J H Kimura

[45] Proteoglycans: Isolation and Characterization

Publisher Summary This chapter presents procedure for extraction and characterization of proteoglycans. In general, tissue or cultures should be processed for extraction quickly and using procedures that minimize endogenous degradation. Fresh tissue should be chilled on ice, cleaned, and minced quickly. Extraction efficiency is often improved when finer tissue pieces are used to expose more surface area to the solvent. Once the proteoglycans are in solution, different fractionation procedures (ion exchange in 7 M urea, rate zonal-velocity, centrifugation molecular-sieve chromatography, electrophoresis in composite polyacrylamide-agarose gels), which take advantage of their macromolecular properties, are used to purify them from other macromolecules in the extract and to begin to separate them from each other. Application of the various techniques described above has identified the different classes of proteoglycans in a starting extract based upon differences in charge density, hydrodynamic size, and buoyant density. Such analyses not only provide information about the physical properties of the proteoglycans but help design effective strategies for purifying at least some of the subpopulations of proteoglycans identified.

Separate effects of exogenous hyaluronic acid on proteoglycan synthesis and deposition in pericellular matrix by cultured chick embryo limb chondrocytes

Abstract The effects of exogenous hyaluronic acid on cell cultures of chick embryo limb chondrocytes are reported in this paper. The evidence shows that exogenous hyaluronic acid (HA) can both depress the incorporation of 35 SO 4 into glycosaminoglycans and cause a displacement of newly synthesized proteoglycan from the cell layer to the culture medium. The results demonstrate that these two effects are mediated by distinct mechanisms. The displacement effect has a rapid onset (by 2 hr) while the effect of exogenous HA on 35 SO 4 incorporation has a long latency (12 hr). The displacement effect is produced by a lower concentration (5 μg/ml) of hyaluronate oligomers than the effect on 35 SO 4 incorporation (50 μg/ml). In addition, displacement is produced only by hyaluronate oligomers that are decasaccharides or larger. The depression of 35 SO 4 incorporation is produced by tetrasaccharides as well as high molecular weight HA. In fact tetrasaccharides can depress 35 SO 4 incorporation without causing the displacement effect.

Modular Arrangement of Cartilage- and Neural Tissue-specificcis-Elements in the Mouse α2(XI) Collagen Promoter

Type XI collagen, a heterotrimer specific to cartilage matrix, plays an important role in cartilage morphogenesis. We analyzed various α2(XI) collagen promoter-lacZreporter gene constructs in transgenic mice to understand tissue-specific transcriptional regulation. The −530 promoter sequence was sufficient to direct reporter gene expression specifically to cartilage. Further deletion to −500 abolished reporter gene expression in cartilage but activated the expression specific to neural tissues such as brain and neural tube. An additional 47-base pair deletion resulted in random tissue expression patterns. A 24-base pair sequence from −530 to −507 of the α2(XI) promoter was able to switch the activity of the heterologous neurofilament light gene promoter from neural tissues to cartilage. These results suggest that the α2(XI) collagen gene is regulated by at least three modular elements: a basal promoter sequence distal to −453, a neural tissue-specific element (−454 to −500), and a cartilage-specific element (−501 to −530), which inhibits expression in neural tissues and induces expression in cartilage.

An enzyme-linked immunoassay for the cartilage proteoglycan.

Abstract Proteoglycan was purified from a rat chondrosarcoma and antiserum prepared. An enzyme-linked immunoassay was designed using this serum. The assay detected rat and murine, but not chick, high-molecular-weight cartilage proteoglycan. It did not detect noncartilage proteoglycan nor the low-molecular-weight proteoglycans found in cartilage. As little as 100 ng/ml of rat cartilage proteoglycan could be detected.

Characterization of fragments produced by clostripain digestion of proteoglycans from the swarm rat chondrosarcoma

Abstract Rat chondrosarcoma proteoglycan aggregate samples were digested with the protease clostripain (from Clostridium histolyticum ) for various times. The progress of digestion was studied by Sepharose 2B chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After complete digestion, the complex of hyaluronic acid-binding region, link protein, and hyaluronic acid was separated from the chondroitin sulfate-peptide clusters released by the enzyme. In this limit complex, the M r of the link protein was 42,000, slightly smaller than the M r of 45,000 observed for intact link protein. The chondroitin sulfate-peptides contained an average of about seven to eight polysaccharide chains per peptide and, after chondroitinase ABC digestion, were found to consist of two size classes of peptides. By comparison, chondroitin sulfate-peptides isolated from trypsin digests contained four to five chains per peptide and contained primarily the smaller size class of peptides. At early digestion times with clostripain, several distinct molecular weight intermediates containing hyaluronic acid-binding sites were identified on sodium dodecyl sulfate-polyacrylamide gels. These intermediates, with M r , values of about 125,000, 100,000, and 85,000, decreased with increasing digestion time to yield a limit polypeptide ( M r = 67,000). Procedures are described for purifying this limit polypeptide and the link protein for further characterization. The results indicate that clostripain can be used to fragment proteoglycan molecules selectively to define different functional regions for study.

[18] Biosynthesis of cartilage proteoglycan and link protein

Publisher Summary This chapter describe three procedures which have proved useful in the isolation and characterization of newly synthesized proteoglycan and link protein. The first deals with the isolation of newly synthesized link protein and core protein precursor, as well as completed forms of the proteoglycan by taking advantage of their ability to be incorporated into aggregates recoverable from the bottom of CsCl density gradients. The second presents refinements of immunoprecipitation procedures, while the third describes the separation of two populations of newly synthesized aggregating proteoglycan by agarose gel electrophoresis. Bovine articular cartilage aggregating proteoglycans exhibit age-related differences. Electrophoresis in acrylamide-agarose composite gels has been used successfully to show that the matrix contains two populations of aggregating proteoglycans which are present in different ratios at different ages. Electrophoresis in 1% agarose gels has recently been found to offer a number of advantages over the acrylamideagarose composite gel procedure.

Removal of protease from Streptomyces hyaluronidase by affinity chromatography

Abstract A simple method is presented for removing protease(s) from commercial sources of Streptomyces hyaluronidase derived from Streptomyces hyalurolyticus using a column of insolubilized chicken ovomucoid. The protease binds to ovomucoid; 91% of the hyaluronidase and no more than 0.7% of the protease passes through the column. A calcium chloride solution is used to remove the bound protease. Purified hyaluronidase cleaves the hyaluronic acid of proteoglycan aggregates without affecting the size of proteoglycan monomers. The ability of digested monomers to bind hyaluronic acid, however, is somewhat diminished.

Effect of puromycin on cartilage proteoglycan structure and capacity to bind hyaluronic acid

Rat chondrosarcoma chondrocytes were cultured in the presence of puromycin to induce premature termination of core protein precursor. The structure and function of intracellular and extracellular proteoglycans were assessed by molecular sieve chromatography and polyacrylamide gel electrophoresis. [3H]Serine incorporation was maximally inhibited by 3 X 10(-4) M puromycin but unaffected by 10(-5) M puromycin. Proteoglycans synthesized in the presence of puromycin exhibited increased monomer size due to increased chondroitin sulfate chain size, typical of proteoglycans synthesized in the presence of protein synthesis inhibitors, but no loss in ability to bind to hyaluronic acid; and no loss in core protein size was observed after treatment with chondroitinase. These data suggest that chondrocytes select only completed or nearly completed core protein molecules to process into proteoglycans.