Abstract
A large proteoglycan (365 kDa), identified with monoclonal antibodies raised against chondroitin sulfate, was isolated from human brain. The isolation required anion-exchange chromatography followed by gel filtration through a Sephacryl S-500 column. The proteoglycan bound specifically to [3H]hyaluronate (HA). The binding was not reduced by high salt concentrations (up to 4 M) and was inhibited at low pH (< 4.0). The binding was inhibited by the octamer and decamer (but not the hexamer) oligosaccharides of HA. Limited proteolysis of the proteoglycan gave rise to a relatively stable polypeptide (80 kDa). The amino-terminal sequence of the 80-kDa polypeptide was identical to the cDNA-derived amino-terminal sequence of versican, a large human fibroblast proteoglycan. A monoclonal antibody raised against bovine proteoglycans and recognizing the versican core protein reacted by immunoblotting with the proteoglycan isolated from human brain. The antibody was used to localize the proteoglycan in acetone-fixed cryostat sections of bovine spinal cord. The localization of the proteoglycan in the central nervous system was identical to that previously reported for glial hyaluronate-binding protein (GHAP), a 60-kDa glycoprotein of the brain extracellular matrix (ECM). However, a major difference was observed with respect to the sensitivity of the two antigens to hyaluronidase. As previously reported, GHAP was released from the tissue by hyaluronidase digestion, whereas the proteoglycan persisted under these conditions. We conclude that the protein-hyaluronate aggregates in brain ECM contain both GHAP and versican, that GHAP is only retained in the ECM by its interaction with hyaluronate, and that the proteoglycan is anchored in some other manner and probably connects cell surfaces with the ECM since it was not released by hyaluronidase digestion.
Highlights
A largeproteoglycan (365 kDa),identifiedwith and cartilage ECMs share common properties because both monoclonal antibodies raised against chondroitin sul- tissues are not permissive to axonal growth
The bindingof the aggregating proteoglycan (Ag-Pg) to ['HIHA was inhibited by from human brain is shown in Fig. IC.In spinal cord white matter, the immunoreactive material surrounded individual myelinated axons (Fig. 5a)
The presence of chondroitin sulfate proteoglycans in the brain has been demonstrated by several studies
Summary
The abbreviations used are: ECM, extracellular matrix; Ag-Pg, (pH 7.4) containing 0.1 mM CaC12in a final volume of 200 p1 a t 37 “C. aggregating proteoglycan; GHAP, glial hyaluronate-binding protein; Deglycosylation-20 or 4 pg of the proteoglycan were incubated for HA, hyaluronate. To determine whether the isolated protein is a proteoglycan, we used apolyclonal antibody against chondroitin sulfate and a monoclonal antibody against the stubs remaining after were covered with a drop of 0.1 M phosphate buffer (pH 5.0) contain- digestion of chondroitin sulfateproteoglycans with chondroiing 0.15 M NaCI, 1mM EDTA, 1mM phenylmethylsulfonyl fluoride, 1pg/ml pepstatin, 1mM iodoacetic acid, 250 pg/ml ovomucoid (type IV-0; Sigma), and 20 turbidity reducing units/ml Streptomyces hyaluronidase.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.