Pevzner Irina B, Agapov Igor I, Niwa Hideaki, Maluchenko Natalia V, Moisenovich Mikhail M, Pfüller Uwe, Tonevitsky Alexander G
Biological Department, MV Lomonosov Moscow State University, Vorobyevy Gory, Moscow 119899, Russia.
Biochim Biophys Acta. 2004 Nov 18;1675(1-3):155-64. doi: 10.1016/j.bbagen.2004.09.002.
Toxic lectins of European mistletoe Viscum album L.--MLI (viscumin), MLII and MLIII--are present in water extracts of this plant. Earlier we have cloned the full-length gene of MLIII precursor [A.G. Tonevitsky, I.I. Agapov, I.B. Pevzner, N.V. Maluchenko, M.M. Mojsenovich, U. Pfueller, M.P. Kirpichnikov, (2004) Biochemistry (Mosc.), 69 (6), 790-800, in press]. Here for the first time we report the cloning and expression in Escherichia coli cells of MLIII gene fragment encoding the carbohydrate-binding subunit. We have proved with our panel of monoclonal antibodies against ML toxins that the cloned fragment encoded MLIII B-subunit. The immunochemical and sugar-binding activities of renatured recombinant MLIII B-subunit were demonstrated in ELISA and ELLA, respectively. The comparative analysis of amino acid sequences of the cloned rMLIIIB and the B-subunits of other type II RIPs--MLI, ricin, abrin and nigrin b--was performed, revealing the main differences in primary structure of MLI and MLIII B-chains, which could determine their sugar specificity. The antigenicity analysis of MLI and MLIII B-subunits showed one epitope 25RDDDFRDGNQ34 in MLIB that is absent in MLIIIB sequence. The role of the toxic lectins and their subunits in immunological properties of mistletoe extracts is discussed.
欧洲槲寄生（Viscum album L.）的毒性凝集素——MLI（槲寄生毒蛋白）、MLII和MLIII——存在于该植物的水提取物中。此前我们已克隆了MLIII前体的全长基因[A.G. 托涅维茨基、I.I. 阿加波夫、I.B. 佩夫兹纳、N.V. 马卢琴科、M.M. 莫伊谢诺维奇、U. 普弗勒、M.P. 基尔皮奇尼科夫，（2004年）《生物化学（莫斯科）》，69（6），790 - 800，待发表]。在此，我们首次报道了编码碳水化合物结合亚基的MLIII基因片段在大肠杆菌细胞中的克隆与表达。我们利用针对ML毒素的单克隆抗体证明，克隆片段编码MLIII B亚基。分别在ELISA和ELLA中证实了复性重组MLIII B亚基的免疫化学活性和糖结合活性。对克隆的rMLIIIB与其他II型核糖体失活蛋白（RIPs）——MLI、蓖麻毒素、相思子毒素和黑麦凝集素b——的B亚基的氨基酸序列进行了比较分析，揭示了MLI和MLIII B链一级结构的主要差异，这可能决定它们的糖特异性。MLI和MLIII B亚基的抗原性分析表明，MLIB中有一个表位25RDDDFRDGNQ34，而MLIIIB序列中不存在该表位。文中讨论了毒性凝集素及其亚基在槲寄生提取物免疫特性中的作用。
