Purification and characterization of heparan sulphate proteoglycan from bovine brain.
A heparan sulphate proteoglycan was purified from adult bovine brain tissues and its structure was characterized. The major heparan sulphate proteoglycan from whole bovine brain had a molecular mass of >200 kDa on denaturing SDS/PAGE and a core protein size of 66 kDa following the removal of glycosaminoglycan chains. Fractionation on DEAE-Sephacel showed that this proteoglycan consisted of three major forms having high, intermediate and low overall charge. All core proteins were identical in size and reacted with heparan sulphate proteoglycan-stub antibody and an antibody made to a synthetic peptide based on rat glypican. The three forms of proteoglycans had identical peptide maps and their amino acid compositional analysis did not match any of the known glypicans. The internal sequence of a major peptide showed only 37.5% sequence similarity with human glypican 5. The glycosaminoglycan chain sizes of the three forms of this proteoglycan, determined after beta-elimination by PAGE, were identical. The disaccharide compositional analysis on the heparan sulphate chains from the three forms of the proteoglycan, determined by treatment with a mixture of heparin lyases followed by high-resolution capillary electrophoresis, showed that they differed primarily by degree of sulphation. The most highly sulphated proteoglycan isolated had a disaccharide composition similar to heparan sulphate glycosaminoglycans found in brain tissue. Based on their sensitivity to low pH nitrous acid treatment, the N-sulphate groups in these proteoglycans were found to be primarily in the smaller glycosaminoglycan chains. The heparan sulphate proteoglycans were also heavily glycosylated with O-linked glycans and no glycosylphosphatidylinositol anchor could be detected.
Park
Y
Y
Division of Medicinal Chemistry, University of Iowa, Iowa City, IA 52242, USA.
Yu
G
G
Gunay
N S
NS
Linhardt
R J
RJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002850:Chromatography, Gel; D004187:Disaccharides / Q000737:chemistry; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D006025:Glycosaminoglycans / Q000737:chemistry; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry / Q000302:isolation & purification*; D010449:Peptide Mapping; D020539:Sequence Analysis, Protein
D000596:Amino Acids; D004187:Disaccharides; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans