Characterization of dystroglycan binding in adhesion of human induced pluripotent stem cells to laminin-511 E8 fragment
Characterization of dystroglycan binding in adhesion of human induced pluripotent stem cells to laminin-511 E8 fragment
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DOI：
10.1038/s41598-019-49669-x
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发表时间：
2019-09-10
影响因子：
4.6
通讯作者：
Kikkawa, Yamato
中科院分区：
文献类型：
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作者：
Sugawara, Yumika;Hamada, Keisuke;Kikkawa, Yamato
人类诱导多能干细胞（hiPSC）在培养中无限生长，并具有再生各种组织的潜力。在细胞培养系统的开发中，发现层粘连蛋白-511（LM 511-E8）的片段改善干细胞的增殖。未分化细胞与LM 511-E8的粘附主要通过整合素α 6 β 1介导。然而，在使用LM 511-E8的干细胞培养中，非整联蛋白受体的参与仍然未知。在这里，我们表明，肌营养不良蛋白聚糖（DG）在hiPSC中强烈表达。完全糖基化的DG对层粘连蛋白结合具有功能活性，尽管已经表明LM 511-E8缺乏DG结合位点，但该片段与DG结合较弱。我们使用合成肽进一步鉴定了LM 511-E8中的DG结合序列，其中，源自层粘连蛋白卷曲螺旋结构域的hE 8A 5 -20（人层粘连蛋白α 5 2688-2699：KTLPQLLAKLSI）表现出DG结合亲和力和细胞粘附活性。缺失和突变研究表明，LLAKLSI是hE 8A 5 -20的活性核心序列，K2696是DG结合的关键氨基酸。我们进一步证明了hiPSC粘附于hE 8A 5 -20缀合的壳聚糖基质。DG结合肽的氨基酸序列将有助于设计未分化和分化干细胞培养体系的基质。
Human induced pluripotent stem cells (hiPSCs) grow indefinitely in culture and have the potential to regenerate various tissues. In the development of cell culture systems, a fragment of laminin-511 (LM511-E8) was found to improve the proliferation of stem cells. The adhesion of undifferentiated cells to LM511-E8 is mainly mediated through integrin alpha 6 beta 1. However, the involvement of non-integrin receptors remains unknown in stem cell culture using LM511-E8. Here, we show that dystroglycan (DG) is strongly expressed in hiPSCs. The fully glycosylated DG is functionally active for laminin binding, and although it has been suggested that LM511-E8 lacks DG binding sites, the fragment does weakly bind to DG. We further identified the DG binding sequence in LM511-E8, using synthetic peptides, of which, hE8A5-20 (human laminin alpha 5 2688-2699: KTLPQLLAKLSI) derived from the laminin coiled-coil domain, exhibited DG binding affinity and cell adhesion activity. Deletion and mutation studies show that LLAKLSI is the active core sequence of hE8A5-20, and that, K2696 is a critical amino acid for DG binding. We further demonstrated that hiPSCs adhere to hE8A5-20-conjugated chitosan matrices. The amino acid sequence of DG binding peptides would be useful to design substrata for culture system of undifferentiated and differentiated stem cells.