Characterization of the heparin-binding properties of human clusterin.
Clusterin is a highly conserved mammalian glycoprotein which has been predicted to contain heparin-binding sites. We tested this prediction by studying the interactions between heparin and clusterin using ELISA and heparin affinity chromatography methodologies. Two forms of biotinylated heparin were used in ELISA: heparin which had been directly biotinylated with a biotin-N-hydroxysuccinimide ester and heparin which had been activated using epichlorohydrin and 1,6-diaminohexane prior to biotinylation. Both gave dose-dependent increases in ELISA signal with increasing concentrations of biotinylated heparin, with the latter giving signals an order of magnitude greater than the former. There was a dose-dependent increase in the ELISA signal from bound biotinylated heparin with increasing concentrations of plate-bound clusterin. The apparent affinity constant for binding of biotinylated heparin to plate-bound clusterin at pH 6.0 was estimated as 0.06 +/- 0.02 microM. Unlabeled heparin blocked the binding of biotinylated heparin to clusterin over a concentration range similar to that of the binding of biotinylated heparin to plate-bound clusterin. The binding of biotinylated heparin to clusterin was independent of the presence or absence of Ca2+. The binding of biotinylated heparin to plate-bound clusterin increased with decreasing pH over the range 5.5-8.0 and was characterized by an apparent pKa of 6.9. Clusterin in human serum bound to heparin-Sepharose at pH 6.0 but not at pH 7.4. Dot-blot experiments showed that one of the polypeptide chains of clusterin which had been reduced and alkylated under denaturing conditions bound to heparin-Sepharose. This chain was identified as the alpha chain from its N-terminal amino acid sequence.
Pankhurst
G J
GJ
Department of Biochemistry University of Sydney, Sydney NSW 2006, Australia.
Bennett
C A
CA
Easterbrook-Smith
S B
SB
D001710:Biotin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D051152:Clusterin; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018832:Molecular Chaperones; D011485:Protein Binding; D012685:Sepharose / Q000378:metabolism
C495242:CLU protein, human; D051152:Clusterin; D006023:Glycoproteins; D018832:Molecular Chaperones; D001710:Biotin; D006493:Heparin; D012685:Sepharose; D002118:Calcium