可活化细胞穿膜肽的设计与酶解研究

王鹏程 齐宪荣

可活化细胞穿膜肽的设计与酶解研究

王鹏程齐宪荣

The design and enzymatic hydrolysis of activatable cell-penetrating peptide

WANG Feng-Cheng

摘要

摘要:
本文设计可活化细胞穿膜肽 (activatable cell-penetrating peptide, ACPP), 对合成的ACPP进行酶解研究。ACPP由3部分连接而成, 寡聚阴离子序列肽-基质金属蛋白酶 (matrix metalloproteinase, MMP) 的底物肽-寡聚阳离子序列肽。采用反相高效液相色谱 (reversed-phase high performance liquid chromatography, RP-HPLC) 监测37 ℃条件下IV型胶原酶 (含MMP-2和MMP-9) 对ACPP的酶解过程。收集酶解成分进行基质辅助激光解析电离飞行时间质谱 (matrix assisted laser desorption ionization orthogonal time of flight mass spectrometry, MALDIO-TOF-MS) 检测, 比对推测酶解碎片的序列。结果表明, ACPP可被IV型胶原酶裂解, 释放出寡聚阳离子序列肽, 即细胞穿膜肽 (cell-penetrating peptide, CPP), ACPP裂解一半的时间约为4 h。酶解发生于目标位点, 但不排除ACPP裂解后肽段的再次断裂。

Abstract:
The paper is aimed to study the enzymatic hydrolysis of the activatable cell-penetrating peptide (ACPP) that was designed and synthesized. The ACPP was composed of three parts, polyanionic sequence peptide, peptide sequence that specifically cleaved by matrix metalloproteinase (MMP) and cell penetrating peptide (CPP). The ACPP was hydrolyzed by type IV collagenase (MMP-2/9) under the condition of 37 ℃ and was monitored by reversed-phase high performance liquid chromatography (RP-HPLC). The efflux of peak was collected and detected by matrix assisted laser desorption ionization orthogonal time of flight mass spectrometry (MALDIO-TOF-MS) to speculate the sequences of the peptide fragments. The results indicated that the ACPP could be cleaved by type IV collagenase at target site as predicted, released CPP. The half life of the cleavage was about 4 h. Meanwhile, the peptide fragments may be cleaved again at other sites by type IV collagenase.

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