scholarly journals Variations in aggrecan structure modulate its susceptibility to aggrecanases

2003 ◽  
Vol 375 (1) ◽  
pp. 183-189 ◽  
Author(s):  
Peter J. ROUGHLEY ◽  
James BARNETT ◽  
Fengrong ZUO ◽  
John S. MORT
Keyword(s):  
Chondroitin Sulphate ◽  
Human Cartilage ◽  
In Vitro Degradation ◽  
Link Protein ◽  
Rich Domain ◽  
Sds Page ◽  
Lower Ability ◽  
Proteoglycan Aggregates ◽  
Fetal Bovine

Proteoglycan aggregates and purified aggrecan from adult and fetal bovine cartilage and adult and neonatal human cartilage were subjected to in vitro degradation by recombinant aggrecanase-1 and aggrecanase-2. The ability of the aggrecanases to cleave within the aggrecan IGD (interglobular domain) and CS2 domain (chondroitin sulphate-rich domain 2) was monitored by SDS/PAGE and immunoblotting. Aggrecanase-2 showed a similar ability to cleave within the IGD of adult and immature aggrecan, whereas aggrecanase-1 was less efficient in cleavage in the IGD of immature aggrecan, for both the bovine and the human substrates. Both aggrecanases showed a similar ability to cleave within the CS2 domain of bovine aggrecan irrespective of age, but showed a much lower ability to cleave within the CS2 domain of human aggrecan. Equivalent results were obtained whether aggrecan was present in isolation or as part of proteoglycan aggregates. When proteoglycan aggregates were used, neither aggrecanase was able to cleave link protein. Thus, for aggrecan cleavage by aggrecanases, variations in cleavage efficiency exist with respect to the species and age of the animal from which the aggrecan is derived and the type of aggrecanase being used.

scholarly journals Metalloproteinase digestion of cartilage proteoglycan. Pattern of cleavage by stromelysin and susceptibility to collagenase

1991 ◽  
Vol 279 (3) ◽  
pp. 733-739 ◽  
Author(s):  
C Hughes ◽  
G Murphy ◽  
T E Hardingham
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Relative Viscosity ◽  
Gel Chromatography ◽  
High Concentration ◽  
Link Protein ◽  
Rapid Fall ◽  
Sds Page ◽  
Proteoglycan Aggregates ◽  
Rabbit Bone

The action of purified rabbit bone stromelysin was investigated on proteoglycan aggregates from pig laryngeal cartilage. The enzyme caused a rapid fall in viscosity of proteoglycan aggregate solution (6 mg/ml), and the products of a partial digest (60% loss of relative viscosity) and a complete digest (95% loss of relative viscosity) were characterized. Analysis by gel chromatography on Sepharose 2B under associative conditions showed that 95% of the glycosaminoglycans in the complete digest were in small-sized fragments, whereas most of the hyaluronan-binding G1 domain and link protein remained intact and bound to hyaluronan. In contrast, there was extensive digestion of the G2 domain which resulted in 76% loss in its detection by immunoassay. Analysis of the partial digest also showed considerable loss (40%) of detection of the G2 domain, but the glycosaminoglycan-rich fragments were much larger than in the complete digest. There was also much less cleavage to create small fragments containing the G1 domain. This was evident on SDS/PAGE analysis where a 58 kDa G1 domain fragment was abundant in the complete digest, but was only present in small amounts in the partial digest. There was also only very limited conversion of link protein from a 44 kDa form to a 40 kDa form. The digestion of proteoglycan aggregate (6 mg/ml) by stromelysin was unaffected by the addition of a high concentration of extra chondroitin sulphate chains (14 mg/ml), and the digestion of proteoglycan monomer showed that the G1 domain was resistant to stromelysin digestion even when not bound to hyaluronan and link protein. The results show that stromelysin degrades the proteoglycan protein core with major cleavages close to, but not within, the G1 domain, and extensive cleavage in other regions. Experiments with purified collagenase, a metalloproteinase structurally related to stromelysin, showed that it too cleaved proteoglycan at several sites within the glycosaminoglycan-rich region of the core protein. Metalloproteinase attack on proteoglycan thus not only occurs with stromelysin but also with collagenase.

The uptake of labelled sulphate by human cartilage cells and its use as a test for viability

1956 ◽  
Vol 144 (917) ◽  
pp. 572-576 ◽  
Keyword(s):  
Chondroitin Sulphate ◽  
Reliable Indicator ◽  
Human Cartilage ◽  
Cartilage Cells

Human cartilage will incorporate labelled sulphate into chondroitin sulphate in vitro . This activity is apparently exhibited only by the living chondrocyte, and it should serve as a reliable indicator of viability of the tissue.

scholarly journals Proteoglycans in normal and severely osteoarthritic human cartilage

1980 ◽  
Vol 187 (3) ◽  
pp. 781-787 ◽  
Author(s):  
N Vasan
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Binding Capacity ◽  
Osteoarthritic Cartilage ◽  
Human Cartilage ◽  
Controlled Pore Glass ◽  
Pore Glass ◽  
Proteoglycan Aggregates ◽  
Two Peaks ◽  
Two Populations

Proteoglycans from osteoarthritic cartilage were compared with those from normal articular cartilage. Normal proteoglycan aggregates are larger in size and more homogeneous than those in osteoarthritis. Proteoglycan monomers from both sources gave two peaks on controlled pore glass-bead chromatography. Although the retarded material from normal cartilage showed an affinity for hyaluronate, the same material from osteoarthritic cartilage did not. The hyaluronate-binding capacity of the material which is partly in the void volume and partly retarded was similar in both types of cartilage. These results suggest that in osteoarthritic cartilage the proteoglycan aggregates are smaller and more heterogeneous and that the chondroitin sulphate side chains are shorter. They also indicate that there are two populations of proteoglycan, one with its hyaluronate-binding-protein region of core protein intact and the other either possessing an inactive binding region or totally lacking it.

scholarly journals Evidence of a defined spatial arrangement of hyaluronate in the central filament of cartilage proteoglycan aggregates

1995 ◽  
Vol 307 (2) ◽  
pp. 595-601 ◽  
Author(s):  
M Mörgelin ◽  
M Paulsson ◽  
D Heinegård ◽  
U Aebi ◽  
J Engel
Keyword(s):  
Electron Microscopy ◽  
Core Protein ◽  
Gel Filtration ◽  
Spatial Arrangement ◽  
Link Protein ◽  
Original Length ◽  
Central Filament ◽  
Proteoglycan Aggregates ◽  
Rotary Shadowing

Aggregates of proteoglycans from the Swarm rat chondrosarcoma reassembled in vitro have been studied by rotary-shadowing electron microscopy, and shown to be similar to native structures that have never been dissociated [Mörgelin, Engel, Heinegård and Paulsson (1992) J. Biol. Chem. 267, 14275-14284]. A hyaluronate with defined chain length (HAshort) has now been prepared by autoclaving high-Mr hyaluronate and fractionation to a narrow size distribution by gel filtration. Proteoglycan monomers, core protein, hyaluronate-binding region and link protein were combined with HAshort. Free chains of HAshort and reconstituted complexes with proteoglycan, link protein and aggrecan fragments were examined by electron microscopy after rotary shadowing. Length measurements showed that the hyaluronate was condensed to about half of its original length on binding intact aggrecan monomers, any aggrecan fragment or link protein alone. This strongly implies that hyaluronate adopts a defined spatial arrangement within the central filament of the aggregate, probably different from its secondary structure in solution. No differences in length were observed between link-free and link-stabilized aggregates.

scholarly journals Matrix metalloproteinases cleave at two distinct sites on human cartilage link protein

1993 ◽  
Vol 295 (2) ◽  
pp. 595-598 ◽  
Author(s):  
Q Nguyen ◽  
G Murphy ◽  
C E Hughes ◽  
J S Mort ◽  
P J Roughley
Keyword(s):  
Matrix Metalloproteinases ◽  
Degradation Products ◽  
Limited Proteolysis ◽  
Human Cartilage ◽  
Link Protein ◽  
Sequencing Studies ◽  
Proteoglycan Aggregates ◽  
Protein Components ◽  
Gelatinases A And B

The actions of human recombinant stromelysins-1 and -2, collagenase, gelatinases A and B and matrilysin on neonatal human proteoglycan aggregates were examined. With the exception of gelatinase B, aggrecan was degraded extensively by most metalloproteinases studied, whereas link protein showed only limited proteolysis. Sequencing studies of modified link protein components revealed that stromelysins-1 and -2, gelatinases A and B and collagenase cleaved specifically between His16 and Ile17, and matrilysin, stromelysin-2 and gelatinase A cleaved between Leu25 and Leu26. Cleavage at the former bond generated a link protein component with the same N-terminus as that isolated from newborn human cartilage. Based on previously determined in situ cleavage sites it is evident that matrix metalloproteinases are not solely responsible for the accumulation of link protein degradation products in adult human cartilage, indicating that additional proteolytic agents are involved in the normal catabolism of human cartilage matrix.

scholarly journals Assembly of proteoglycan aggregates in cultures of chondrocytes from bovine tracheal cartilage

1981 ◽  
Vol 199 (1) ◽  
pp. 17-29 ◽  
Author(s):  
S Björnsson ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Cell Cultures ◽  
Low Ph ◽  
Link Protein ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycan ◽  
Proteoglycan Aggregates ◽  
Cartilage Proteoglycans ◽  
Free Monomer

The assembly of proteoglycan aggregates in chondrocyte cell cultures was examined in pulse-chase experiments with the use of [35S]sulphate for labelling. Rate-zonal centrifugation in linear sucrose density gradients (10-50%, w/v) was used to separate the aggregated proteoglycans from monomers and to assess the size of the newly formed aggregates. The proportion of aggregates stabilized by link protein was assessed by competition with added exogenous aggregate components. The capacity of the proteoglycans synthesized in culture to compete with exogenous nasal-cartilage proteoglycans for binding was studied in dissociation-reassociation experiments. The results were as follows. (a) The proteoglycan monomers and the hyaluronic acid are exported separately and combined extracellularly. (b) The size of the aggregates increases gradually with time as the proportion of monomers bound to hyaluronic acid increases. (c) All of the aggregates present at a particular time appear to be link-stabilized and therefore not dissociated by added excess of nasal-cartilage proteoglycan monomer or hyaluronic acid oligomers. (d) The free monomer is apparently present as a complex with link protein. The monomer-link complexes are then aggregated to the hyaluronic acid. (e) The aggregates synthesized in vitro and the nasal-cartilage aggregates differ when tested for link-stabilization by incubation at low pH. The aggregates synthesized in vitro were completely dissociated whereas the cartilage proteoglycans remained aggregated. The results obtained from dissociation-reassociation experiments performed at low pH indicate that the proteoglycan monomer synthesized in vitro does not bind the hyaluronic acid or the link protein as strongly as does the nasal-cartilage monomer.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

In Vitro Degradation Of beta-Amyloid[25-35] Peptide

2001 ◽  
Vol 8 (6) ◽  
pp. 423-428 ◽  
Author(s):  
Krisztina Jost ◽  
Jozsef Varga ◽  
Botond Pence ◽  
Marta Zarandi
Keyword(s):  
Beta Amyloid ◽  
In Vitro Degradation

scholarly journals In Vitro Degradation of Absorbable Zinc Alloys in Artificial Urine

Materials ◽  
2019 ◽  
Vol 12 (2) ◽  
pp. 295 ◽  
Author(s):  
Sébastien Champagne ◽  
Ehsan Mostaed ◽  
Fariba Safizadeh ◽  
Edward Ghali ◽  
Maurizio Vedani ◽  
...  
Keyword(s):  
Surface Characterization ◽  
Urinary Tract Obstruction ◽  
Calcium Content ◽  
Corrosion Layer ◽  
Uniform Corrosion ◽  
In Vitro Degradation ◽  
Pure Zinc ◽  
Zinc Alloys ◽  
Artificial Urine

Absorbable metals have potential for making in-demand rigid temporary stents for the treatment of urinary tract obstruction, where polymers have reached their limits. In this work, in vitro degradation behavior of absorbable zinc alloys in artificial urine was studied using electrochemical methods and advanced surface characterization techniques with a comparison to a magnesium alloy. The results showed that pure zinc and its alloys (Zn–0.5Mg, Zn–1Mg, Zn–0.5Al) exhibited slower corrosion than pure magnesium and an Mg–2Zn–1Mn alloy. The corrosion layer was composed mostly of hydroxide, carbonate, and phosphate, without calcium content for the zinc group. Among all tested metals, the Zn–0.5Al alloy exhibited a uniform corrosion layer with low affinity with the ions in artificial urine.

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