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首页> 外文期刊>Biochemistry >Inclusion of an RGD Motif Alters Invasin Integrin-Binding Affinity and Specificity
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Inclusion of an RGD Motif Alters Invasin Integrin-Binding Affinity and Specificity

机译：包含RGD母题会更改Invasin整合素结合的亲和力和特异性。

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Invasin is a key adhesin displayed on the outer membrane of Yersinia enterocolitica and Y pseudotuberculosis that mediates the initial stages of infection. Invasin specifically targets microfold (M) cells in the small intestine by binding beta(1) integrins and is sufficient to trigger eukaryotic uptake of invasin-coated particles, including Yersinia, Escherichia coli, and latex beads. As a result, invasin has generated interest to mediate oral delivery of vaccines and other biologics. Integrin binding affinity has been shown to correlate with particle uptake; thus we hypothesized that invasin variants with higher affinity would confer enhanced internalization. We first performed alanine scanning of surface-exposed tyrosine residues to identify those contributing to integrin binding. We identified two residues, which, when substituted with alanine, reduced binding to soluble alpha(5)beta(1), integrin. Next, we constructed four targeted mutagenesis libraries spanning these and other residues known to contribute to binding, followed by enrichment of variants able to mediate Caco-2 cellular invasion and to bind soluble alpha(5)beta(1) integrin. We identified three amino acid substitutions that increased alpha(5)beta(1) integrin binding affinity as measured by flow cytonietry and ELISA assays, two of which created a novel RGD motif surrounding the D911 residue critical for binding. This variant confers enhanced internalization into CHO cells but not Caco-2 cells when expressed on the E. coli surface. Further analysis showed that inclusion of an RGD expands invasin-integrin specificity, thereby impacting cellular selectivity. This work provides a molecular explanation for the lack of an RGD motif in invasin that is present in many other adhesins.
机译：侵染素是展示在小肠结肠炎耶尔森氏菌和假结核耶尔森氏菌外膜上的关键粘附素，介导感染的初始阶段。 Invasin通过结合beta（1）整合素特异性靶向小肠中的微褶（M）细胞，足以触发真核生物摄取被Invasin包被的颗粒，包括耶尔森菌，大肠杆菌和乳胶珠。结果，入侵素引起了介导疫苗和其他生物制剂的口服传递的兴趣。整联蛋白结合亲和力已被证明与颗粒摄取有关。因此，我们假设具有更高亲和力的入侵素变体将赋予增强的内在化。我们首先对暴露于表面的酪氨酸残基进行丙氨酸扫描，以鉴定那些有助于整合素结合的残基。我们确定了两个残基，当被丙氨酸取代时，减少了对可溶性α（5）beta（1），整联蛋白的结合。接下来，我们构建了四个靶向诱变文库，跨越这些和其他已知有助于结合的残基，随后富集了能够介导Caco-2细胞入侵并结合可溶性α（5）β（1）整联蛋白的变体。我们确定了三个氨基酸取代，可通过流式细胞仪和ELISA分析测定增加α（5）beta（1）整联蛋白结合亲和力的氨基酸，其中两个产生了围绕D911残基对结合至关重要的新型RGD基序。当在大肠杆菌表面表达时，该变体赋予进入CHO细胞的增强内化作用，而不是Caco-2细胞。进一步的分析表明，RGD的加入扩大了血管紧张素原整合素的特异性，从而影响了细胞的选择性。这项工作为许多其他粘附素中存在的invasin中缺少RGD基序提供了分子解释。

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