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A Fluorescence-Labeled Heptapeptide, (FITC)KP6, as an Efficient Probe for the Specific Detection of Oxidized and Minimally Modified Low-Density Lipoprotein

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Abstract

Two oxidized forms of low-density lipoprotein (LDL), oxidized LDL (ox-LDL) and minimally modified LDL (MM-LDL), are believed to play a major role in the pathogenesis of atherosclerosis. Recently, we reported that a heptapeptide (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled through the ε-amino group of N-terminus Lys to fluorescein isothiocyanate, (FITC)KP6, bound to ox-LDL but not to LDL. In the present study, we investigated whether (FITC)KP6 could be used as a fluorescent probe for the specific detection of MM-LDL and ox-LDL. Results from polyacrylamide gel electrophoresis and surface plasmon resonance proved that (FITC)KP6 could efficiently bind to MM-LDL as well as ox-LDL in a dose-dependent manner and with high affinity (K D = 3.16 and 3.54 ng/mL protein for MM-LDL and ox-LDL, respectively). (FITC) KP6 bound to lysophosphatidylcholine and oxidized phosphatidylcholine, both present abundantly in ox-LDL and MM-LDL, respectively. In vitro, (FITC)KP6 was detected on the surface and/or in the cytosol of human THP-1-derived macrophages incubated with ox-LDL and MM-LDL, but not LDL. These results suggest that (FITC)KP6 could be an efficient fluorescent probe for the specific detection of ox-LDL and MM-LDL and can therefore contribute to the identification, diagnosis, prevention, and treatment of atherosclerosis.

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Acknowledgments

This work was supported by the Adaptable and Seamless Technology Transfer Program through Target-driven R&D from the Japan Science and Technology Agency (to A.S.), and the Intelligent Cosmos Scientific Foundation (to A.S.). We thank Ms. Hiromi Yoshida (Center of Research Instruments, Institute of Development, Aging and Cancer, Tohoku University) for SPR measurements. We thank Allisere Co. Ltd. (Tokyo, Japan) for forming tissue slice by microtome. We thank Mr. Hikaru Yamanaka, Mr. Keitaro Oe, and Ms. Izumi Yokoyama (Faculty of Pharmacy, Iwaki Meisei University) for experimental help. We thank Editage by Cactus Communications Inc. (Tokyo, Japan) for providing assistance with the English language.

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  1. Faculty of Pharmacy, Iwaki Meisei University, 5-5-1, Chuodai-Iino, Iwaki, Fukushima, 970-8551, Japan

    Akira Sato, Chiemi Ueda, Ryu Kimura, Chisato Kobayashi, Yoji Yamazaki & Keiichi Ebina

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  1. Akira Sato
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  2. Chiemi Ueda
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Correspondence to Akira Sato.

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Sato, A., Ueda, C., Kimura, R. et al. A Fluorescence-Labeled Heptapeptide, (FITC)KP6, as an Efficient Probe for the Specific Detection of Oxidized and Minimally Modified Low-Density Lipoprotein. J Fluoresc 26, 1141–1150 (2016). https://doi.org/10.1007/s10895-016-1808-1

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