Catabolism of aggrecan, decorin and biglycan in tendon

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.

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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

Biochemical Journal ◽

10.1042/bj3400353 ◽

1999 ◽

Vol 340 (2) ◽

pp. 353-357 ◽

Cited By ~ 3

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.

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The immunologic detection and characterization of cartilage proteoglycan degradation products in synovial fluids of patients with arthritis

Arthritis & Rheumatism ◽

10.1002/art.1780300506 ◽

1987 ◽

Vol 30 (5) ◽

pp. 519-529 ◽

Cited By ~ 77

Author(s):

James Witter ◽

Peter J. Roughley ◽

Carolyn Webber ◽

Nancy Roberts ◽

Edward Keystone ◽

...

Keyword(s):

Degradation Products ◽

Cartilage Proteoglycan ◽

Synovial Fluids ◽

Proteoglycan Degradation

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Molecular Characterization of a NovelStaphylococcus aureus Serine Protease Operon

Infection and Immunity ◽

10.1128/iai.69.3.1521-1527.2001 ◽

2001 ◽

Vol 69 (3) ◽

pp. 1521-1527 ◽

Cited By ~ 82

Author(s):

Samantha B. Reed ◽

Carla A. Wesson ◽

Linda E. Liou ◽

William R. Trumble ◽

Patrick M. Schlievert ◽

...

Keyword(s):

Amino Acid ◽

Serine Protease ◽

Degradation Products ◽

Regulatory System ◽

Pcr Analysis ◽

Sequence Identity ◽

Serine Protease Family ◽

Operon Expression ◽

Amino Acid Signal

ABSTRACT The present study identified and characterized a unique operon (spl) encoding six serine protease-like proteins. In addition, native Spl proteins were isolated and characterized. Typical of most exoproteins, the spl gene products contain putative 35- or 36-amino-acid signal peptides. The Spl proteins share 44 to 95% amino acid sequence identity with each other and 33 to 36% sequence identity with V8 protease. They also contain amino acids found in catalytic triads of enzymes in the trypsin-like serine protease family, and SplB and SplC were shown to degrade casein. The sploperon is transcribed on a 5.5-kb transcript, but several nonrandom degradation products of this transcript were also identified. Similar to other S. aureus exoprotein genes, the sploperon is maximally expressed during the transition into stationary phase and is positively controlled by the Agr virulence factor regulator. The Sar regulatory system did not affect sploperon expression. PCR analysis revealed the presence of thespl operon in 64% of the S. aureus isolates tested, although one spl operon-negative isolate was shown to contain at least two of the spl genes. Finally, intraperitoneal injection of an spl operon deletion mutant revealed no major differences in virulence compared to the parental strain.

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Characterization of proteoglycan degradation by calpain

Biochemical Journal ◽

10.1042/bj2850857 ◽

1992 ◽

Vol 285 (3) ◽

pp. 857-862 ◽

Cited By ~ 32

Author(s):

K Suzuki ◽

K Shimizu ◽

T Hamamoto ◽

Y Nakagawa ◽

T Murachi ◽

...

Keyword(s):

Core Protein ◽

Cysteine Proteinase ◽

Limited Proteolysis ◽

Side Chains ◽

Proteoglycan Degradation ◽

Cartilage Proteoglycans ◽

Neutral Conditions ◽

Calpain Ii ◽

Enzyme Ratio

Degradation of cartilage proteoglycans was investigated under neutral conditions (pH 7.5) by using pig kidney calpain II (EC 3.4.22.17; Ca(2+)-dependent cysteine proteinase). Aggregate and monomer degradation reached a maximum in 5 min at 30 degrees C when the substrate/enzyme ratio was less than 1000:1. The mode of degradation was limited proteolysis of the core protein; the size of the products was larger than that of papain-digested products and comparable with that of trypsin-digested products. The hyaluronic acid-binding region was lost from the major glycosaminoglycan-bearing region after incubation with calpain II. Calpains thus may affect the form of proteoglycans in connective tissue. Ca(2+)-dependent proteoglycan degradation was unique in that proteoglycans adsorb large amounts of Ca2+ ions rapidly before activation of calpain II: 1 mg of pig cartilage proteoglycan monomer adsorbed 1.3-1.6 mu equiv. of Ca2+ ions before activation of calpain II, which corresponds to half the sum of anion groups in glycosaminoglycan side chains. This adsorption of Ca2+ was lost after solvolysis of proteoglycan monomer with methanol/50 mM-HCl, which was used to desulphate glycosaminoglycans. Therefore cartilage proteoglycans are not merely the substrates of proteolysis, but they may regulate the activation of Ca(2+)-dependent enzymes including calpains through tight chelation of Ca2+ ions between glycosaminoglycan side chains.

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Selective inhibition of proteoglycan and hyaluronate synthesis in chondrocyte cultures by cyclofenil diphenol, a non-steroidal weak oestrogen

Biochemical Journal ◽

10.1042/bj2230401 ◽

1984 ◽

Vol 223 (2) ◽

pp. 401-412 ◽

Cited By ~ 11

Author(s):

R M Mason ◽

J D Lineham ◽

M A Phillipson ◽

C M Black

Keyword(s):

Culture Medium ◽

Core Protein ◽

Chondroitin Sulphate ◽

Primary Cultures ◽

Normal Subjects ◽

Dermal Fibroblasts ◽

Proteoglycan Synthesis ◽

Dependent Manner ◽

Glycoprotein Synthesis ◽

Chondrocyte Cultures

Cyclofenil diphenol, a weak non-steroidal oestrogen, binds to albumin. In the presence of concentrations of albumin just sufficient to keep cyclofenil diphenol in solution, the compound inhibited the synthesis of [35S]proteoglycans, [3H]glycoproteins, [3H]hyaluronate and [3H]proteins in primary cultures of chondrocytes from the Swarm rat chondrosarcoma in a dose-dependent manner. When excess albumin was present, conditions were found (90 micrograms of cyclofenil diphenol and 4 mg of albumin per ml of culture medium) which completely inhibited [35S]proteoglycan and [3H]hyaluronate synthesis but had little effect on [3H]protein or [3H]glycoprotein synthesis. The time of onset of inhibition of [35S]proteoglycan synthesis by cyclofenil diphenol was very rapid (t1/2 less than 25 min) and incompatible with an action mediated through suppression of proteoglycan core protein synthesis. Cyclofenil diphenol inhibited the synthesis of [35S]chondroitin sulphate chains onto p-nitrophenyl beta-D-xyloside in the cultures. Cyclofenil diphenol had little effect on the secretion from chondrocytes of [35S]proteoglycans synthesized immediately prior to treatment. Chondrocyte cultures treated with cyclofenil diphenol recovered their biosynthetic activities almost completely within 3 h of removing the compound from the culture medium. Cyclofenil diphenol had a similar inhibitory action on the synthesis of [35S]proteoglycans in secondary cultures of human dermal fibroblasts from both normal subjects and patients with systemic sclerosis. It is proposed that cyclofenil diphenol inhibits the synthesis of [35S]proteoglycans by interfering with the formation of the glycosaminoglycan side chains of these molecules in the Golgi apparatus of cells. The action may be due to disturbance of Golgi membrane organization by the compound.

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Treatment of cartilage proteoglycan aggregate with hydrogen peroxide. Relationship between observed degradation products and those that occur naturally during aging

Biochemical Journal ◽

10.1042/bj2470349 ◽

1987 ◽

Vol 247 (2) ◽

pp. 349-357 ◽

Cited By ~ 26

Author(s):

C R Roberts ◽

J S Mort ◽

P J Roughley

Keyword(s):

Transition Metal ◽

Metal Ion ◽

Core Protein ◽

Degradation Products ◽

Transition Metal Ions ◽

Human Articular Cartilage ◽

Human Aging ◽

Human Cartilage ◽

Cartilage Proteoglycan ◽

Age Related

The effects of treatment of purified neonatal human articular-cartilage proteoglycan aggregate with H2O2 were studied. (1) Exposure of proteoglycan aggregate to H2O2 resulted in depolymerization of the aggregate and modification of the core protein of both the proteoglycan subunits and the link proteins. (2) Treatment of the proteoglycan aggregate with H2O2 rendered the proteoglycan subunits unable to interact with hyaluronic acid, with minimal change in their hydrodynamic size. (3) Specific cleavages of the neonatal link proteins occurred. The order in which the major products were generated and their electrophoretic mobilities resembled the pattern observed during human aging. (4) The proteolytic changes in the link proteins were inhibited in the presence of transition-metal-ion chelators, thiourea or tetramethylurea, suggesting that generation of hydroxyl radicals from H2O2 by trace transition-metal ions via a site-specific Fenton reaction may be responsible for the selective cleavages observed. (5) Cleavage of the link proteins in proteoglycan aggregates by H2O2 was shown to have a limited effect on the susceptibility of these proteins to cleavage by trypsin. (6) The relationship between these changes and those observed in cartilage during human aging suggests that some of the age-related changes in the structure of human cartilage proteoglycan aggregate may be the result of radical-mediated damage.

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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.

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Endocytosis of hyaluronan in rat Kupffer cells

Biochemical Journal ◽

10.1042/bj2860519 ◽

1992 ◽

Vol 286 (2) ◽

pp. 519-526 ◽

Cited By ~ 12

Author(s):

J Alston-Smith ◽

H Pertoft ◽

T C Laurent

Keyword(s):

Kupffer Cells ◽

Culture Medium ◽

Degradation Products ◽

Chondroitin Sulphate ◽

Fluid Phase ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Intracellular Degradation ◽

Acetyl Group ◽

A Minor

The binding, uptake and degradation of hyaluronan (HA) labelled with 3H in its acetyl group were studied in cultured rat Kupffer cells (KC). At 4 degrees C the binding increased with increasing concentrations of HA in the culture medium up to at least 1 microgram/ml, when saturation occurred. Binding could be prevented efficiently by the addition of an excess of unlabelled HA, and to a lesser extent by chondroitin sulphate and oligosaccharide fragments of HA, consisting of four sugars or more. The labelled HA bound to the cells could be removed by incubating the cells with Streptomyces hyaluronidase, or trypsin, indicating that the HA-binding sites are located on the cell surface. At 37 degrees C HA was internalized in a concentration-dependent manner, and degradation products appeared in the supernatant after 1-5 h, depending on the concentration applied. At 50 ng of free HA/ml, each KC accumulated 60 ag of the polysaccharide/min in the first 1 h, and degraded a total amount of 10 fg of HA during an 8 h period. Addition of the negatively charged polysaccharide dextran sulphate reduced binding, and to an even greater extent internalization, of HA in KC, while no effect was observed with dextran. Depletion of intracellular potassium caused a marked reduction in the rate of endocytosis of cell-membrane-associated HA into KC, without affecting binding. Addition of KCl to the culture medium returned endocytosis of [3H]HA to normal levels. There was no effect on binding and a partial effect on internalization by depletion of bivalent cations or in the presence of EDTA. The degradation of [3H]HA by KC cultures was abolished in the presence of weak bases, NH4Cl and chloroquine, supporting the idea that HA is endocytosed into lysosomes prior to degradation. The fluid-phase marker [14C]sucrose was internalized in the cells at much lower rate than was HA. Rates of binding, internalization and degradation of HA in KC point therefore to a specific endocytosis followed by an intracellular degradation to low-M(r) compounds. It was estimated that, under physiological conditions, KC only clear a minor proportion of circulating HA.

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Genome-Wide Identification and Characterization of JAZ Protein Family in Two Petunia Progenitors

Plants ◽

10.3390/plants8070203 ◽

2019 ◽

Vol 8 (7) ◽

pp. 203 ◽

Cited By ~ 1

Author(s):

Shaoze Tian ◽

Siyu Liu ◽

Yu Wang ◽

Kun Wang ◽

Chaoqun Yin ◽

...

Keyword(s):

Expression Patterns ◽

Purifying Selection ◽

Protein Family ◽

Pcr Analysis ◽

Petunia Inflata ◽

Flower Bud ◽

Genome Wide ◽

Meja Treatment ◽

Jaz Proteins

Jasmonate ZIM-domain (JAZ) family proteins are the key repressors in the jasmonate signaling pathway and play crucial roles in plant development, defenses, and responses to stresses. However, our knowledge about the JAZ protein family in petunia is limited. This research respectively identified 12 and 16 JAZ proteins in two Petunia progenitors, Petunia axillaris and Petunia inflata. Phylogenetic analysis showed that the 28 proteins could be divided into four groups (Groups A–D) and further classified into six subgroups (A1, A2, B1, B3, C, and D1); members in the same subgroup shared some similarities in motif composition and sequence structure. The Ka/Ks ratios of seven paralogous pairs were less than one, suggesting the petunia JAZ family might have principally undergone purifying selection. Quantitative real-time PCR (qRT-PCR) analysis revealed that PaJAZ genes presented differential expression patterns during the development of flower bud and anther in petunia, and the expression of PaJAZ5, 9, 12 genes was generally up-regulated after MeJA treatment. Subcellular localization assays demonstrated that proteins PaJAZ5, 9, 12 were localized in nucleus. Yeast two hybrid (Y2H) elucidated most PaJAZ proteins (PaJAZ1-7, 9, 12) might interact with transcription factor MYC2. This study provides insights for further investigation of functional analysis in petunia JAZ family proteins.

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Expression Patterns, Molecular Characterization, and Response to Host Stress of CYP Genes from Phenacoccus solenopsis (Hemiptera: Pseudococcidae)

Insects ◽

10.3390/insects10090264 ◽

2019 ◽

Vol 10 (9) ◽

pp. 264

Author(s):

Lingyu Xi ◽

Dan Liu ◽

Lei Ma ◽

Ying Zhang ◽

Ruirui Sheng ◽

...

Keyword(s):

Molecular Characterization ◽

Host Plants ◽

Adult Stage ◽

Insect Pest ◽

Expression Patterns ◽

Pcr Analysis ◽

Broad Host Range ◽

Phenacoccus Solenopsis ◽

P450 Monooxygenase

The quarantine insect pest Phenacoccus solenopsis (Hemiptera: Pseudococcidae) has a broad host range and is distributed worldwide. Each year, P. solenopsis causes significant crop losses. The detoxification of various xenobiotic compounds involves the cytochrome P450 monooxygenase (CYP) superfamily of enzymes. However, the functions of CYPs in P. solenopsis are poorly understood. In the present study, P. solenopsis was reared from the egg to the adult stage on three host plants: Tomato, cotton, and hibiscus. Thirty-seven P. solenopsis CYP genes were identified and their phylogenetic relationships were analyzed. Eleven CYP genes (PsCYP4NT1, PsCYP4G219, PsCYP6PZ1, PsCYP6PZ5, PsCYP301B1, PsCYP302A1, PsCYP305A22, PsCYP315A1, PsCYP353F1, PsCYP3634A1, and PsCYP3635A2) were selected for quantitative real-time PCR analysis. The results demonstrated marked differences in CYP expression levels in P. solenopsis grown on different host plants. The results will aid the molecular characterization of CYPs and will increase our understanding of CYP expression patterns in P. solenopsis during development and growth on different hosts.

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