Isolation and characterization of proteoglycans.
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Animals↗
Biomedical subjects
Y Sommarin
Publications and source records attributed to Y Sommarin.
Two novel matrix proteins isolated from articular cartilage show wide distributions among connective tissues.
Two proteins of Mr = 58,000 and 59,000, respectively, were purified from 4 M guanidinium chloride extracts of articular cartilage by dissociative CsCl-density gradient centrifugation followed by gel chromatography on Sephadex G-200 and ion exchange chromatography on DEAE-cellulose. The two proteins differ in ionic properties and only the one with Mr = 59,000 bound to the ion exchanger. Although the two proteins showed dissimilar peptide patterns after proteolysis, their amino acid composition was similar, with very high contents of leucine and aspartic acid/asparagine. The two proteins showed no cross-reactivity in radioimmunoassays. By use of these assays, the proteins were demonstrated in extracts of most connective tissues, with high contents of about 0.1% of tissue wet weight determined in several types of cartilage. Among the non-cartilage connective tissues, tendon and sclera had the highest contents of the proteins, i.e. about 0.1% of the tissue wet weight. Bone extracts, on the other hand, contained insignificant amounts of the proteins. Only the Mr = 59,000 protein was detected in serum, its concentration being about 33 micrograms/l. Both proteins were shown to be localized in the extracellular matrix of cartilage, predominantly in the territorial matrix, by using indirect immunofluorescence.
Amino Acids↗
Four classes of cell-associated proteoglycans in suspension cultures of articular-cartilage chondrocytes.
The characteristics of cell-associated proteoglycans were studied and compared with those from the medium in suspension cultures of calf articular-cartilage chondrocytes. By including hyaluronic acid or proteoglycan in the medium during [35S]sulphate labelling the proportion of cell-surface-associated proteoglycans could be decreased from 34% to about 15% of all incorporated label. A pulse-chase experiment indicated that this decrease was probably due to blocking of the reassociation with the cells of proteoglycans exported to the medium. Three peaks of [35S]sulphate-labelled proteoglycans from cell extracts and two from the medium were isolated by gel chromatography on Sephacryl S-500. These were characterized by agarose/polyacrylamide-gel electrophoresis, by SDS/polyacrylamide-gel electrophoresis of core proteins, by glycosaminoglycan composition and chain size as well as by distribution of glycosaminoglycans in proteolytic fragments. The results showed that associated with the cells were (a) large proteoglycans, typical for cartilage, apparently bound to hyaluronic acid at the cell surface, (b) an intermediate-size proteoglycan with chondroitin sulphate side chains (this proteoglycan, which had a large core protein, was only found associated with the cells and is apparently not related to the large proteoglycans), (c) a small proteoglycan with dermatan sulphate side chains with a low degree of epimerization, and (d) a somewhat smaller proteoglycan containing heparan sulphate side chains. The medium contained a large aggregating proteoglycan of similar nature to the large cell-associated proteoglycan and small proteoglycans with dermatan sulphate side chains with a higher degree of epimerization than those of the cells, i.e. containing some 20% iduronic acid.
Animals↗
Common structures of the core proteins of interstitial proteoglycans.
Connective tissues, with few exceptions, contain easily distinguishable large and small proteoglycans with chondroitin sulphate or dermatan sulphate side-chains. One group consists of the large aggregating proteoglycans that have the capacity to interact specifically with hyaluronate, thereby forming very large aggregates. These proteoglycans can be divided into two families which can be separated by electrophoresis. Preliminary results indicate that one of these may be derived from the other by processing in the extracellular matrix. Although most prominent in cartilage, similar proteoglycans are present in many types of tissue, such as aorta, sclera and tendon. Another population are the large non-aggregating proteoglycans, identified in cartilage. These proteoglycans show structural features partially different from any of the others. They may represent a distinct population of molecules present in many connective tissues. Many tissues contain major populations of small, non-aggregating proteoglycans. These can be divided into two major groups, differing in the composition of their core proteins, while having similar types of side-chain constituents. One group is represented by proteoglycans from nasal cartilage and aorta, while the other is represented by proteoglycans from tendon, bone, sclera and cornea.
Animals↗
Assay of proteoglycan populations using agarose-polyacrylamide gel electrophoresis.
The agarose-polyacrylamide gel electrophoresis procedure for the analysis of proteoglycans originally described by C. A. McDevitt and H. Muir (1971, Anal. Biochem. 44, 612-622) has been modified to minimize trailing and to allow the analysis of crude samples, i.e., tissue extracts. A slab gel system was used, permitting reproducible analysis of many samples. Procedures are described that can be used to separate and quantify several subpopulations of proteoglycans and also to quantify the proportion of proteoglycans capable of aggregating with hyaluronic acid. Applications of the procedure include transfer to nitrocellulose paper followed by immunological detection of proteoglycans as well as fluorography of separated, radiolabeled proteoglycans.
Animals↗
Separation and characterization of two populations of aggregating proteoglycans from cartilage.
Intermediary gel immunoelectrophoresis was used to show that purified aggregating cartilage proteoglycans from 2-year-old steers contain two distinct populations of molecules and that only one of these is immunologically related to non-aggregating cartilage proteoglycans. The two types of aggregating proteoglycans were purified by density-gradient centrifugation in 3.5M-CsCl/4M-guanidinium chloride and separated by zonal rate centrifugation in sucrose gradients. The higher-buoyant-density faster-sedimenting proteoglycan represented 43% of the proteoglycans in the extract. It had a weight-average Mr of 3.5 X 10(6), did not contain a well-defined keratan sulphate-rich region, had a quantitatively dominant chondroitin sulphate-rich region and contained 5.9% protein and 23% hexosamine. The lower-buoyant-density, more slowly sedimenting, proteoglycan represented 15% of the proteoglycans in the extract. It had a weight-average Mr of 1.3 X 10(6), contained both the keratan sulphate-rich and the chondroitin sulphate-rich regions and contained 7.3% protein and 23% hexosamine. Each of the proteoglycan preparations showed only one band on agarose/polyacrylamide-gel electrophoresis. The larger proteoglycan had a lower mobility than the smaller. The distribution of chondroitin sulphate chains along the chondroitin sulphate-rich region was similar for the two types of proteoglycans. The somewhat larger chondroitin sulphate chains of the larger proteoglycan could not alone account for the larger size of the proteoglycan. Peptide patterns after trypsin digestion of the proteoglycans showed great similarities, although the presence of a few peptides not shared by both populations indicates that the core proteins are partially different.
Amino Acids↗
Specific interaction between cartilage proteoglycans and hyaluronic acid at the chondrocyte cell surface.
Binding of exogenous [35S]sulphate-labelled cartilage proteoglycans to cells was studied with suspension cultures of calf articular-cartilage chondrocytes. Proteoglycans interact with hyaluronic acid at the cell surface via their hyaluronic acid-binding region. The binding is time-dependent and saturable, but does not appear to be freely reversible. The bound 35S-labelled proteoglycans are located at the cell surface, and only small proportions of the proteoglycans are internalized.
Animals↗
Metabolism of cartilage proteins in cultured tissue sections.
The biosynthesis and turnover of cartilage proteins was studied in organ cultures of bovine tracheal-cartilage sections. In cultures labelled with [3H]leucine, more than 99% of the labelled macromolecules were retained in the sections. About half of the [3H]leucine-labelled protein was extracted with 4M-guanidinium chloride. The incorporation of [3H]leucine into protein extractable with guanidinium chloride was linear with time, after an initial delay of 20-25 min. The 148kDa and 36kDa cartilage proteins were major labelled components in this extract. The elimination of the proteins was studied by using a pulse-chase protocol. The 148kDa protein was found to have a very slow turnover, similar to that of proteoglycans, whereas the 36kDa protein was eliminated more rapidly.
Animals↗
Proteoglycans and matrix proteins in cartilage.
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Animals↗
ATP-A activity in cultured human glioma cells.
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Cells, Cultured↗
Characterization of monoclonal antibodies to proteinase-3 and application in the study of epitopes for classical anti-neutrophil cytoplasm antibodies.
Wegener's granulomatosis is associated with autoantibodies (classical antineutrophil cytoplasm antibodies, c-ANCAs) to a serine protease called proteinase-3. In this study three IgG class monoclonal antibodies, designated 4A3, 4A5 and 6A6, against proteinase-3 were generated to study the immune response of c-ANCA-positive patients. All monoclonal antibodies were tested by immunofluorescence staining of human granulocytes and gave a staining pattern identical to the pattern obtained with sera from patients with Wegener's granulomatosis. On protein transfer blots of neutrophil alpha-granule extract, all monoclonal antibodies stained a 29-kD protein band corresponding to proteinase-3. Also, in a direct binding ELISA with alpha-granule extract as antigen, binding of the monoclonal antibodies to the antigen could be completely inhibited by adding a pure preparation of proteinase-3. In the ELISA type of competition experiments, none of the monoclonal antibodies could substantially inhibit binding of any of the other antibodies to the antigen, indicating that all monoclonal antibodies recognize separate epitopes on the antigen. The same conclusion was reached from experiments by real-time competition analysis using a Pharmacia BIAcore system. The monoclonal antibodies were used to study whether some epitopes on proteinase-3 are preferred by patient autoantibodies. A total of 36 patients sera was tested by competing for autoantibody binding to proteinase-3 with the monoclonal antibodies in an ELISA. Autoantibody binding to proteinase-3 could be partially or completely inhibited by either the 4A5 (50% of the sera) or by the 6A6 antibody (11%). The 4A3 antibody could only partially inhibit 8 of the sera (22%).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗