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 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 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.

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 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 A Dynamic Scaffold of Pre-snoRNP Factors Facilitates Human Box C/D snoRNP Assembly

2007 ◽  
Vol 27 (19) ◽  
pp. 6782-6793 ◽  
Author(s):  
Kenneth Scott McKeegan ◽  
Charles Maurice Debieux ◽  
Séverine Boulon ◽  
Edouard Bertrand ◽  
Nicholas James Watkins
Keyword(s):  
Protein Interactions ◽  
Core Protein ◽  
The Novel ◽  
Protein Protein Interactions ◽  
Core Complex ◽  
The Core ◽  
Snornp Biogenesis ◽  
The Individual ◽  
Vitro Protein

ABSTRACT The box C/D small nucleolar RNPs (snoRNPs) are essential for the processing and modification of rRNA. The core box C/D proteins are restructured during human U3 box C/D snoRNP biogenesis; however, the molecular basis of this is unclear. Here we show that the U8 snoRNP is also restructured, suggesting that this may occur with all box C/D snoRNPs. We have characterized four novel human biogenesis factors (BCD1, NOP17, NUFIP, and TAF9) which, along with the ATPases TIP48 and TIP49, are likely to be involved in the formation of the pre-snoRNP. We have analyzed the in vitro protein-protein interactions between the assembly factors and core box C/D proteins. Surprisingly, this revealed few interactions between the individual core box C/D proteins. However, the novel biogenesis factors and TIP48 and TIP49 interacted with one or more of the core box C/D proteins, implying that they mediate the assembly of the pre-snoRNP. Consistent with this, we show that NUFIP bridges interactions between the core box C/D proteins in a partially reconstituted pre-snoRNP. Restructuring of the core complex probably reflects the conversion of the pre-snoRNP, where core protein-protein interactions are maintained by the bridging biogenesis factors, to the mature snoRNP.

scholarly journals Catabolism of aggrecan, decorin and biglycan in tendon

2000 ◽  
Vol 350 (1) ◽  
pp. 181-188 ◽  
Author(s):  
Sarah G. REES ◽  
Carl R. FLANNERY ◽  
Chris B. LITTLE ◽  
Clare E. HUGHES ◽  
Bruce CATERSON ◽  
...  
Keyword(s):  
Culture Medium ◽  
Core Protein ◽  
Degradation Products ◽  
Chondroitin Sulphate ◽  
Expression Patterns ◽  
Collagen Matrix ◽  
Pcr Analysis ◽  
Age Related ◽  
Proteoglycan Degradation

We have examined the catabolism of the proteoglycans aggrecan, decorin and biglycan in fresh tendon samples and in explant cultures of tissue from the tensional and compressed regions of young and mature bovine tendons. A panel of well-characterized antibodies that recognize glycosaminoglycan or protein (linear or neoepitope) sequences was used to detect proteoglycans and proteoglycan degradation products that were both retained within the tissue and released into the culture medium. In addition, a reverse-transcriptase-mediated PCR analysis was used to examine the mRNA expression patterns of tendon proteoglycans and aggrecanases. The results of this study indicate a major role for aggrecanase(s) in the catabolism of aggrecan in bovine tendon. The study also provides a characterization of glycosaminoglycan epitopes associated with the proteoglycans of tendon, illustrating age-related changes in the isomers of chondroitin sulphate disaccharides that remain attached to the core protein glycosaminoglycan linkage region after digestion with chondroitinase ABC. Evidence for a rapid turnover of the small proteoglycans decorin and biglycan was also observed, indicating additional molecular pathways that might compromise the integrity of the collagen matrix and potentially contribute to tendon dysfunction after injury and during disease.

scholarly journals Involvement of the core protein in the first β-N-acetylgalactosamine transfer to the glycosaminoglycan–protein linkage-region tetrasaccharide and in the subsequent polymerization: the critical determining step for chondroitin sulphate biosynthesis

1999 ◽  
Vol 340 (2) ◽  
pp. 353-357 ◽  
Author(s):  
Satomi NADANAKA ◽  
Hiroshi KITAGAWA ◽  
Fumitaka GOTO ◽  
Jun-ichi TAMURA ◽  
Klaus W. NEUMANN ◽  
...  
Keyword(s):  
Culture Medium ◽  
Glucuronic Acid ◽  
Core Protein ◽  
Enzyme System ◽  
Chondroitin Sulphate ◽  
Human Melanoma ◽  
The Core ◽  
A Cell ◽  
Chain Polymerization

α-Thrombomodulin (α-TM) with a truncated glycosaminoglycan-protein linkage tetrasaccharide, GlcAβ1-3Galβ1-3Galβ1-4Xyl, was tested as an acceptor together with a sugar donor, UDP-N-[3H]acetylgalactosamine, using a cell-free enzyme system prepared from the serum-free culture medium of a human melanoma cell line. The truncated tetrasaccharide on α-TM served as an acceptor, whereas the linkage tetrasaccharide-serine did not. Our characterization of the radioactively labelled product by enzymic digestion revealed that the N-[3H]acetylgalactosamine residue was transferred to α-TM through a β1,4-linkage. The substrate competition experiments with the chondro-hexasaccharide and α-TM reinforced our speculation that a common N-acetylgalactosaminyltransferase catalysed the transfer of N-acetylgalactosamine to both the linkage tetrasaccharide and the longer chondroitin oligosaccharides. Moreover, chondroitin polymerization was demonstrated on the tetrasaccharide of α-TM using both UDP-glucuronic acid and UDP-N-acetylgalactosamine as sugar donors. Much longer chains were synthesized on α-TM than on the linkage penta- and hexa-saccharide-serines. Together, these results indicated that the core protein is required for the transfer of the first N-acetylgalactosamine residue through a β1,4-linkage and also for subsequent efficient chain polymerization reactions, and that the critical determining step for chondroitin sulphate biosynthesis is the transfer of the first N-acetylgalactosamine residue.

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 Domain structure of cartilage proteoglycans revealed by rotary shadowing of intact and fragmented molecules

1984 ◽  
Vol 224 (1) ◽  
pp. 331-333 ◽  
Author(s):  
H Wiedemann ◽  
M Paulsson ◽  
R Timpl ◽  
J Engel ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Side Chain ◽  
Keratan Sulphate ◽  
Molecular Electron ◽  
Attachment Region ◽  
Proteoglycan Aggregates ◽  
Cartilage Proteoglycans ◽  
Rotary Shadowing

The rotary-shadowing technique for molecular electron microscopy was used to study cartilage proteoglycan structure. The high resolution of the method allowed demonstration of two distinct globular domains as well as a more strand-like portion in the core protein of large aggregating proteoglycans. Studies of proteoglycan aggregates and fragments showed that the globular domains represent the part of the proteoglycans that binds to the hyaluronic acid, i.e. the hyaluronic acid-binding region juxtapositioned to the keratan sulphate-attachment region. The strand-like portion represents the chondroitin sulphate-attachment region. Low-Mr proteoglycans from cartilage could be seen as a globule connected to one or two side-chain filaments of chondroitin sulphate.

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