Algal lectin binding to core (α1-6) fucosylated N-glycans: Structural basis for specificity and production of recombinant protein
Algal lectin binding to core (α1-6) fucosylated N-glycans: Structural basis for specificity and production of recombinant protein
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DOI：
10.1093/glycob/cwv002
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发表时间：
2015-06-01
期刊：
影响因子：
4.3
通讯作者：
Varrot, Annabelle
中科院分区：
文献类型：
--
作者：
do Nascimento, Antonia S. F.;Serna, Sonia;Varrot, Annabelle
我们确定了BTL的特异性，凝集素从红色的海洋植物Bryothamnion triquetrum，对岩藻糖基化低聚糖。BTL对核心α 1，6-岩藻糖基化显示出严格的特异性，这是癌症发生和治疗性抗体质量控制的重要标志物。还识别了双岩藻糖基化α 1，6和α 1，3，但在仅存在α 1，3-岩藻糖基化的情况下，结合完全消除。对BTL特异性的更详细分析显示，偏好双触角和三触角非平分N-聚糖。N-聚糖非还原端的唾液酸化或岩藻糖基化并不影响凝集素的识别。BTL表现出很强的亲和力的核心α 1，6-岩藻糖基化八糖的K-D为12 μ M的滴定微量热法。通过STD-NMR对BTL与八糖的相互作用进行了结构表征。它证明了一个扩展的识别表位，包括岩藻糖残基，远端GlcNAc和一个甘露糖残基。在大肠杆菌中获得重组rBTL并进行表征。利用血凝试验和聚糖阵列分析研究了其对碳水化合物的结合特性。rBTL只有在用木瓜蛋白酶和胰蛋白酶处理后才能凝集兔红细胞，具有较强的血凝活性，表明其配体不能直接在细胞表面接触。rBTL的血凝特性证实了蛋白质的正确折叠和功能状态。结果显示BTL作为癌症诊断的有效候选物以及作为用于制备和质量控制缺乏核心α 1，6-岩藻糖基化N-聚糖的抗体的试剂。
We determined the specificity of BTL, a lectin from the red marine alga Bryothamnion triquetrum, toward fucosylated oligosaccharides. BTL showed a strict specificity for the core alpha 1,6-fucosylation, which is an important marker for cancerogenesis and quality control of therapeutical antibodies. The double fucosylation alpha 1,6 and alpha 1,3 was also recognized, but the binding was totally abolished in the sole presence of the alpha 1,3-fucosylation. A more detailed analysis of the specificity of BTL showed a preference for bi- and tri-antennary nonbisected N-glycans. Sialylation or fucosylation at the nonreducing end of N-glycans did not affect the recognition by the lectin. BTL displayed a strong affinity for a core alpha 1,6-fucosylated octasaccharide with a K-d of 12 mu M by titration microcalorimetry. The structural characterization of the interaction between BTL and the octasaccharide was obtained by STD-NMR. It demonstrated an extended epitope for recognition that includes the fucose residue, the distal GlcNAc and one mannose residue. Recombinant rBTL was obtained in Escherichia coli and characterized. Its binding properties for carbohydrates were studied using hemagglutination tests and glycan array analysis. rBTL was able to agglutinate rabbit erythrocytes with strong hemagglutination activity only after treatment with papain and trypsin, indicating that its ligands were not directly accessible at the cell surface. The hemagglutinating properties of rBTL confirm the correct folding and functional state of the protein. The results show BTL as a potent candidate for cancer diagnosis and as a reagent for the preparation and quality control of antibodies lacking core alpha 1,6-fucosylated N-glycans.