Immunochemical analysis of cartilage proteoglycans. Radioimmunoassay of the molecules and the substructures.
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ABSTRACT:
SUBMITTER: Wieslander J
PROVIDER: S-EPMC1162452 | biostudies-other | 1980 Jun
REPOSITORIES: biostudies-other
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Project description: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.
| S-EPMC1186592 | biostudies-other
Project description:Antibodies directed against whole bovine nasal-cartilage proteoglycan and against the hyaluronic acid-binding region and chondroitin sulphate peptides from the same molecule were used in immunodiffusion and immunoelectromigration experiments. Proteoglycans from bovine nasal and tracheal cartilage showed immunological identity, with all three antisera. Proteoglycans from pig hip articular cartilage, dog hip articular cartilage, human tarsal articular cartilage and rat chondrosarcoma reacted with all the antisera and showed immunological identity with the corresponding structures isolated from bovine nasal-cartilage proteoglycans. In contrast, proteoglycans from rabbit articular cartilage, rabbit nasal cartilage and cultured chick limb buds did not react with the antibodies directed against the hyaluronic acid-binding region, though reacting with antibodies raised against whole proteoglycan monomer and against chondroitin sulphate peptides. All the proteoglycans gave two precipitation lines with the anti-(chondroitin sulphate peptide) antibodies. Similarly, the proteoglycans reacting with the anti-(hyaluronic acid-binding region) antibodies gave two precipitation lines. The results indicate the presence of at least two populations of aggregating proteoglycan monomers in cartilage. The relative affinity of the antibodies for cartilage proteoglycans and proteoglycan substructures from various species was determined by radioimmunoassay. The affinity of the anti-(hyaluronic acid-binding region) antibodies for the proteoglycans decreased in the order bovine, dog, human and pig cartilage. Rat sternal-cartilage and rabbit articular-cartilage proteoglycans reacted weakly, whereas chick limb-bud and chick sternal-cartilage proteoglycans did not react. In contrast, the affinity of antibodies to chondroitin sulphate peptides for proteoglycans increased in the order bovine cartilage, chick limb bud and chick sternal cartilage, dog cartilage, rat chondrosarcoma, human cartilage, pig cartilage, rat sternal cartilage and rabbit cartilage.
| S-EPMC1163337 | biostudies-other
Project description:Proteoglycan monomers from guinea-pig costal cartilage, bovine nasal and bovine tracheal cartilage were observed in the electron microscope after being spread in a monomolecular layer with cytochrome c. The proteoglycan molecule appeared as an extended central core filament to which side-chain filaments were attached at various intervals. The molecules from the three sources displayed great ultrastructural similarities. On average, the core filament was about 290 nm long, there were about 25 side-chain filaments per core filament, the side-chain filaments were about 45 nm long, and the distance between the attachment points of the side-chain filaments to the core filament was about 11 nm. With regard to the overall size of the molecules, no evidence of distinct subpopulations was obtained. Good correlation was found between ultrastructural data for the proteoglycan molecules and chemical data obtained by enzyme digestions and gel chromatography. Together these data strongly support the interpretation of the electron-microscopic pictures as indicating a central filament corresponding to the protein core and side-chain filaments corresponding to the chondroitin sulphate chain clusters of the proteoglycan monomers.
| S-EPMC1172338 | biostudies-other
Project description:In drug development, late stage toxicity issues of a compound are the main cause of failure in clinical trials. In silico methods are therefore of high importance to guide the early design process to reduce time, costs and animal testing. Technical advances and the ever growing amount of available toxicity data enabled machine learning, especially neural networks, to impact the field of predictive toxicology. In this study, cytotoxicity prediction, one of the earliest handles in drug discovery, is investigated using a deep learning approach trained on a highly consistent in-house data set of over 34,000 compounds with a share of less than 5% of cytotoxic molecules. The model reached a balanced accuracy of over 70%, similar to previously reported studies using Random Forest. Albeit yielding good results, neural networks are often described as a black box lacking deeper mechanistic understanding of the underlying model. To overcome this absence of interpretability, a Deep Taylor Decomposition method is investigated to identify substructures that may be responsible for the cytotoxic effects, the so-called toxicophores. Furthermore, this study introduces cytotoxicity maps which provide a visual structural interpretation of the relevance of these substructures. Using this approach could be helpful in drug development to predict the potential toxicity of a compound as well as to generate new insights into the toxic mechanism. Moreover, it could also help to de-risk and optimize compounds.
| S-EPMC7292813 | biostudies-literature
Project description:Because cartilage lacks nerves, blood vessels, and lymphatic vessels, it is thought to contain factors that inhibit the growth and development of those tissues. Chondroitin sulfate proteoglycans (CSPGs) are a major extracellular component in cartilage. CSPGs contribute to joint flexibility and regulate extracellular signaling via their attached glycosaminoglycan, chondroitin sulfate (CS). CS and CSPG inhibit axonal regeneration; however, their role in blood vessel formation is largely unknown. To clarify the function of CSPG in blood vessel formation, we tested salmon nasal cartilage proteoglycan (PG), a member of the aggrecan family of CSPG, for endothelial capillary-like tube formation. Treatment with salmon PG inhibited endothelial cell adhesion and in vitro tube formation. The anti-angiogenic activity was derived from CS in the salmon PG but not the core protein. Salmon PG also reduced matrix metalloproteinase expression and inhibited angiogenesis in the chick chorioallantoic membrane. All of these data support an anti-angiogenic role for CSPG in cartilage.
| S-EPMC5614546 | biostudies-literature
Project description: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.
| S-EPMC1144431 | biostudies-other
Project description:Electronegative LDL (LDL(-)) is a minor subfraction of modified LDL present in plasma. Among its atherogenic characteristics, low affinity to the LDL receptor and high binding to arterial proteoglycans (PGs) could be related to abnormalities in the conformation of its main protein, apolipoprotein B-100 (apoB-100). In the current study, we have performed an immunochemical analysis using monoclonal antibody (mAb) probes to analyze the conformation of apoB-100 in LDL(-). The study, performed with 28 anti-apoB-100 mAbs, showed that major differences of apoB-100 immunoreactivity between native LDL and LDL(-) concentrate in both terminal extremes. The mAbs Bsol 10, Bsol 14 (which recognize the amino-terminal region), Bsol 2, and Bsol 7 (carboxyl-terminal region) showed increased immunoreactivity in LDL(-), suggesting that both terminal extremes are more accessible in LDL(-) than in native LDL. The analysis of in vitro-modified LDLs, including LDL lipolyzed with sphingomyelinase (SMase-LDL) or phospholipase A(2) (PLA(2)-LDL) and oxidized LDL (oxLDL), suggested that increased amino-terminal immunoreactivity was related to altered conformation due to aggregation. This was confirmed when the aggregated subfractions of LDL(-) (agLDL(-)) and oxLDL (ag-oxLDL) were isolated and analyzed. Thus, Bsol 10 and Bsol 14 immunoreactivity was high in SMase-LDL, ag-oxLDL, and agLDL(-). The altered amino-terminal apoB-100 conformation was involved in the increased PG binding affinity of agLDL(-) because Bsol 10 and Bsol 14 blocked its high PG-binding. These observations suggest that an abnormal conformation of the amino-terminal region of apoB-100 is responsible for the increased PG binding affinity of agLDL(-).
| S-EPMC3020719 | biostudies-literature
Project description:BackgroundIn osteoarthritis (OA), cartilage matrix is lost despite vigorous chondrocyte anabolism. In this study, we attempted to determine whether altered matrix synthesis is involved in this paradox in disease progression through gene expression analysis and ultrastructural analysis of collagen fibrils within the cartilage matrix.MethodsCartilage tissues were obtained from 29 end-stage OA knees and 11 control knees. First, cDNA microarray analysis was performed and the expression of 9 genes involved in collagen fibrillogenesis was compared between OA and control cartilages. Then their expression was investigated in further detail by a quantitative polymerase chain reaction (qPCR) analysis combined with laser capture microdissection. Finally, collagen fibril formation was compared between OA and control cartilage by transmission electron microscopy.ResultsThe result of the microarray analysis suggested that the expression of type IX and type XI collagens and fibrillogenesis-related small leucine-rich proteoglycans (SLRPs) may be reduced in OA cartilage relative to the type II collagen expression. The qPCR analysis confirmed these results and further indicated that the relative reduction in the minor collagen and SLRP expression may be more obvious in degenerated areas of OA cartilage. An ultrastructural analysis suggested that thicker collagen fibrils may be formed by OA chondrocytes possibly through reduction in the minor collagen and SLRP expression.ConclusionsThis may be the first study to report the possibility of altered collagen fibrillogenesis in OA cartilage. Disturbance in collagen fibril formation may be a previously unidentified mechanism underlying the loss of cartilage matrix in OA.
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Project description:In this paper, we report a novel normal-mode analysis for supramolecular complexes, named fSUB. The method models a complex as a group of flexible substructures. The low-frequency substructure modes are first determined with substructures in isolation, and the motions of the whole complex are then calculated on the basis of substructure modes and substructure-substructure interactions. The calculation of modes in fSUB requires modal analysis without initial energy minimization, which is essential for maintaining energetic and structural consistency between substructures and whole complex. Compared with other coarse-grained methods, such as the RTB method, fSUB delivers much more accurate modes for the complex and allows for the choice of much larger substructures. The method can also accommodate any type of substructure arrangement including covalent bonds across the interface. In tests on molecular chaperonin GroEL (7350 residues) and HK97 capsid complex (118,092 residues), fSUB was shown to be much more efficient in terms of combined accuracy and demand of computing resources. Our results clearly demonstrated the vital importance of including substructure flexibility in complex modal analysis, as the deformational patterns of substructures were found to play an important role even in the lowest frequency modes of the whole complex.
| S-EPMC3547612 | biostudies-literature
Project description:Proteoglycans were extracted from normal human articular cartilage of various ages with 4M-guanidinium chloride and were purified and characterized by using preformed linear CsCl density gradients. With advancing age, there was a decrease in high-density proteoglycans of low protein/uronic acid weight ratio and an increase in the proportion of lower-density proteoglycans, richer in keratan sulphate and protein. Proteoglycans of each age were also shown to disaggregate in 4M-guanidinium chloride and at low pH and to reaggregate in the presence of hyaluronic acid and/or low-density fractions. Osteoarthrotic-cartilage extracts had an increased content of higher-density proteoglycans compared with normal cartilage of the same age, and results also suggested that these were not mechanical or enzymic degradation products, but were possibly proteoglycans of an immature nature.
| S-EPMC1184201 | biostudies-other