Conformational study reveals amino acid residues essential for hemagglutinating and anti-proliferative activities of Clematis montana lectin

Bangmin Lu, Bin Zhang, Wei Qi, Yanan Zhu, Yan Zhao, Nan Zhou, Rong Sun, Jinku Bao, and Chuanfang Wu

School of Life Sciences and Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, State Key Laboratory of Biotherapy, Sichuan University, Chengdu 610064, China

Clematis montana lectin (CML), a novel mannose-binding lectin purified from C. montana Buch.-Ham stem (Ranunculaceae), has been proved to have hemagglutinating activity in rabbit erythrocytes and apoptosis-inducing activity in tumor cells. However, the biochemical properties of CML have not revealed and its structural information still needs to be elucidated. In this study, it was found that CML possessed quite good thermostability and alkaline resistance, and its hemagglutinating activity was bivalent metal cation dependent. In addition, hemagglutination test and fluorescence spectroscopy proved that GuHCl, urea, and sodium dodecyl sulfate could change the conformation of CML and further caused the loss of hemagglutination activity. Moreover, the changes of fluorescence spectrum indicated that the tryptophan (Trp) microenvironment conversion might be related to the conformation and bioactivities of CML. In addition, it was also found that Trp residues, arginine (Arg) residues, and sulfhydryl were important for the hemagglutinating activity of CML, but only Trp was proved to be crucial for the CML conformation. Furthermore, the Trp, Arg, and sulfhydryl-modified CML exhibited 97.17%, 76.99%, and 49.64% loss of its anti-proliferative activity, respectively, which was consistent with the alterations of its hemagglutinating activity. Given these findings, Trp residues on the surface of CML are essential for the active center to form substrate-accessible conformation and suitable environment for carbohydrate binding.

图例: 温度对绣球藤凝集素(CML)活力和频谱的影响

Acta Biochim Biophys Sin (Shanghai). 2014 Nov;46(11):923-34. doi: 10.1093/abbs/gmu085.

全文: http://abbs.oxfordjournals.org/content/46/11/923.full