Pinedo Marcela, Genoula Melanie, Silveyra María Ximena, De Oliveira Carvalho André, Regente Mariana, Del Río Marianela, Ribeiro Soares Júlia, Moreira Gomes Valdirene, De La Canal Laura
Instituto de Investigaciones Biológicas (IIB), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Mar del Plata, Funes 3250, 7600 Mar del Plata, Argentina.
Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes 28013-602, Brazil.
Int J Mol Sci. 2017 Jan 10;18(1):92. doi: 10.3390/ijms18010092.
According to their sugar recognition specificity, plant lectins are proposed as bioactive proteins with potential in cancer treatment and diagnosis. Helja is a mannose-specific jacalin-like lectin from sunflower which was shown to inhibit the growth of certain fungi. Here, we report its recombinant expression in a prokaryotic system and its activity in neurobalstoma cells. Helja coding sequence was fused to the pET-32 EK/LIC, the enterokinase/Ligation-independent cloning vector and a 35 kDa protein was obtained in representing Helja coupled to thioredoxin (Trx). The identity of this protein was verified using anti-Helja antibodies. This chimera, named Trx-rHelja, was enriched in the soluble bacterial extracts and was purified using Ni-Sepharose and d-mannose-agarose chromatography. Trx-rHelja and the enterokinase-released recombinant Helja (rHelja) both displayed toxicity on human SH-SY5Y neuroblastomas. rHelja decreased the viability of these tumor cells by 75% according to the tetrazolium reduction assay, and microscopic analyses revealed that the cell morphology was disturbed. Thus, the stellate cells of the monolayer became spheroids and were isolated. Our results indicate that rHelja is a promising tool for the development of diagnostic or therapeutic methods for neuroblastoma cells, the most common solid tumors in childhood.
根据其糖识别特异性，植物凝集素被认为是具有癌症治疗和诊断潜力的生物活性蛋白。Helja是一种来自向日葵的甘露糖特异性类jacalin凝集素，已证明它能抑制某些真菌的生长。在此，我们报告其在原核系统中的重组表达及其在神经母细胞瘤细胞中的活性。将Helja编码序列与pET-32 EK/LIC（肠激酶/不依赖连接的克隆载体）融合，获得了一个35 kDa的蛋白，它代表与硫氧还蛋白（Trx）偶联的Helja。使用抗Helja抗体验证了该蛋白的身份。这种嵌合体名为Trx-rHelja，在可溶性细菌提取物中富集，并通过镍琼脂糖凝胶亲和层析和D-甘露糖琼脂糖层析进行纯化。Trx-rHelja和肠激酶释放的重组Helja（rHelja）对人SH-SY5Y神经母细胞瘤均表现出毒性。根据四氮唑还原试验，rHelja使这些肿瘤细胞的活力降低了75%，显微镜分析显示细胞形态受到干扰。因此，单层的星状细胞变成球体并分离出来。我们的结果表明，rHelja是开发神经母细胞瘤细胞（儿童期最常见的实体瘤）诊断或治疗方法的一个有前景的工具。
