Assessment of the fifth ligand-binding repeat (LR5) of the LDL receptor as an analytical reagent for LDL binding.
The fifth ligand binding repeat (LR5) of the low density lipoprotein (LDL) receptor was assessed ex vivo as an 'analytical reagent' to distinguish LDL state, in atherosclerosis risk monitoring. LR5 was immobilized to mercaptoundecanoic acid modified gold surfaces via a glycine linker. Surface plasmon resonance (SPR) was used to monitor LDL binding. Unfolded LR5 was ineffectual as an affinity ligand for LDL but refolded LR5 showed a high affinity for native LDL but little affinity for oxidized LDL. LR5 refolded in the presence of calcium or EDTA gave the equivalent LDL binding capacity. However, EDTA-LR5 was less stable than Ca-LR5 at pH 5 and, from tryptophan fluorescence evidence, they appeared to involve different regions of LR5 and/or LDL in the binding. Involvement of amino acid residues of the calcium cage of LR5 was tested in LDL binding by monitoring calcium ion release with a calcium ionophore. The results were consistent with approximately 7-8 LR5 binding per LDL, of which only some induce calcium release (a maximum of approximately 25 mol% calcium, based on LR5, was released during LDL binding). For LDL binding to the LDL receptor in vivo more than one ligand-binding repeat is needed and this may be consistent with LR5 acting here also at binding sites which other LRs normally occupy in the LDL-LDL receptor complex. This initial study is encouraging for the use of a minimum peptide repeat array based on the conserved region of the LRs as an affinity surface for atherosclerosis risk monitoring.
Gaus
K
K
Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, UK CB2 1QT.
Basran
A
A
Hall
E A
EA
D000595:Amino Acid Sequence; D001161:Arteriosclerosis / Q000175:diagnosis* / Q000378:metabolism; D002118:Calcium; D004492:Edetic Acid; D006801:Humans; D007202:Indicators and Reagents; D008024:Ligands; D008077:Lipoproteins, LDL / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism
D007202:Indicators and Reagents; D008024:Ligands; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D004492:Edetic Acid; D002118:Calcium