Taniguchi Masayuki, Aida Ryousuke, Saito Kazuki, Ochiai Akihito, Takesono Satoshi, Saitoh Eiichi, Tanaka Takaaki
Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan.
Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan.
J Biosci Bioeng. 2019 Apr;127(4):472-478. doi: 10.1016/j.jbiosc.2018.09.016. Epub 2018 Oct 16.
In this study, we investigated the lipopolysaccharide (LPS)-neutralizing and angiogenic activities of cationic peptides derived from the traditional Japanese fermented product Natto, which is made by fermenting cooked soybeans using Bacillus subtilis. Initially, we prepared 20 fractions of Natto extracts with various isoelectric points (pI's) using ampholyte-free isoelectric focusing (autofocusing). Cationic peptides were then purified from fractions 19 and 20, whose pH values were greater than 12, using reversed-phase high-performance liquid chromatography, and were identified using matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Among the 13 identified cationic peptides, seven (KFNKYGR, FPFPRPPHQK, GQSSRPQDRHQK, QRFDQRSPQ, ERQFPFPRPPHQK, GEIPRPRPRPQHPE, and EQPRPIPFPRPQPR) had pI's greater than 9.5, positive net charges, and differing molecular weights. These peptides were then chemically synthesized and applied to chromogenic LPS-neutralizing assays using Limulus amebocyte lysates, and 50% effective (neutralizing) concentrations of 2.6-5.5 μM were demonstrated. In addition, tube formation assays in human umbilical vein endothelial cells revealed angiogenic activities for all but one (GEIPRPRPRPQHPE) of these seven cationic peptides, with increases in relative tube lengths of 23-31% in the presence of peptides at 10 μM. Subsequent experiments showed negligible hemolytic activity of these peptides at concentrations of up to 500 μM in mammalian red blood cells. Collectively, these data demonstrate that six cationic peptides from Natto extracts, with the exception of GEIPRPRPRPQHPE, have LPS-neutralizing and angiogenic activities but do not induce hemolysis.
在本研究中，我们调查了源自日本传统发酵食品纳豆的阳离子肽的脂多糖（LPS）中和及血管生成活性。纳豆是通过用枯草芽孢杆菌发酵煮熟的大豆制成的。最初，我们使用无两性电解质等电聚焦（自动聚焦）制备了20个具有不同等电点（pI）的纳豆提取物级分。然后使用反相高效液相色谱法从pH值大于12的第19和20级分中纯化阳离子肽，并使用基质辅助激光解吸/电离飞行时间质谱法进行鉴定。在鉴定出的13种阳离子肽中，有7种（KFNKYGR、FPFPRPPHQK、GQSSRPQDRHQK、QRFDQRSPQ、ERQFPFPRPPHQK、GEIPRPRPRPQHPE和EQPRPIPFPRPQPR）的pI大于9.5，带正净电荷且分子量不同。然后对这些肽进行化学合成，并应用于使用鲎试剂的显色LPS中和试验，结果显示50%有效（中和）浓度为2.6 - 5.5μM。此外，在人脐静脉内皮细胞中进行的管形成试验显示，这7种阳离子肽中除一种（GEIPRPRPRPQHPE）外均具有血管生成活性，在存在10μM肽的情况下，相对管长度增加23% - 31%。随后的实验表明，这些肽在高达500μM的浓度下对哺乳动物红细胞的溶血活性可忽略不计。总体而言，这些数据表明，除GEIPRPRPRPQHPE外，纳豆提取物中的6种阳离子肽具有LPS中和及血管生成活性，但不会诱导溶血。
