Mannose-binding type Jacalin-related lectins (mJRLs) bind to branched N-glycans via conserved sugar-binding sites. Despite, significant 3D structural similarities, each mJRL is known to have a unique binding preference toward various N-glycans. However, the molecular basis of varying binding preference is substantially unknown. Here, we report a detailed comparison of N-glycan-binding preference for two mJRLs, Orysata and Calsepa using frontal affinity chromatography (FAC), X-ray and molecular modeling. The FAC analysis using a panel of N-glycans shows difference in N-glycan-binding preference between the lectins. Orysata shows broader specificity toward most high-mannose-type glycans as well as biantennary complex-type glycans bearing an extension on the Manα1–6 branch. Whereas, Calsepa shows narrow specificity to complex-type glycans with bisecting GlcNAc. The X-ray crystallographic structure reveals that two Orysata lectins bind to one biantennary N-glycan (2:1 binding) where one lectin binds to mannose of the α1–3 branch, while the other interacts with an N-acetylglucosamine of the α1–6 branch. In contrast, Calsepa shows 1:1 binding where α1–3 branch and core chitobiose region N-glycan interacts with lectin, while α1–6 branch is flipped-back to the chitobiose core. Molecular dynamics study of Orysata bound to N-glycans substantiate possibility of two-binding modes for each N-glycan. Binding free energies calculated separately for α1–3 and α1–6 branches of each N-glycan suggest both branches can bind to Orysata. Overall these results suggest that each branch of N-glycan has a distinct role in binding to mJRLs and the nonbinding branch can contribute significantly to the binding affinity and hence to the specificity.

中文翻译：

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每个N-聚糖分支与甘露糖结合型Jacalin相关凝集素Orysata和Calsepa相互作用的不同作用

甘露糖结合型Jacalin相关凝集素（mJRLs）通过保守的糖结合位点结合至分支的N-聚糖。尽管存在显着的3D结构相似性，但已知每种mJRL对各种N-聚糖都有独特的结合偏好。然而，改变结合偏好的分子基础基本上是未知的。在这里，我们报告使用额叶亲和色谱（FAC），X射线和分子建模对两种mJRL，稻米和Calsepa的N-聚糖结合偏好的详细比较。使用一组N聚糖进行的FAC分析显示了N的差异-凝集素之间的-聚糖结合偏好。Orysata对大多数高甘露糖型聚糖以及在Manα1-6分支上延伸的双天线复合型聚糖显示出更广泛的特异性。而卡尔塞帕（Calsepa）对二等分GlcNAc的复合型聚糖显示出狭窄的特异性。X射线晶体学结构表明，两种Orysata凝集素与一个双触角N-聚糖结合（2：1结合），其中一种凝集素与α1-3分支的甘露糖结合，而另一种与α1-3的N-乙酰氨基葡糖相互作用。 6个分支。相反，Calsepa显示1：1结合，其中α1–3分支和核心壳二糖区域N-聚糖与凝集素相互作用，而α1–6分支倒转回到壳二糖核心。Orysata结合的分子动力学研究。N-聚糖证实了每种N-聚糖具有两种结合模式的可能性。对每个N-聚糖的α1-3和α1-6分支分别计算的结合自由能表明，两个分支都可以与Orysata结合。总的来说，这些结果表明，N-聚糖的每个分支在与mJRLs的结合中具有独特的作用，并且非结合分支可以显着地促进结合亲和力并因此对特异性起作用。