Isolation and partial characterization of heparan sulphate proteoglycan from the human glomerular basement membrane.
Heparan sulphate proteoglycan was solubilized from human glomerular basement membranes by guanidine extraction and purified by ion-exchange chromatography and gel filtration. The yield of proteoglycan was approx. 2 mg/g of basement membrane. The glycoconjugate had an apparent molecular mass of 200-400 kDa and consisted of about 75% protein and 25% heparan sulphate. The amino acid composition was characterized by a high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulphonic acid yielded core proteins of 160 and 110 kDa (and minor bands of 90 and 60 kDa). Alkaline NaBH4 treatment of the proteoglycan released heparan sulphate chains with an average molecular mass of 18 kDa. HNO2 oxidation of these chains yielded oligosaccharides of about 5 kDa, whereas heparitinase digestion resulted in a more complete degradation. The data suggest a clustering of N-sulphate groups in the peripheral regions of the glycosaminoglycan chains. A polyclonal antiserum raised against the intact proteoglycan showed reactivity against the core protein. It stained all basement membranes in an intense linear fashion in immunohistochemical studies on frozen kidney sections from man and various mammalian species.
van den Heuvel
L P
LP
Department of Biochemistry, University of Nijmegen, The Netherlands.
van den Born
J
J
van de Velden
T J
TJ
Veerkamp
J H
JH
Monnens
L A
LA
Schroder
C H
CH
Berden
J H
JH
D000596:Amino Acids / Q000032:analysis; D001485:Basement Membrane / Q000032:analysis; D015153:Blotting, Western; D011508:Chondroitin Sulfate Proteoglycans / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006025:Glycosaminoglycans / Q000302:isolation & purification*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000302:isolation & purification*; D006595:Hexosamines / Q000032:analysis; D006801:Humans; D007672:Kidney Cortex / Q000166:cytology; D007678:Kidney Glomerulus / Q000032:analysis* / Q000166:cytology; D008970:Molecular Weight; D011509:Proteoglycans / Q000302:isolation & purification*
D000596:Amino Acids; D011508:Chondroitin Sulfate Proteoglycans; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D006595:Hexosamines; D011509:Proteoglycans