A Fluorescence-Labeled Heptapeptide, (FITC)KP6, as an Efficient Probe for the Specific Detection of Oxidized and Minimally Modified Low-Density Lipoprotein
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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DOI：
10.1007/s10895-016-1808-1
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发表时间：
2016-05-01
影响因子：
2.7
通讯作者：
Ebina, Keiichi
中科院分区：
文献类型：
--
作者：
Sato, Akira;Ueda, Chiemi;Ebina, Keiichi
低密度脂蛋白（LDL）的两种氧化形式，氧化型LDL（ox-LDL）和最小修饰型LDL（MM-LDL），被认为在动脉粥样硬化的发病机制中起主要作用。最近，我们报道了一种通过N-末端赖氨酸的ε-氨基与异硫氰酸荧光素（FITC）偶联的七肽（Lys-Trp-Tyr-Lys-Asp-Gly-Asp，KP_6）与ox-LDL结合，但不与LDL结合。在本研究中，我们研究了（FITC）KP 6是否可以作为荧光探针特异性检测MM-LDL和ox-LDL。聚丙烯酰胺凝胶电泳和表面等离子体共振（SPR）结果表明，（FITC）KP 6能与MM-LDL和ox-LDL有效结合，并呈剂量依赖性，且具有高亲和力（K（D）分别为3.16和3.54 ng/mL蛋白）。（FITC）KP 6结合溶血磷脂酰胆碱和氧化磷脂酰胆碱，这两者分别大量存在于ox-LDL和MM-LDL中。在体外，（FITC）KP 6检测到的表面上和/或在人THP-1衍生的巨噬细胞与ox-LDL和MM-LDL孵育，但不是LDL的胞质溶胶。这些结果表明，（FITC）KP 6可能是一个有效的荧光探针特异性检测ox-LDL和MM-LDL，因此可以有助于识别，诊断，预防和治疗动脉粥样硬化。
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 epsilon-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.