Engineered cystine-knot peptides that bind alpha(v)beta(3) integrin with antibody-like affinities.
Engineered cystine-knot peptides that bind alpha(v)beta(3) integrin with antibody-like affinities.
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DOI：
10.1016/j.jmb.2008.11.004
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发表时间：
2009-01-30
影响因子：
5.6
通讯作者：
Cochran, Jennifer R.
中科院分区：
文献类型：
--
作者：
Silverman, Adam P.;Levin, Aron M.;Lahti, Jennifer L.;Cochran, Jennifer R.
αvβ3整联蛋白受体是一个重要的癌症靶点，因为它在许多实体瘤和肿瘤新生血管中过表达，并且在转移和血管生成中起作用。我们使用Agouti相关蛋白（AgRP）的截短形式，一种具有四个二硫键和四个溶剂暴露环的4 kDa胱氨酸结肽，作为工程肽的支架，以高亲和力和特异性结合αvβ3整联蛋白。通过用含有Arg-Gly-Asp（RGD）整合素识别基序和随机侧翼残基的9-氨基酸环取代AgRP的6-氨基酸环，产生酵母展示的胱氨酸结肽文库。通过荧光激活细胞分选（FACS）以高通量方式筛选突变胱氨酸结肽，以鉴定对去污剂溶解的αvβ3整联蛋白受体具有高亲和力的克隆。选择整合素结合肽在巴斯德毕赤酵母中重组表达，并测试其与表达各种整合素受体的人癌细胞结合的能力。这些研究表明，工程化的AgRP肽与表达αvβ3整联蛋白的细胞结合，亲和力范围为15 nM至780 pM。此外，工程化肽显示特异性结合αvβ3整联蛋白，并且仅具有最小的或不结合αvβ5、α5β1和α 11b β3整联蛋白。工程化的AgRP肽还显示出抑制细胞粘附至细胞外基质蛋白玻连蛋白，玻连蛋白是αvβ3和其他整联蛋白的天然存在的配体。接下来，为了评估AgRP的其他三个环是否可以调节整联蛋白特异性，我们通过在高亲和力整联蛋白结合变体之一中单独随机化这些环来制备第二代文库。针对αvβ3整联蛋白筛选这些环随机化文库产生了保留对αvβ3的高亲和力并且对αvβ3的特异性高于αiibβ3整联蛋白的肽。总的来说，这些数据验证了AgRP作为蛋白质工程的支架，并证明了单个环的修饰可以导致基于AgRP的肽与其靶标具有抗体样亲和力。
The αvβ3 integrin receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature, and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4 kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to αvβ3 integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a 6-amino acid loop of AgRP with a 9-amino acid loop containing the Arg-Gly-Asp (RGD) integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting (FACS) to identify clones with high affinity to detergent-solubilized αvβ3 integrin receptor. Select integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various integrin receptors. These studies showed that the engineered AgRP peptides bound to cells expressing αvβ3 integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown bind specifically to αvβ3 integrins, and had only minimal or no binding to αvβ5, α5β1, and αiibβ3 integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally-occurring ligand for αvβ3 and other integrins. Next, to evaluate whether the other three loops of AgRP could modulate integrin specificity, we made second generation libraries by individually randomizing these loops in one of the high affinity integrin-binding variants. Screening of these loop-randomized libraries against αvβ3 integrins resulted in peptides that retained high affinities for αvβ3 and had increased specificities for αvβ3 over αiibβ3 integrins. Collectively, these data validate AgRP as a scaffold for protein engineering and demonstrate that modification of a single loop can lead to AgRP-based peptides with antibody-like affinities for their target.
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影响因子：
7.8
作者：
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