Department of Environmental Health Sciences, Fielding School of Public Health, University of California, Los Angeles, CA 90095, USA.
Max Perutz Laboratories, Department of Structural and Computational Biology, University of Vienna, A-1030 Vienna, Austria.
Molecules. 2021 Jun 13;26(12):3621. doi: 10.3390/molecules26123621.
Glycan-targeting antibodies and pseudo-antibodies have been extensively studied for their stoichiometry, avidity, and their interactions with the rapidly modifying glycan shield of influenza A. Broadly neutralizing antiviral agents bind in the same order when they neutralize enveloped viruses regardless of the location of epitopes to the host receptor binding site. Herein, we investigated the binding of cyanovirin-N (CV-N) to surface-expressed glycoproteins such as those of human immunodeficiency virus (HIV) gp120, hemagglutinin (HA), and Ebola (GP)1,2 and compared their binding affinities with the binding response to the trimer-folded gp140 using surface plasmon resonance (SPR). Binding-site knockout variants of an engineered dimeric CV-N molecule (CVN2) revealed a binding affinity that correlated with the number of (high-) affinity binding sites. Binding curves were specific for the interaction with N-linked glycans upon binding with two low-affinity carbohydrate binding sites. This biologically active assembly of a domain-swapped CVN2, or monomeric CV-N, bound to HA with a maximum K of 2.7 nM. All three envelope spike proteins were recognized at a nanomolar K, whereas binding to HIV neutralizing 2G12 by targeting HA and Ebola GP1,2 was measured in the µM range and specific for the bivalent binding scheme in SPR. In conclusion, invariant structural protein patterns provide a substrate for affinity maturation in the membrane-anchored HA regions, as well as the glycan shield on the membrane-distal HA top part. They can also induce high-affinity binding in antiviral CV-N to HA at two sites, and CVN2 binding is achieved at low-affinity binding sites.
糖基靶向抗体和假抗体因其化学计量、亲和力以及与流感 A 的快速修饰糖盾的相互作用而得到广泛研究。广谱中和抗病毒药物在中和包膜病毒时以相同的顺序结合，无论表位位于宿主受体结合位点的位置如何。在此，我们研究了氰基维生素 N（CV-N）与表面表达的糖蛋白（如人类免疫缺陷病毒（HIV）gp120、血凝素（HA）和埃博拉（GP）1、2 的 gp）的结合，并用表面等离子体共振（SPR）比较了它们与三聚体折叠 gp140 结合的亲和力。工程二聚体 CV-N 分子（CVN2）的结合位点敲除变体揭示了与（高）亲和力结合位点数量相关的结合亲和力。结合曲线特异性地与 N 连接糖基的相互作用，在与两个低亲和力碳水化合物结合位点结合时。这种具有生物学活性的域交换 CVN2 或单体 CV-N 的组装，与 HA 的最大 K 为 2.7 nM。所有三种包膜刺突蛋白都以纳摩尔 K 的浓度被识别，而通过靶向 HA 与 HIV 中和 2G12 的结合则在微摩尔范围内测量，并且在 SPR 中特异性针对二价结合方案。总之，不变的结构蛋白模式为膜锚定的 HA 区域以及膜远端 HA 顶部的糖盾中的亲和力成熟提供了底物。它们还可以诱导抗病毒 CV-N 在两个位点与 HA 发生高亲和力结合，并且 CVN2 结合是在低亲和力结合位点实现的。
