Characterization of the Heparin-Binding Properties of Human Clusterin

Abstract

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.

Two Domains within theMycoplasma hyopneumoniaeCilium Adhesin Bind Heparin

“…The binding affinities of both F1 P97 (0.27 Ϯ 0.02 M) and F2 P97 (1.89 Ϯ 0.33 M) for heparin are within the range reported for other heparin-binding proteins (0.3 nM to 4 M) such as antithrombin (2), interleukin-2 and -12 (14,24), clusterin (26), and the mycobacterial heparin-binding hemagglutinin (29), suggesting that these interactions are of physiological significance. The ability of the M. hyopneumoniae cilium adhesin to bind to components of the ECM, as well as to cilia, indicates that this molecule plays a multifunctional role in adherence to the porcine respiratory tract.…”

P159 is a proteolytically processed, surface adhesin ofMycoplasma hyopneumoniae: defined domains of P159 bind heparin and promote adherence to eukaryote cells

“…F3 P159 and F4 P159 were also shown to bind to heparin-agarose showing that these P159 recombinant fragments bind both soluble and bound heparin. These values are comparable to other biologically significant heparin-protein binding interactions (Pethe et al, 2000) and fall in the midrange of other reported constants for heparin-protein interactions (Pankhurst et al, 1998). The ability of F2 P159 to promote the adherence of latex beads to PK15 cells despite an inability to bind heparin suggests that different regions within P159 use different mechanisms to bind eukaryote cells.…”

Drusen proteome analysis: An approach to the etiology of age-related macular degeneration

“…Indeed AGEs stimulate vascular endothelial growth factor expression (35) and angiogenesis in vivo (36) and may play a role in choroidal neovascularization in AMD. A plausible drusen biogenesis scenario might first involve proteins like TIMP3, clusterin, and vitronectin, which normally interact with sulfated glycosaminoglycans (GAGs) (37)(38)(39), to noncovalently bind to the GAGs in Bruch's membrane. As oxidative stress defense mechanisms deteriorate with age, oxidative modifications may gradually lock these and other proteins to Bruch's membrane with crosslinks, preventing normal turnover and initiating drusen development.…”