Centro de Investigación y de Estudios Avanzados Unidad Irapuato, Departamento de Biotecnología y Bioquímica, Irapuato 36821, Guanaj uato, Mexico.
University of Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France.
Biomolecules. 2020 Apr 23;10(4):654. doi: 10.3390/biom10040654.
Herein, we report the production of a recombinant Tepary bean lectin (TBL-1), its three-dimensional (3D) structure, and its differential recognition for cancer-type glycoconjugates. TBL-1 was expressed in yielding 316 mg per liter of culture, and was purified by nickel affinity chromatography. Characterization of the protein showed that TBL-1 is a stable 120 kDa homo-tetramer folded as a canonical leguminous lectin with two divalent cations (Ca and Mn) attached to each subunit, confirmed in its 3D structure solved by X-ray diffraction at 1.9 Å resolution. Monomers also presented a ~2.5 kDa -linked glycan located on the opposite face of the binding pocket. It does not participate in carbohydrate recognition but contributes to the stabilization of the interfaces between protomers. Screening for potential TBL-1 targets by glycan array identified 14 positive binders, all of which correspond to β1-6 branched -glycans' characteristics of cancer cells. The presence of α1-6 core fucose, also tumor-associated, improved carbohydrate recognition. TBL-1 affinity for a broad spectrum of mono- and disaccharides was evaluated by isothermal titration calorimetry (ITC); however, no interaction was detected, corroborating that carbohydrate recognition is highly specific and requires larger ligands for binding. This would explain the differential recognition between healthy and cancer cells by Tepary bean lectins.
本文报告了重组豌豆凝集素（TBL-1）的生产、三维结构及其对癌症型糖缀合物的差异识别。TBL-1在大肠杆菌中表达，每升培养物产生 316 毫克，通过镍亲和层析进行纯化。该蛋白的特性表明，TBL-1 是一种稳定的 120 kDa 同源四聚体，折叠成典型的豆科凝集素，每个亚基上附着有两个二价阳离子（Ca 和 Mn），这在其通过 X 射线衍射以 1.9 Å 分辨率解决的 3D 结构中得到证实。单体还呈现出位于结合口袋对面的~2.5 kDa 连接聚糖。它不参与碳水化合物识别，但有助于稳定原聚体之间的界面。通过糖芯片筛选潜在的 TBL-1 靶标，鉴定出 14 个阳性结合物，它们都对应于癌细胞中β1-6 分支聚糖的特征。存在α1-6 核心岩藻糖，也与肿瘤相关，提高了碳水化合物的识别。通过等温滴定量热法（ITC）评估了 TBL-1 对广泛的单糖和二糖的亲和力；然而，没有检测到相互作用，这证实了碳水化合物识别具有高度特异性，需要更大的配体才能结合。这可以解释豌豆凝集素对健康细胞和癌细胞的差异识别。
