Bimolecular integrin-ligand interactions quantified using peptide-functionalized dextran-coated microparticles.
Bimolecular integrin-ligand interactions quantified using peptide-functionalized dextran-coated microparticles.
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DOI：
10.1039/c1ib00085c
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发表时间：
2012-01
期刊：
影响因子：
--
通讯作者：
Litvinov RI
中科院分区：
文献类型：
--
作者：
Sun JE;Vranic J;Composto RJ;Streu C;Billings PC;Bennett JS;Weisel JW;Litvinov RI
整合素在细胞-细胞和细胞-基质相互作用中起关键作用。人工表面移植整合素配体，模仿天然界面，已被用来研究整合素介导的细胞粘附。在这里，我们报告使用一种新的化学工程技术结合单分子纳米力学测量来量化肽结合整合素。我们用共价连接的葡聚糖制备乳胶珠。然后用生物活性肽、环状RGDFK（cRGD）和纤维蛋白原γ C-十二肽（H12）（对应于纤维蛋白原与血小板整联蛋白αIIbβ3结合的活性位点）对珠进行功能化。使用基于光镊的力谱来测量非特异性蛋白质-蛋白质相互作用，我们发现葡聚糖包被的珠对纤维蛋白原无反应性，从而为生物特异性修饰提供了惰性平台。使用高碘酸盐氧化，然后还原胺化，我们用cRGD或H12功能化珠连接的葡聚糖，并使用肽接枝珠测量与纯化的αIIbβ3的单分子相互作用。双分子力光谱表明，肽功能化的珠与固定化的αIIbβ3高度和特异性反应。此外，cRGD-和H12-官能化的珠显示出显著的相互作用曲线，具有高达90 pN的双峰力分布。cRGD-αIIbβ3相互作用的结合强度大于H12-αIIbβ3，表明它们在机械上更稳定和耐受，与含RGD配体的血小板反应性一致。因此，本文报告的结果描述了αIIbβ3-配体相互作用的机制特征，证实了肽官能化乳胶珠用于分子识别定量分析的实用性。
Integrins play a key role in cell–cell and cell–matrix interactions. Artificial surfaces grafted with integrin ligands, mimicking natural interfaces, have been used to study integrin-mediated cell adhesion. Here we report the use of a new chemical engineering technology in combination with single-molecule nanomechanical measurements to quantify peptide binding to integrins. We prepared latex beads with covalently-attached dextran. The beads were then functionalized with the bioactive peptides, cyclic RGDFK (cRGD) and the fibrinogen γC-dodecapeptide (H12), corresponding to the active sites for fibrinogen binding to the platelet integrin αIIbβ3. Using optical tweezers-based force spectroscopy to measure non-specific protein–protein interactions, we found the dextran-coated beads nonreactive towards fibrinogen, thus providing an inert platform for biospecific modifications. Using periodate oxidation followed by reductive amination, we functionalized the bead-attached dextran with either cRGD or H12 and used the peptide-grafted beads to measure single-molecule interactions with the purified αIIbβ3. Bimolecular force spectroscopy revealed that the peptide-functionalized beads were highly and specifically reactive with the immobilized αIIbβ3. Further, the cRGD- and H12-functionalized beads displayed a remarkable interaction profile with a bimodal force distribution up to 90 pN. The cRGD–αIIbβ3 interactions had greater binding strength than that of H12–αIIbβ3, indicating that they are more stable and resistant mechanically, consistent with the platelet reactivity of RGD-containing ligands. Thus, the results reported here describe the mechanistic characteristics of αIIbβ3–ligand interactions, confirming the utility of peptide-functionalized latex beads for the quantitative analysis of molecular recognition.
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影响因子：
14
作者：
Lee, MH;Brass, DA;Ducheyne, P
通讯作者：
Ducheyne, P
影响因子：
20.3
作者：
Litvinov, RI;Nagaswami, C;Weisel, JW
通讯作者：
Weisel, JW
影响因子：
3.4
作者：
Evans, E;Ritchie, K
通讯作者：
Ritchie, K
影响因子：
4.9
作者：
Lee, Mark H.;Adams, Christopher S.;Ducheyne, Paul
通讯作者：
Ducheyne, Paul
DOI：
10.1163/156856202320401997
发表时间：
2002-01-01
影响因子：
3.6
作者：
Machy, D;Carteron, P;Jozefonvicz, J
通讯作者：
Jozefonvicz, J