediates in the chondrocyte-mediated degradation of aggrecan by aggrecanase(s). Degradation products were identified by Western-blot analysis with antibodies to cleavage-site neoepitopes and to peptides within the globular domains. Rat chondrosarcoma tumour contained full-length aggrecan and all of the individual peptides expected from single independent cleavages at each of the four aggrecanase sites in the chondroitin sulphate (CS) domain. Kinetic analysis of the products present in rat chondrosarcoma cell cultures treated with interleukin-1b showed that the first aggrecanase-mediated cleavages occurred at the four sites within the CS attachment region to generate two stable intermediates, Val1–Glu1459 and Val1–Glu1274. These species were subsequently cleaved at the Glu373 site in the interglobular domain to form the terminal products, Val1–Glu373, Ala374–Glu1274 and Ala374–Glu1459. It therefore appears that the aggrecanase-mediated processing of native aggrecan by chondrocytes insitu is initiated within the CS-attachment region and completed by cleavage within the interglobular domain. Since it has been shown that digestion of aggrecan monomer in solution with recombinant ADAMTS-4 [Tortorella, Pratta, Liu, Austin, Ross, Abbaszade, Burn and Arner (2000) Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4). J. Biol. Chem. 275, 18566–18573] exhibits similar kinetics, it appears that preferential proteinase cleavage in the CS-rich region is determined by properties inherent in the aggrecan monomer itself, such as preferred peptide sequences for enzyme binding or enhanced accessibility to the core protein at these sites.

scholarly journals Proteoglycans of the intervertebral disc. Absence of degradation during the isolation of proteoglycans from the intervertebral disc

1979 ◽  
Vol 179 (3) ◽  
pp. 573-578 ◽  
Author(s):  
R L Stevens ◽  
P G Dondi ◽  
H Muir
Keyword(s):  
Intervertebral Disc ◽  
Density Gradient ◽  
Core Protein ◽  
Density Gradient Centrifugation ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Hydrodynamic Size ◽  
Laryngeal Cartilage ◽  
Cartilage Proteoglycans

Proteoglycans extracted with 4M-guanidinium chloride from pig intervetebral discs, and purified by equilibrium density-gradient centrifugation in CsCl, were of smaller hydrodynamic size than those extracted and purified in the same way from the laryngeal cartilage of the same animal. Whether this difference in size arose from degradation during the extraction and purification of the proteoglycans of the disc was investigated. Purified proteoglycans labelled either in the chondroitin sulphate chains or in the core protein were obtained from laryngeal cartilage by short-term organ culture. These labelled proteoglycans were added at the beginning of the extraction of the disc proteoglycans, and labelled cartilage and unlabelled disc proteoglycans were isolated and purified together. There was no appreciable loss of radioactivity after density-gradient centrifugation nor decrease in hydrodynamic size of the labelled cartilage proteoglycans on chromatography on Sepharose 2B, when these were present during the extraction of disc proteoglycans. It is concluded that disc proteoglycans are intrinsically of smaller size than cartilage proteoglycans and this difference in size does not arise from degradation during the extraction.

scholarly journals Physical properties of chondroitin sulphate/dermatan sulphate proteoglycans from bovine aorta

1986 ◽  
Vol 240 (2) ◽  
pp. 575-583 ◽  
Author(s):  
R Kapoor ◽  
C F Phelps ◽  
T N Wight
Keyword(s):  
Uronic Acid ◽  
Glucuronic Acid ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Polymer Concentration ◽  
Dermatan Sulphate ◽  
Guanidinium Chloride ◽  
Structural Units ◽  
The Core ◽  
Covalently Linked

Bovine aortic chondroitin sulphate/dermatan sulphate proteoglycans (PG-25, PG-35 and PG-50) were differentially precipitated with ethanol and analysed by a variety of chemical and physical techniques. The glycosaminoglycan chains of PG-25 and PG-35 contained a mixture of glucuronic acid and iduronic acid, whereas the uronic acid component of PG-50 was primarily glucuronic acid. In addition, various amounts of oligosaccharides containing small amounts of mannose, a galactose/hexosamine ratio of 1:1 and an absence of uronic acid were covalently linked to the core protein of all proteoglycans. The weight-average Mr (Mw) values of the proteoglycans determined by light-scattering in 4 M-guanidinium chloride were 1.3 × 10(6) (PG-25), 0.30 × 10(6) (PG-35) and 0.88 × 10(6) (PG-50). The s0 values of the proteoglycans were distributed between 7 and 8 S, and the reduced viscosities, eta sp./c, of all proteoglycans were dependent on the shear rate and polymer concentration. Electron microscopy of spread molecules revealed that PG-25 contained small structural units that appeared to self-associate into large aggregates, whereas PG-35 and PG-50 appeared mainly as monomers consisting of a core with various numbers of side projections. Hyaluronic acid-proteoglycan complexes occurred only with a small proportion of the molecules present in PG-35, and their formation could be inhibited by oligosaccharides. These results suggest the presence in the aorta of subspecies of chondroitin sulphate and dermatan sulphate proteoglycans, which show large variations in their physicochemical and inter- and intra-molecular association properties.

scholarly journals Extended and globular protein domains in cartilage proteoglycans

1987 ◽  
Vol 245 (3) ◽  
pp. 763-772 ◽  
Author(s):  
M Paulsson ◽  
M Mörgelin ◽  
H Wiedemann ◽  
M Beardmore-Gray ◽  
D Dunham ◽  
...  
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Average Length ◽  
Globular Domain ◽  
Protein Core ◽  
Nasal Cartilage ◽  
Cdna Sequences ◽  
Multidomain Structure ◽  
Variable Extent ◽  
Cartilage Proteoglycans

Electron microscopy after rotary shadowing and negative staining of the large chondroitin sulphate proteoglycan from rat chondrosarcoma, bovine nasal cartilage and pig laryngeal cartilage demonstrated a unique multidomain structure for the protein core. A main characteristic is a pair of globular domains (diameter 6-8 nm), one of which forms the N-terminal hyaluronate-binding region. They are connected by a 25 nm-long rod-like domain of limited flexibility. This segment is continued by a 280 nm-long polypeptide strand containing most chondroitin sulphate chains (average length 40 nm) in a brush-like array and is terminated by a small C-terminal globular domain. The core protein showed a variable extent of degradation, including the loss of the C-terminal globular domain and sections of variable length of the chondroitin sulphate-bearing strand. The high abundance (30-50%) of the C-terminal domain in some extracted proteoglycan preparations indicated that this structure is present in the cartilage matrix rather than being a precursor-specific segment. It may contain the hepatolectin-like segment deduced from cDNA sequences corresponding to the 3′-end of protein core mRNA [Doege, Fernandez, Hassell, Sasaki & Yamada (1986) J. Biol. Chem. 261, 8108-8111; Sai, Tanaka, Kosher & Tanzer (1986) Proc. Natl. Acad. Sci. 83, 5081-5085; Oldberg, Antonsson & Heinegård (1987) Biochem. J. 243, 255-259].

scholarly journals Cell-free translation of messenger RNA for chondroitin sulphate proteoglycan core protein in rat cartilage

1984 ◽  
Vol 217 (1) ◽  
pp. 259-263 ◽  
Author(s):  
B M Vertel ◽  
W B Upholt ◽  
A Dorfman
Keyword(s):  
Messenger Rna ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Protein Product ◽  
Neonatal Rat ◽  
The Core ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan ◽  
Specific Antisera ◽  
Free Translation

Total RNA was extracted from the cartilage tissues rat Swarm chondrosarcoma, neonatal-rat breastplate and embryonic-chicken sterna and translated in wheat-germ cell-free reactions. The core protein of the chondroitin sulphate proteoglycan subunit was identified among translation products of rat mRNA by its apparent Mr of 330 000 and by its immunoprecipitation with specific antisera prepared against rat or chicken proteoglycan antigens. The apparent Mr of the rat proteoglycan core protein is 8000-10000 less than that of the equivalent chicken cartilage core-protein product.

scholarly journals Cartilage proteoglycans. Assembly with hyaluronate and link protein as studied by electron microscopy

1988 ◽  
Vol 253 (1) ◽  
pp. 175-185 ◽  
Author(s):  
M Mörgelin ◽  
M Paulsson ◽  
T E Hardingham ◽  
D Heinegård ◽  
J Engel
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Core Protein ◽  
Protein Domains ◽  
Globular Domain ◽  
Binding Region ◽  
Link Protein ◽  
The Core ◽  
Cartilage Proteoglycan ◽  
Cartilage Proteoglycans

Aggregates formed by the interaction of cartilage proteoglycan monomers and fragments thereof with hyaluronate were studied by electron microscopy by use of rotary shadowing [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333]. The differences in shape and packing of the proteins bound along the hyaluronate strand in aggregates formed in the presence and in the absence of link protein were examined in detail. The high resolution of the method allowed examination of the involvement in hyaluronate binding of the globular core-protein domains G1, G2 and G3 [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333; Paulsson, Mörgelin, Wiedemann, Beardmore-Gray, Dunham, Hardingham, Heinegård, Timpl & Engel (1987) Biochem. J. 245, 763-772]. Fragments comprising the globular hyaluronate-binding region G1 form complexes with hyaluronate with an appearance of necklace-like structures, statistically interspaced by free hyaluronate strands. The closest centre-to-centre distance found between adjacent G1 domains was 12 nm. Another fragment comprising the binding region G1 and the adjacent second globular domain G2 attaches to hyaluronate only by one globule. Also, the core protein obtained by chondroitinase digestion of proteoglycan monomer binds only by domain G1, with domain G3 furthest removed from the hyaluronate. Globule G1 shows a statistical distribution along the hyaluronate strands. In contrast, when link protein is added, binding is no longer random, but instead uninterrupted densely packed aggregates are formed.

scholarly journals A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7

1996 ◽  
Vol 318 (3) ◽  
pp. 1051-1056 ◽  
Author(s):  
Dagmar-Christiane FISCHER ◽  
Hans-Dieter HAUBECK ◽  
Kirsten EICH ◽  
Susanne KOLBE-BUSCH ◽  
Georg STÖCKER ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Human Articular Cartilage ◽  
Rich Region ◽  
Keratan Sulphate ◽  
The Core ◽  
Gel Shift Assay ◽  
Chemical Deglycosylation

Monoclonal antibodies (mAbs) were prepared against aggrecan which has been isolated from human articular cartilage and purified by several chromatographic steps. One of these mAbs, the aggrecan-specific mAb 3D12/H7, was selected for further characterization. The data presented indicate that this mAb recognizes a novel domain of keratan sulphate chains from aggrecan: (1) immunochemical staining of aggrecan is abolished by treatment with keratanase/keratanase II, but not with keratanase or chondroitin sulphate lyase AC/ABC; (2) after chemical deglycosylation of aggrecan no staining of the core-protein was observed; (3) different immunochemical reactivity was observed against keratan sulphates from articular cartilage, intervertebral disc and cornea for the mAbs 3D12/H7 and 5D4. For further characterization of the epitope, reduced and 3H-labelled keratan sulphate chains were prepared. In an IEF–gel-shift assay it was shown that the 3H-labelled oligosaccharides obtained after keratanase digestion of reduced and 3H-labelled keratan sulphate chains were recognized by the mAb 3D12/H7. Thus it can be concluded that the mAb 3D12/H7 recognizes an epitope in the linkage region present in, at least some, keratan sulphate chains of the large aggregating proteoglycan from human articular cartilage. Moreover, this domain seems to be expressed preferentially on those keratan sulphate chains which occur in the chondroitin sulphate-rich region of aggrecan, since the antibody does not recognize the keratan sulphate-rich region obtained after combined chondroitinase AC/ABC and trypsin digestion of aggrecan.

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