Highly Stable, Fluorescence‐Labeled Heptapeptides Substituted with a D‐Amino Acid for the Specific Detection of Oxidized Low‐Density Lipoprotein in Plasma

Abstract

Probes that can detect oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques can be useful for the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that two heptapeptides (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled to fluorescein isothiocyanate (FITC) through the ε-amino group of N-terminus Lys in the absence/presence of 6-amino-n-caproic acid (AC) linker to FITC-(FITC)KP6 and (FITC-AC)KP6-can be useful as fluorescent probes for the specific detection of ox-LDL. In this study, to develop the fluorescent peptides with high plasma stability for the specific detection of ox-LDL, we investigated the interaction of (FITC)KP6 and (FITC-AC)KP6 substituted with D-Lys at the N-terminus-(FITC)dKP6 and (FITC-AC)dKP6-with ox-LDL, and the in vitro stability of these peptides in mouse plasma. (FITC)dKP6 and (FITC-AC)dKP6 bound with high specificity to ox-LDL in a dose-dependent manner, and also to ox-LDL in the mouse plasma. Furthermore, (FITC)dKP6 was more stable than (FITC)KP6 in mouse plasma (102.1% versus 69.0% remained after 1 h). These findings strongly suggest that (FITC)dKP6 and (FITC-AC)dKP6 may be effective fluorescent probes with higher plasma stability than (FITC)KP6 and (FITC-AC)KP6 for the specific detection of ox-LDL.

A fluorescently labeled undecapeptide derived from a protein in royal jelly of the honeybee—royalisin—for specific detection of oxidized low‐density lipoprotein

“…Besides, we have recently found that these synthetic peptides having the reverse sequence of a Tyr‐Lys‐Asp (ie, an Asp‐Lys‐Tyr sequence) can bind to ox‐LDL (data not shown). Lately, we identified 2 heptapeptides (Lys‐Trp‐Tyr‐Lys‐Asp‐Gly‐Asp, KP6) coupled to fluorescein isothiocyanate (FITC) through the ε‐amino group of N‐terminus Lys, with/without a 6‐amino‐ n ‐caproic acid (AC) linker to minimize the effects of potential steric hindrance, termed (FITC)KP6 and (FITC‐AC)KP6, respectively, both of which bind with high specificity to ox‐LDL in a dose‐dependent manner through the binding to LPC and ox‐PC …”

Biotinylated heptapeptides substituted with a d-amino acid as platelet-activating factor inhibitors

“…However, currently there are no clinical anti-inflammatory drugs targeting PAF. We previously reported that fluorescence-labelled heptapeptides substituted with a D -amino acid at the N-terminus are stable in the plasma (Sato et al, 2015).…”

“…Alternatively, we previously reported that synthetic biotinylated peptides derived from Asp-hemolysin specifically inhibited the bioactivities of ox-LDL, the atherogenic lipoprotein existing in atherosclerotic arteries (Rosenfeld, 1991;Steinberg et al, 1989;Witztum, 1993), its major lipid component LPC, and PAF (Kudo et al, 2002;Kumagai et al, 2005Kumagai et al, , 2006Tsutsumi et al, 2006;Sato et al, 2012Sato et al, , 2013 through the binding of a Tyr-Lys-Asp-Gly region in the peptide to ox-LDL, LPC, and PAF. Additionally, two heptapeptides (Lys-Trp-Tyr-Lys-Asp-Gly-Asp), (FITC)KP6 and (FITC)dKP6, which were coupled to a FITC through ε-amino acid of N-terminus Lys and D-amino acid substituted at the N-terminus, were found to bind to ox-LDL (Sato et al, 2014(Sato et al, , 2015; (FITC)dKP6 was found to be more stable than (FITC)KP6 in plasma (Sato et al, 2015). BP21, one of the Asp-hemolysin-related biotinylated peptides, which directly binds to PAF and lyso-PAF, efficiently inhibits PAF-induced rat paw oedema even at doses 150-to 300-fold lower than the doses of PAF antagonists CV-3988 and alprazolam.…”

“…The first limitation is that it is unknown whether biotinylated peptides used in this study have prolonged effects in vivo. Fluorescence-labelled peptides substituted with a D-amino acid reportedly have higher plasma stability than those entirely made up of L-amino acids in vitro (Sato et al, 2015). The second limitation is that the conformational changes of peptides coupled to biotin and substituted with a D-amino acid are unknown.…”

“…Thus, biotinylated peptides are useful in the treatment of many inflammatory diseases mediated by PAF, but the in vivo stability of the peptides remains unknown because peptides are generally degraded by peptidases. Recently, we have reported that both a heptapeptide (Lys-Trp-Tyr-Lys-Aspand lysophosphatidylcholine (LPC), which are PAF-like lipids, in oxidized low-density lipoprotein (ox-LDL); ox-LDL is known to be a proinflammatory and proatherogenic factor found in atherosclerotic plaques (Sato et al, 2014(Sato et al, , 2015Tokumura et al, 2000).…”