Isolation of a large aggregating proteoglycan from human brain.
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.
Perides
G
G
Department of Pathology, Harvard Medical School, Boston, Massachusetts.
Rahemtulla
F
F
Lane
W S
WS
Asher
R A
RA
Bignami
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D011508:Chondroitin Sulfate Proteoglycans / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000737:chemistry; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D006821:Hyaluronoglucosaminidase; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011509:Proteoglycans / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D013116:Spinal Cord / Q000166:cytology*
D011508:Chondroitin Sulfate Proteoglycans; D010446:Peptide Fragments; D011509:Proteoglycans; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase