Tavelin Staffan, Hashimoto Kei, Malkinson John, Lazorova Lucia, Toth Istvan, Artursson Per
Department of Pharmacy, Uppsala University, PO Box 580, SE-751 23 Uppsala, Sweden.
Mol Pharmacol. 2003 Dec;64(6):1530-40. doi: 10.1124/mol.64.6.1530.
The aim of this study was to investigate whether peptides from the extracellular loops of the tight junction protein occludin could be used as a new principle for tight junction modulation. Peptides of 4 to 47 amino acids in length and covering the two extracellular loops of the tight junction protein occludin were synthesized, and their effect on the tight junction permeability in Caco-2 cells was investigated using [14C]mannitol as a para-cellular marker. Lipopeptide derivatives of one of the active occludin peptides (OPs), synthesized by adding a lipoamino acid containing 14 carbon atoms (C14-) to the N terminus of the peptide, were also investigated. Peptides corresponding to the N terminus of the first extracellular loop of occludin increased the permeability of the tight junctions without causing short-term toxicity. However, the peptides had an effect only when added to the basolateral side of the cells, which could be partly explained by degradation by apical peptidases and aggregate formation. By contrast, the lipopeptide C14-OP90-103, which protects the peptide from degradation and aggregation, displayed a rapid apical effect. The l- and d-diastereomers of C14-OP90-103 had distinctly different effects. The d-isomer, which releases intact OP90-103 from the lipoamino acid, displayed a rapid and transient increase in tight junction permeability. The l-isomer, which releases OP90-103 more rapidly, gave a more sustained increase in tight junction permeability. In conclusion, C14-OP90-103 represents a prototype of a new class of tight junction modulators that act on the extracellular domains of tight junction proteins.
本研究的目的是调查紧密连接蛋白闭合蛋白胞外环的肽段是否可作为紧密连接调节的新原理。合成了长度为4至47个氨基酸且覆盖紧密连接蛋白闭合蛋白两个胞外环的肽段，并使用[14C]甘露醇作为细胞旁标记物研究了它们对Caco-2细胞紧密连接通透性的影响。还研究了其中一种活性闭合蛋白肽（OPs）的脂肽衍生物，其通过在肽的N端添加含14个碳原子的脂氨基酸（C14-）合成。与闭合蛋白第一个胞外环N端对应的肽段增加了紧密连接的通透性，且未引起短期毒性。然而，这些肽段仅在添加到细胞基底外侧时才有作用，这部分可通过顶端肽酶的降解和聚集体形成来解释。相比之下，能保护肽段不被降解和聚集的脂肽C14-OP90-103显示出快速的顶端效应。C14-OP90-103的l-和d-非对映异构体具有明显不同的作用。能从脂氨基酸释放完整OP90-103的d-异构体使紧密连接通透性快速短暂增加。释放OP90-103更快的l-异构体使紧密连接通透性有更持续的增加。总之，C14-OP90-103代表了一类作用于紧密连接蛋白胞外结构域的新型紧密连接调节剂的原型。
