Interaction and functional association of protein disulfide isomerase with αVβ3 integrin on endothelial cells
Interaction and functional association of protein disulfide isomerase with αVβ3 integrin on endothelial cells
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DOI：
10.1111/j.1742-4658.2008.06339.x
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发表时间：
2008-04-01
期刊：
影响因子：
5.4
通讯作者：
Cierniewski, Czeslaw S.
中科院分区：
文献类型：
--
作者：
Swiatkowska, Maria;Szymanski, Jacek;Cierniewski, Czeslaw S.
内皮细胞的粘附特性受巯基二硫平衡的影响。然而，这种效应的分子机制尚不清楚，尽管最近的观察表明整合素受体可能是氧化还原调节的直接靶点。本研究的目的是检查蛋白质二硫键异构酶（PDI）是否直接参与这一过程。由于已知锰离子影响硫醇二硫化物平衡并将整合素活化至最大亲和力，因此我们在Mn 2+处理的内皮细胞中寻找PDI与整合素的相互作用，特别是与α（V）β（3）的相互作用。通过采用共聚焦显微镜、流式细胞术和免疫共沉淀实验，我们表明内皮细胞暴露于Mn 2+导致：（a）表面蛋白巯基的出现，其可以在PDI和α（V）β（3）中发现，并且两种蛋白质共定位在细胞表面上;和（B）PDI-α（V）β（3）复合物的形成，其在还原时解离。此外，与α（V）β（3）复合的PDI诱导整联蛋白转化为配体感受态高亲和力状态，如玻连蛋白结合增加所证明。膜不可渗透的巯基阻断剂3-N-马来酰亚胺基丙酰基生物胞素3-N-马来酰亚胺基丙酰基生物胞素和对氯汞苯磺酸盐，以及PDI抑制剂杆菌肽，MA 3 018和MA 3 019，消除了玻连蛋白和LM 609与由Mn 2+激活的内皮细胞的结合。同样，LM 609几乎完全阻断玻连蛋白与这些细胞的结合。通过表面等离子体共振分析进一步证明了PDI-alpha（V）beta（3）化学计量复合物的形成，其显示在Mn 2+存在下，PDI的初始可逆结合变得不可逆，可能由二硫键介导。因此，我们发现Mn 2+同时调节与α（V）β（3）结合的PDI的巯基异构酶活性，并诱导其转变为配体感受态，这表明了整合素调节的另一种机制。
Adhesive properties of endothelial cells are influenced by the thioldisulfide balance. However, the molecular mechanism of this effect is unclear, although recent observations indicate that integrin receptors may be direct targets for redox modulation. The purpose of this study was to examine whether protein disulfide isomerase (PDI) is directly involved in this process. As manganese ions are known to affect the thioldisulfide balance and activate integrins to maximal affinity, we searched for PDI interactions with integrins, particularly with alpha(V)beta(3), in Mn2+-treated endothelial cells. By employing confocal microscopy, flow cytometry and coimmunoprecipitation experiments, we showed that exposure of endothelial cells to Mn2+ resulted in: (a) the appearance of surface protein thiol groups, which can be found in PDI and alpha(V)beta(3), and both proteins colocalizing on the cellular surface; and (b) the formation of the PDI-alpha(V)beta(3) complex, which dissociates upon reduction. In addition, PDI in a complex with alpha(V)beta(3) induces conversion of the integrin to the ligand-competent high-affinity state, as evidenced by increased binding of vitronectin. The membrane-impermeable sulfhydryl blockers 3-N-maleimidylpropionyl biocytin 3-N-maleimidylpropionyl biocytin and p-chloromercuriphenyl sulfonate, as well as the PDI inhibitors bacitracin, MA3 018, and MA3 019, abolished the binding of vitronectin and LM609 to endothelial cells that is activated by Mn2+. Consistently, LM609 almost completely blocked binding of vitronectin to such cells. The formation of the PDI-alpha(V)beta(3) stoichiometric complex was further demonstrated by surface plasmon resonance analysis, which showed that the initial reversible binding of PDI becomes irreversible in the presence of Mn2+, probably mediated by disulfide bonds. Thus, we show that Mn2+ simultaneously modulates the thiol isomerase activity of PDI that is bound to alpha(V)beta(3) and induces its transition to the ligand-competent state, suggesting an alternative mechanism of integrin regulation.