Interaction of a brain extracellular matrix protein with hyaluronic acid.
A glial hyaluronate-binding protein (GHAP) was isolated from bovine spinal cord and partially characterized. Bovine GHAP consisted of three immunologically related polypeptides with molecular masses of 76, 64, and 54 kDa and isoelectric points of 4.1, 4.2, and 4.4, respectively. Peptide mapping and partial amino acid sequencing showed that all three polypeptides derive from the same protein. The protein was localized immunohistochemically with rabbit antisera in the white matter surrounding the myelinated axons. Sugar analyses indicated that the three polypeptides are glycosylated and the sugar residues account for at least 30% of their weight. After enzymatic deglycosylation, the apparent molecular mass of the bovine GHAP was reduced to 43 kDa. The biochemical properties of bovine GHAP were compared to those of human GHAP. Initial peptide mapping indicated similarities between bovine and human GHAP. Partial amino acid sequencing of bovine GHAP showed a striking identity (up to 90%) with human GHAP and with the hyaluronate binding domain of the large human fibroblast proteoglycan, versican. Bovine and human GHAP were demonstrated to bind specifically to hyaluronic acid (HA) with one protein molecule binding to an average 17 disaccharide repeating units. The binding of bovine and human GHAP was inhibited by oligosaccharides of HA and specifically by the octamer. Salt concentrations of up to 1 M NaCl had very little effect on the binding of the GHAP to HA. The GHAP-HA interaction was pH dependent. Dissociation only took place at low pH (less than 3.5). Analysis of several polypeptides derived from GHAP by limited proteolysis allowed us to conclude that one of the tandem repeated sequences is sufficient for HA binding and that the aminoterminal domain (which contains an immunoglobulin-like fold) is not involved in the GHAP-HA-binding event.
Perides
G
G
Department of Pathology, Harvard Medical School, West Roxbury, MA.
Biviano
F
F
Bignami
A
A
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D005109:Extracellular Matrix / Q000737:chemistry*; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism
D000596:Amino Acids; D002352:Carrier Proteins; D016326:Extracellular Matrix Proteins; D018960:Hyaluronan Receptors; D010455:Peptides; D006820:Hyaluronic Acid