Physical-chemical interaction of heparin and human plasma low-density lipoproteins

Abstract

This study characterizes the physical-chemical interactions of heparin with human plasma low-density lipoproteins (LDL). A high reactive heparin (HRH) specific for the surface determinants of LDL was isolated by chromatography of commercial bovine lung heparin on LDL immobilized to AffiGel-10. HRH was derivatized with fluoresceinamine and repurified by affinity chromatography, and its interaction with LDL in solution was monitored by steady-state fluorescence polarization. Binding of LDL to fluoresceinamine-labeled HRH (FL . HRH) was saturable, reversible, and specific; HRH stoichiometrically displaced FL . HRH from the soluble complex, and acetylation of lysine residues on LDL blocked heparin binding. Titration of FL.HRH with excess LDL yielded soluble complexes with two LDL molecules per heparin chain (Mr 13,000) characterized by an apparent Kd of 1 microM. Titration of LDL with excess HRH resulted in two classes of heparin binding with two and five heparin molecules bound per LDL and apparent Kd values of 1 and 10 microM, respectively. At physiological pH and ionic strength, the soluble HRH-LDL complexes were maximally precipitated with 20-50 mM Ca2+. Insoluble complexes contained 2-10 HRH molecules per LDL with the final product stoichiometry dependent on the ratio of the reactants. The affinity of HRH for LDL in the insoluble complexes was estimated between 1 and 10 microM. Insoluble LDL-heparin complexes were readily dissociated with 1.0 M NaCl, and their formation was prevented by acetylation of the lysine residues on LDL.

Molecular modeling of protein-glycosaminoglycan interactions.

“…15 - 19 The LDL receptor-binding region in apo E 56 corresponds to a heparin-binding region 18 ' 18 and is structurally similar to the heparin-binding region defined by residues 3352 to 3378 of apo B-100. 1 «' 17 Studies with monoclonal antibodies to apo B-100 that block LDL binding to the receptor have been interpreted as a single domain for receptor recognition.…”

“…1 «' 17 Studies with monoclonal antibodies to apo B-100 that block LDL binding to the receptor have been interpreted as a single domain for receptor recognition. 68 ' 69 ' 70 In contrast, studies on the interaction of heparin with LDL show that there are five to seven heparin contact sites on the LDL surface, 15 and approximately this number of heparin-binding peptides have been purified from apo B-IOO. 1617 It is known that heparin releases LDL from cell receptors.…”

“…We showed that LDL contains five to seven heparin-binding sites on the lipoprotein surface. 15 The heparin-binding cyanogen bromide peptides of apo B-100 were isolated, and their amino acid sequences were determined. 16 These peptides account for five domains of high positive charge density that we suggest mediate the lipoprotein's interaction with glycosaminoglycans.…”

Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription.

“…Peptide regions of Fos (FB-1, residues 139-161) and the Jun (JB, residues 257-279) representing the DNA-binding domains were synthesized . These peptides were selected as the most likely domains to bind heparin based on a comparison of their amino acid sequences to those of known heparin-binding proteins (Cardin et al ., 1987;Jackson et al, 1991) . Furthermore, if heparin bound to these domains in the intact Fos and Jun proteins it would be expected to block DNA binding competitively.…”

Time course of 125I-labeled LDL accumulation in the healing, balloon-deendothelialized rabbit aorta.

“…13 - 15 To see if defined domains of apo B were important for LDL accumulation in the injured arterial wall, we synthesized short peptides, with sequences corresponding to specific regions of apo B, and examined their ability to accumulate in the healing rabbit aorta. 16 (An amino-terminal tyrosine was added to all of the peptides to facilitate 125 I-labeling.)…”