Articular cartilage cultured with interleukin 1. Increased release of link protein, hyaluronate-binding region and other proteoglycan fragments

Pig articular cartilage was maintained in culture for 3 days with and without porcine interleukin 1. The proteoglycans remaining in the cartilage and those released into the medium were analysed by using radioimmunoassays for the hyaluronate-binding region, link protein and keratan sulphate. In interleukin 1-treated cultures after 3 days there was 38% release of total glycosaminoglycans into the medium, 18% release of binding region, 14% release of link protein and 20% release of keratan sulphate epitope, whereas in control cultures the proportions released were much less (16, 9, 10 and 7% respectively). Characterization of the proteoglycans in the media after 1.5 days and 3 days of culture showed that interleukin 1 promoted the release of proteoglycan of large average size and also the release of link protein and of low-Mr binding region which was unattached to proteoglycan. Both the link protein and binding region released were able to bind to exogenously added hyaluronate, whereas the proteoglycan in the medium was not. The proteoglycans extracted from cultured cartilage were similar to those from fresh cartilage: they contained a high proportion of aggregating proteoglycans and some low-Mr binding region. The proportion of this binding region extracted from the interleukin 1-treated cartilage was increased. The presence of interleukin 1 in the cultures therefore appeared to increase the rate of proteolytic degradation of proteoglycan in the matrix and to lead to a more rapid loss of intact binding region, of link protein and of large proteoglycan fragments into the medium.

Download Full-text

Chondrocyte-mediated depletion of articular cartilage proteoglycans in vitro

Biochemical Journal ◽

10.1042/bj2250493 ◽

1985 ◽

Vol 225 (2) ◽

pp. 493-507 ◽

Cited By ~ 111

Author(s):

J A Tyler

Keyword(s):

Articular Cartilage ◽

Core Protein ◽

Fold Increase ◽

Acid Proteinase ◽

Binding Region ◽

Guanidinium Chloride ◽

The Core ◽

The Matrix ◽

Average Size

The degradation of proteoglycan was examined in cultured slices of pig articular cartilage. Pig leucocyte catabolin (10 ng/ml) was used to stimulate the chondrocytes and induce a 4-fold increase in the rate of proteoglycan loss from the matrix for 4 days. Material in the medium of both control and depleted cultures was mostly a degradation product of the aggregating proteoglycan. It was recovered as a very large molecule slightly smaller than the monomers extracted with 4M-guanidinium chloride and lacked a functional hyaluronate binding region. The size and charge were consistent with a very limited cleavage or conformational change of the core protein near the hyaluronate binding region releasing the C-terminal portion of the molecule intact from the aggregate. The ‘clipped’ monomer diffuses very rapidly through the matrix into the medium. The amount of proteoglycan extracted with 4M-guanidinium chloride decreased during culture from both the controls and depleted cartilage, and the average size of the molecules initially remained the same. However, the proportion of molecules with a smaller average size increased with time and was predominant in explants that had lost more than 70% of their proteoglycan. All of this material was able to form aggregates when mixed with hyaluronate, and glycosaminoglycans were the same size and charge as normal, indicating either that the core protein had been cleaved in many places or that larger molecules were preferentially released. A large proportion of the easily extracted and non-extractable proteoglycan remained in the partially depleted cartilage and the molecules were the same size and charge as those found in the controls. There was no evidence of detectable glycosidase activity and only very limited sulphatase activity. A similar rate of breakdown and final distribution pattern was found for newly synthesized proteoglycan. Increased amounts of latent neutral metalloproteinases and acid proteinase activities were present in the medium of depleted cartilage. These were not thought to be involved in the breakdown of proteoglycan. Increased release of proteoglycan ceased within 24h of removal of the catabolin, indicating that the effect was reversible and persisted only while the stimulus was present.

Download Full-text

Turnover of proteoglycans in articular-cartilage cultures. Characterization of proteoglycans released into the medium

Biochemical Journal ◽

10.1042/bj2590021 ◽

1989 ◽

Vol 259 (1) ◽

pp. 21-25 ◽

Cited By ~ 29

Author(s):

M A Campbell ◽

C J Handley ◽

S E D'Souza

Keyword(s):

Articular Cartilage ◽

Core Protein ◽

Chondroitin Sulphate ◽

Polyacrylamide Gel Electrophoresis ◽

Keratan Sulphate ◽

Bovine Articular Cartilage ◽

Definitive Evidence ◽

Core Proteins ◽

The Matrix

By using an e.l.i.s.a. method it was demonstrated that the majority of proteoglycans released into the medium of both control and retinoic acid-treated explant cultures of bovine articular cartilage did not contain a hyaluronate-binding region. This supports our previous findings [Campbell & Handley (1987) Arch. Biochem. Biophys. 258, 143-155] that proteoglycans released into the medium of both cultures were of smaller hydrodynamic size, more polydisperse and unable to form aggregates with hyaluronate. Analysis of 35S-labelled core proteins associated with proteoglycans released into the medium of both cultures by using SDS/polyacrylamide-gel electrophoresis and fluorography indicated the presence of a series of core-protein bands (Mr approx. 300,000, 230,000, 215,000, 200,000, 180,000, 140,000, 135,000, 105,000, 85,000 and 60,000) compared with three core proteins derived from the proteoglycans remaining in the matrix (Mr 300,000, 230,000 and 215,000). Further analysis of the core proteins released into the medium indicated that the larger core proteins associated with medium proteoglycans contain both chondroitin sulphate and keratan sulphate glycosaminoglycans whereas the smaller core proteins contain only chondroitin sulphate chains. These experiments provide definitive evidence that the loss of proteoglycans from the matrix involves proteolytic cleavage at various sites along the proteoglycan core protein.

Download Full-text

N-terminal sequence of proteoglycan fragments isolated from medium of interleukin-1-treated articular-cartilage cultures. Putative site(s) of enzymic cleavage

Biochemical Journal ◽

10.1042/bj2840589 ◽

1992 ◽

Vol 284 (2) ◽

pp. 589-593 ◽

Cited By ~ 91

Author(s):

P Loulakis ◽

A Shrikhande ◽

G Davis ◽

C A Maniglia

Keyword(s):

Hyaluronic Acid ◽

Articular Cartilage ◽

Amino Acid ◽

Interleukin 1 ◽

Human Articular Cartilage ◽

Terminal Amino Acid ◽

Protein Core ◽

Nasal Cartilage ◽

The Media ◽

Terminal Amino

Bovine articular cartilage was cultured both in the presence and in the absence of human recombinant interleukin-1 alpha (IL-1) (100 units/ml). Addition of this cytokine stimulated matrix degradation approx. 3-fold. This increased degradation permitted characterization of the large chondroitin sulphate proteoglycan (aggrecan) fragments accumulating in the media. When compared with controls, the proteoglycans isolated from the medium of cultures treated with IL-1 exhibited a decrease in the Kav. (control 0.25; IL-1-treated 0.37), determined by Sepharose CL-2B chromatography. This decrease in proteoglycan size was accompanied by a decreased ability of these monomers to associate with hyaluronic acid. Thus only 20% of the proteoglycans isolated from the medium of IL-1-treated cultures, compared with 39% for control cultures, had the capacity to form high-M(r) aggregates with hyaluronic acid. SDS/PAGE analysis of the proteoglycans from the media of IL-1-treated cultures demonstrated several large proteoglycan protein-core bands (M(r) 144,000-380,000). The protein-core bands with M(r) 144,000-266,000 exhibited a significantly decreased reactivity with monoclonal antibody 1-C-6 (specific for domains G1 and G2). The N-terminal amino acid sequence of four of these protein-core bands (M(r) 144,000, 173,000, 214,000 and 266,000) yielded sequences LGQRPPV-Y-PQLF(E), AGEGP(S)GILEL-GAP(S)-AP(D)M, GLG-VEL-LPGE and (A)RGSVIL-AKPDFEV-P-A. A comparison of these N-terminal amino acid sequences with the published proteoglycan sequence for bovine nasal cartilage [Oldberg, Antonsson & Heinegård (1987) Biochem. J. 243, 255-259], rat chondrosarcoma [Doege, Sasaki, Horigan, Hassell & Yamada (1987) J. Biol. Chem. 262, 17757-17769] and human articular cartilage [Doege, Sasaki, Kimura & Yamada (1991) J. Biol. Chem. 266, 894-902] permitted assignment of their relative positions on the core protein. Furthermore, on the basis of this similarity to published sequence, putative sites of enzymic cleavage were constructed. These theoretical cleavage sites revealed a glutamic acid residue in the P1 position and an uncharged polar or non-polar residue in the P1′ position.

Download Full-text

Localization of proteoglycan monomer and link protein in the matrix of bovine articular cartilage: An immunohistochemical study.

Journal of Histochemistry & Cytochemistry ◽

10.1177/28.7.6156200 ◽

1980 ◽

Vol 28 (7) ◽

pp. 621-635 ◽

Cited By ~ 68

Author(s):

A R Poole ◽

I Pidoux ◽

A Reiner ◽

L H Tang ◽

H Choi ◽

...

Keyword(s):

Articular Cartilage ◽

Articular Surface ◽

Guanidine Hydrochloride ◽

Cartilage Matrix ◽

Binding Studies ◽

Superficial Zone ◽

Surface Culture ◽

Bovine Articular Cartilage ◽

Link Protein ◽

The Matrix

Using monospecific antisera and immunofluorescence microscopy, proteoglycan monomer (PG), and link proteins were demonstrated throughout the extracellular matrix of bovine articular cartilage. A narrow band of strong pericellular staining was usually observed for both molecules, indicating a pericellular concentration of proteoglycan monomer: this conclusion was supported by dye-binding studies. Whereas PG was evenly distributed throughout the remaining matrix, more link protein was detectable in interterritorial sites in middle and deep zones. Well-defined zones of weaker territorial staining for link protein stained strongest for chondroitin sulfate. Trypsin treatment of cartilage resulted in a loss of most of the PG staining, but some selective retention of link protein, particularly around chondrocytes in the superficial zone at and near the articular surface. This residual staining was largely removed if sections were fixed after chondroitinase treatment. After extraction of cartilage with 4M guanidine hydrochloride, only PG remained and this was concentrated in the superficial zone. These observations are shown to support the concept of aggregation of PG and link protein with hyaluronic acid (HA) in cartilage matrix, and the binding of PG and link protein to HA, which is attached to the chondrocyte surface. Culture of cartilage depleted of PG and link protein by trypsin demonstrated that individual chondrocytes can secrete both PG and link proteins and that the organization of cartilage matrix can be regenerated in part over a period of 4 days.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj3181051 ◽

1996 ◽

Vol 318 (3) ◽

pp. 1051-1056 ◽

Cited By ~ 10

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.

Download Full-text

The action of human articular-cartilage metalloproteinase on proteoglycan and link protein. Similarities between products of degradation in situ and in vitro

Biochemical Journal ◽

10.1042/bj2370117 ◽

1986 ◽

Vol 237 (1) ◽

pp. 117-122 ◽

Cited By ~ 31

Author(s):

I K Campbell ◽

P J Roughley ◽

J S Mort

Keyword(s):

Hyaluronic Acid ◽

Articular Cartilage ◽

Interleukin 1 ◽

Human Articular Cartilage ◽

Link Protein ◽

Protein Components ◽

Stimulation Of ◽

Hyaluronic Acid Binding

Interleukin 1 stimulation of human articular cartilage in organ culture produced the concomitant release of proteoglycan fragments and latent metalloproteinase. The released fragments ranged in size from that of almost intact proteoglycan subunits to the product of limiting digestion generated by the activated metalloproteinase. None of the fragments possessed the ability to interact with hyaluronic acid. Analysis of proteoglycan aggregate digested with the activated metalloproteinase showed that isolated hyaluronic acid-binding regions were produced from the proteoglycan subunits, and that the two higher-Mr link-protein components (Mr 48,000 and 44,000) were converted into the lowest-Mr component (Mr 41,000). Link protein extracted from cartilage under stimulation with interleukin 1 showed a similar conversion. These results suggest that interleukin 1 stimulates the release of latent metalloproteinase from chondrocytes and that a proportion of the enzyme is activated in situ in the cartilage matrix. The mode of action of the activated enzyme is compatible with a role in the changes in proteoglycan structure seen in aging.

Download Full-text

Immunochemical analysis of cartilage proteoglycans. Antigenic determinants of substructures

Biochemical Journal ◽

10.1042/bj1790035 ◽

1979 ◽

Vol 179 (1) ◽

pp. 35-45 ◽

Cited By ~ 64

Author(s):

J Wieslander ◽

D Heinegård

Keyword(s):

Hyaluronic Acid ◽

Chondroitin Sulphate ◽

Antigenic Site ◽

Trypsin Digestion ◽

Antigenic Determinants ◽

Binding Region ◽

Keratan Sulphate ◽

Link Protein ◽

Before And After ◽

Hyaluronic Acid Binding

Antibodies were raised in rabbits by injection of cartilage proteoglycan monomers, isolated hyaluronic acid-binding region, polysaccharide-peptides prepared by trypsin digestion of proteoglycans and link-protein. The rabbits injected with the proteoglycan monomers made antibodies reacting with the intact proteoglycan. The antiserum contained antibodies specific for, and also reacting with, the isolated hyaluronic acid-binding region and the keratan sulphate-rich region. In addition there were probably antibodies reacting with other structures of the proteoglycan monomer. When isolated hyaluronic acid-binding region was used for immunization the antibodies obtained reacted specifically with the hyaluronic acid-binding region. The antibodies obtained from rabbits immunized with the polysaccharide-peptides reacted with the proteoglycan monomers and showed a reaction identical with that of the chondroitin sulphate-peptides isolated after trypsin digestion of proteoglycans. The antibodies prepared with the link-protein as the antigen reacted only with the link-protein and not with any preparation from the proteoglycan monomer. Neither did any of the antisera raised against the proteoglycan monomer or its substructures react with the link-protein. Separately it was shown that the peptide ‘maps’ prepared from trypsin digests of the link-protein and the hyaluronic acid-binding region were different. Therefore it appears that the link-protein is not structurally related to the proteoglycan or the hyaluronic acid-binding region. Digestion of proteoglycan monomers or isolated hyaluronic acid-binding region with trypsin did not destroy the antigenic sites of the hyaluronic acid-binding region. In contrast trypsin digests of previously reduced and alkylated preparations did not react with the anti-(hyaluronic acid-binding region). The trypsin digests, however, reacted with both the antibodies directed against the chondroitin sulphate-peptides and those against the keratan sulphate-peptides. Trypsin digestion of the link-proteins destroyed the antigenic site and the reactivity with the antibodies. By combining immunoassay of proteoglycan preparations before and after trypsin digestion it is feasible to quantitatively determine its substructures by using the antisera described above.

Download Full-text

Variations in the composition of bovine hip articular cartilage with distance from the articular surface

Biochemical Journal ◽

10.1042/bj1950535 ◽

1981 ◽

Vol 195 (3) ◽

pp. 535-543 ◽

Cited By ~ 69

Author(s):

A Franzén ◽

S Inerot ◽

S O Hejderup ◽

D Heinegård

Keyword(s):

Hyaluronic Acid ◽

Articular Cartilage ◽

Chondroitin Sulphate ◽

Articular Surface ◽

Full Thickness ◽

Rich Region ◽

Keratan Sulphate ◽

Average Size ◽

Almost All ◽

Cartilage Proteoglycans

Punch biopsies of bovine hip articular cartilage was sectioned according to depth and the proteoglycans were isolated. The mid-sections of the cartilage contained more proteoglycans than did either the superficial or the deepest portions of the cartilage proteoglycans than did either the superficial or the deepest portions of the cartilage. The most superficial 40 micrometer of the cartilage contained relatively more glucosaminoglycans compared with the remainder of the cartilage. The proteoglycans recovered from the surface 200 micrometer layer contained less chondroitin sulphate, were smaller and almost all of these molecules were able to interact with hyaluronic acid to form aggregates. From about 200 micrometer and down to 1040 micrometer from the surface, the proteoglycans became gradually somewhat smaller, probably owing to decreasing size of the chondroitin sulphate-rich region. The proportion of molecules that were able to interact with the hyaluronic acid was about 90% and remained constant with depth. The proteoglycans from the deepest layer near the cartilage-bone junction contained a large proportion of non-aggregating molecules, and the average size of the proteoglycans was somewhat larger. The alterations of proteoglycan structure observed with increasing depth of the articular cartilage beneath the surface layer (to 200 micrometer) are of the same nature as those observed with increasing age in full-thickness articular cartilage. The articular-cartilage proteoglycans were smaller and had much higher keratan sulphate and protein contents that did molecules isolated from bovine nasal or tracheal cartilage.

Download Full-text

Germanium Nanostructures Fabricated by PLD

MRS Proceedings ◽

10.1557/proc-536-329 ◽

1998 ◽

Vol 536 ◽

Author(s):

K. M. Hassan ◽

A. K. Sharma ◽

J. Narayan ◽

J. F. Muth ◽

C. W. Teng

Keyword(s):

Pulsed Laser Deposition ◽

Electronic Transport ◽

Pulsed Laser ◽

Single Layer ◽

Laser Deposition ◽

Transmission Electron ◽

The Matrix ◽

Average Size ◽

Substrate Temperatures

AbstractQuantum confined nanostructures of semiconductors such as Ge and Si are being actively studied due to their interesting optical and electronic transport properties. We fabricated Ge nanostructures buried in the matrix of polycrystalline-AIN grown on Si(111) by pulsed laser deposition at lower substrate temperatures than that used in previous studies. The characterization of these structures was performed using high resolution transmission electron microscopy (HRTEM), photoluminescence and Raman spectroscopy. HRTEM observations show that the Ge islands are single crystal with a pyramidal shape. The average size of Ge islands was determined to be 15 nm, considerably smaller than that produced by other techniques. The Raman spectrum reveals a peak downward shift, upto 295 cm−1, of the Ge-Ge mode caused by quantum confinement in the Ge-dots. Photoluminescence (PL) was observed both with a single layer of Ge nanodots embedded in the AlN matrix and from ten layers of dots interspersed with AIN. The PL of the dots was blue shifted by ˜0.266 eV from the bulk Ge value of 0.73 eV at 77 K, resulting in a distinct peak at ˜1.0 eV. The full width at half maximum (FWHM) of the peak was 13 meV, for the single layer and 8 meV for the ten layered sample, indicating that the Ge nanodots are fairly uniform in size, which was found to be consistent with our HRTEM results. The importance of pulsed laser deposition (PLD) in fabricating novel nanostructures is discussed.

Download Full-text

Characterization of a non-reducing terminal fragment from bovine articular cartilage keratan sulphates containing α(2-3)-linked sialic acid and α(1-3)-linked fucose. A sulphated variant of the VIM-2 epitope

Biochemical Journal ◽

10.1042/bj3190137 ◽

1996 ◽

Vol 319 (1) ◽

pp. 137-141 ◽

Cited By ~ 15

Author(s):

Gavin M. BROWN ◽

Thomas N. HUCKERBY ◽

Beverley L. ABRAM ◽

Ian A. NIEDUSZYNSKI

Keyword(s):

Articular Cartilage ◽

Sialic Acid ◽

Spectroscopic Analysis ◽

Anion Exchange Column ◽

Keratan Sulphate ◽

Bovine Articular Cartilage ◽

Sialyl Lex ◽

Strong Anion Exchange ◽

Keratanase Ii

Alkaline-borohydride-reduced keratan sulphate chains were isolated from bovine articular cartilage (6–8-year-old animals) and digested with keratanase II, an endo-β-N-acetylglucosaminidase. The resulting oligosaccharides were borohydride-reduced and fractionated on a strong anion-exchange column. 1H-NMR spectroscopic analysis of the products revealed one containing both α(2-3)-linked sialic acid and α(1-3)-linked fucose which was shown to have the structure (I) shown. This structure is a sulphated variant of the VIM-2 epitope (CD65), a putative ligand of E-selectin. No oligosaccharide containing the sialyl-Lex structure [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-] was identified in this study.

Download Full-text