Department of Biochemistry, Faculty of Chemistry, University of Belgrade, 11000 Belgrade, Serbia.
Institute of Virology, Vaccines and Sera, 11152 Belgrade, Serbia.
Biomolecules. 2021 Jan 28;11(2):180. doi: 10.3390/biom11020180.
Fluorescently labeled lectins are useful tools for in vivo and in vitro studies of the structure and function of tissues and various pathogens such as viruses, bacteria, and fungi. For the evaluation of high-mannose glycans present on various glycoproteins, a three-dimensional (3D) model of the chimera was designed from the crystal structures of recombinant banana lectin (BanLec, Protein Data Bank entry (PDB): 5EXG) and an enhanced green fluorescent protein (eGFP, PDB 4EUL) by applying molecular modeling and molecular mechanics and expressed in . BanLec-eGFP, produced as a soluble cytosolic protein of about 42 kDa, revealed β-sheets (41%) as the predominant secondary structures, with the emission peak maximum detected at 509 nm (excitation wavelength 488 nm). More than 65% of the primary structure was confirmed by mass spectrometry. Competitive BanLec-eGFP binding to high mannose glycans of the influenza vaccine (Vaxigrip) was shown in a fluorescence-linked lectin sorbent assay (FLLSA) with monosaccharides (mannose and glucose) and wild type BanLec and H84T BanLec mutant. BanLec-eGFP exhibited binding to mannose residues on different strains of in flow cytometry, with especially pronounced binding to a Typhi clinical isolate. BanLec-eGFP can be a useful tool for screening high-mannose glycosylation sites on different microorganisms.
荧光标记的凝集素是研究组织和各种病原体（如病毒、细菌和真菌）结构和功能的体内和体外研究的有用工具。为了评估各种糖蛋白上存在的高甘露糖聚糖，通过应用分子建模和分子力学，从重组香蕉凝集素（BanLec，蛋白数据库条目（PDB）：5EXG）和增强型绿色荧光蛋白（eGFP，PDB 4EUL）的晶体结构设计了嵌合体的三维（3D）模型，并在 中表达。BanLec-eGFP 作为约 42 kDa 的可溶性细胞质蛋白产生，显示出β-折叠（41%）作为主要的二级结构，发射峰最大值检测到 509nm（激发波长 488nm）。通过质谱法确认了超过 65%的一级结构。在荧光连接的凝集素吸附测定（FLLSA）中，用单糖（甘露糖和葡萄糖）和野生型 BanLec 和 H84T BanLec 突变体显示了 BanLec-eGFP 与流感疫苗（Vaxigrip）上高甘露糖聚糖的竞争结合。BanLec-eGFP 在流式细胞术中与不同株的 结合，与 Typhi 临床分离株的结合尤其明显。BanLec-eGFP 可以成为筛选不同微生物上高甘露糖糖基化位点的有用工具。
