Computationally guided conversion of the specificity of E-selectin to mimic that of Siglec-8.
Computationally guided conversion of the specificity of E-selectin to mimic that of Siglec-8.
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DOI：
10.1073/pnas.2117743119
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发表时间：
2022-10-11
影响因子：
11.1
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中科院分区：
文献类型：
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合理的设计辅助计算方法是一个强大的和有效的工具，创造特异性的识别新的聚糖蛋白质。我们设计了E-选择素的双突变体，以消除对内源性配体（sLex）的特异性，同时引入对该配体的6′-硫酸化形式（6′-sulfo-sLex）的亲和力。通过聚糖阵列筛选定义的特异性完全模拟不相关蛋白Siglec-8的特异性，其天然偏好结合6′-磺基-sLex及其未岩藻糖基化形式（6′-磺基-sLacNAc）。我们发现，这种高度特异性识别6′-硫酸化的合理设计需要三个核心特征：1）去除6′-硫酸化的不利相互作用，2）引入6′-硫酸化的有利相互作用，3）去除有利于与内源性配体结合的相互作用。已发现硫酸化聚糖与多种疾病相关，因此在分子病理学中作为生物标志物具有显著的潜力。虽然凝集素是检测聚糖的有用试剂，但硫酸盐识别凝集素缺乏，并且存在的那些凝集素，如来自怀槐的凝集素，显示混合特异性。重组凝集素工程为通过改变和/或增强内源性特异性来产生新的聚糖识别提供了一种新兴的工具。目前的研究表明，使用计算方法在工程的突变形式的E-选择素，显示高度特异性识别6′-磺基-唾液酸刘易斯X（6′-磺基-sLex），与其内源性非硫酸化配体，sLex的结合可以忽略不计。这种新的特异性模拟了不相关蛋白Siglec-8的特异性，其中6′-sulfo-sLex是其优选配体。分子动力学模拟和能量计算预测，需要两个点突变（E92 A/E107 A）来稳定与E-选择素的硫酸化寡糖的结合。除了消除带负电荷的侧链和硫酸酯部分之间的推定排斥外，突变还消除了与内源性配体的有利相互作用。重组表达蛋白的聚糖微阵列筛选证实了预测的特异性变化，但也鉴定了对6′-磺基-sLex（6′-磺基-sLacNAc）的非岩藻糖基化形式的意外亲和力的引入。在这种情况下，证明了硫酸化寡糖工程特异性的三个关键要求：1）去除与6′-硫酸酯的不利相互作用，2）引入硫酸酯的有利相互作用，3）去除与内源性配体的有利相互作用。
Rational design assisted by computational approaches is a powerful and efficient tool for creating proteins specific for the recognition of novel glycans. We engineered a double mutant of E-selectin to eliminate specificity for the endogenous ligand (sLex) while introducing affinity for the 6′-sulfated form of that ligand (6′-sulfo-sLex). The specificity, defined by glycan array screening, identically mimics that of the unrelated protein Siglec-8, which naturally prefers to bind 6′-sulfo-sLex and its unfucosylated form (6′-sulfo-sLacNAc). We show that the rational design for this highly specific recognition for 6′-sulfation requires three core features: 1) removal of unfavorable interactions for 6′-sulfation, 2) introduction of favorable interactions for 6′-sulfation, and 3) removal of interactions that favor binding to the endogenous ligand. Sulfated glycans have been found to be associated with various diseases and therefore have significant potential in molecular pathology as biomarkers. Although lectins are useful reagents for detecting glycans, there is a paucity of sulfate-recognizing lectins, and those that exist, such as from Maackia amurensis, display mixed specificities. Recombinant lectin engineering offers an emerging tool for creating novel glycan recognition by altering and/or enhancing endogenous specificities. The present study demonstrated the use of computational approaches in the engineering of a mutated form of E-selectin that displayed highly specific recognition of 6′-sulfo-sialyl Lewis X (6′-sulfo-sLex), with negligible binding to its endogenous nonsulfated ligand, sLex. This new specificity mimics that of the unrelated protein Siglec-8, for which 6′-sulfo-sLex is its preferred ligand. Molecular dynamics simulations and energy calculations predicted that two point mutations (E92A/E107A) would be required to stabilize binding to the sulfated oligosaccharide with E-selectin. In addition to eliminating putative repulsions between the negatively charged side chains and the sulfate moiety, the mutations also abolished favorable interactions with the endogenous ligand. Glycan microarray screening of the recombinantly expressed proteins confirmed the predicted specificity change but also identified the introduction of unexpected affinity for the unfucosylated form of 6′-sulfo-sLex (6′-sulfo-sLacNAc). Three key requirements were demonstrated in this case for engineering specificity for sulfated oligosaccharide: 1) removal of unfavorable interactions with the 6′-sulfate, 2) introduction of favorable interactions for the sulfate, and 3) removal of favorable interactions with the endogenous ligand.
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影响因子：
5.5
作者：
Goetz, Andreas W.;Williamson, Mark J.;Xu, Dong;Poole, Duncan;Le Grand, Scott;Walker, Ross C.
通讯作者：
Walker, Ross C.
影响因子：
4.6
作者：
Gao W;Xu Y;Liu J;Ho M
通讯作者：
Ho M
影响因子：
3.8
作者：
Fernández-Vega I;García O;Crespo A;Castañón S;Menéndez P;Astudillo A;Quirós LM
通讯作者：
Quirós LM
影响因子：
14.8
作者：
Gama, Cristal I.;Tully, Sarah E.;Hsieh-Wilson, Linda C.
通讯作者：
Hsieh-Wilson, Linda C.
DOI：
10.1073/pnas.032068299
发表时间：
2002-04-02
影响因子：
11.1
作者：
Honke, K;Hirahara, Y;Taniguchi, N
通讯作者：
Taniguchi, N