Chan Yau Sang, Yu Huimin, Xia Lixin, Ng Tzi Bun
State Key Laboratory of Respiratory Disease for Allergy at Shenzhen University, School of Medicine, Shenzhen University, Nanhai Ave 3688, 518060 Shenzhen, Guangdong People's Republic of China.
School of Biomedical Sciences, Lo Kwee Seong Integrated Biomedical Sciences Building, The Chinese University of Hong Kong, Shatin, New Territories Hong Kong, People's Republic of China.
Chin Med. 2015 Sep 8;10:25. doi: 10.1186/s13020-015-0057-6. eCollection 2015.
The green speckled lentil seed (Lens culinaris) lectin (GSLL) exhibits hemagglutinating activity, and possesses some properties distinct from those of other lentil lectins (e.g., molecular size, biological activities) that deserve further investigation. This study aims to investigate the basic properties (e.g., molecular size, amino acid sequence, sugar specificity) and biological activities (e.g., antiproliferative activity) of GSLL.
GSLL was purified by successive fractionation on SP-Sepharose, Affi-gel blue gel, Mono Q, and Superdex 75. The biochemical properties of GSLL were investigated by SDS-PAGE, mass spectrometry, N-terminal amino acid sequencing, and sugar inhibition tests. For the biological activities, purified lyophilized GSLL was sterilized, adjusted to concentrations from 1 to 0 mg/mL (by twofold serial dilution) in Dulbecco's modified Eagle's medium with fetal bovine serum, and examined by using the MTT assay, flow cytometry, and western blotting after treatment of nasopharyngeal carcinoma CNE1 and CNE2 cell lines with the lectin.
GSLL appeared as a 21-kDa band in non-reducing SDS-PAGE. It was composed of two subunits with molecular sizes of 17 and ~4 kDa. It exhibited specificity in binding to glucose and mannose, as well as glucosides and mannosides. Mass spectrometry and N-terminal amino acid sequencing revealed similarity of GSLL to L. culinaris lectin (LcL), especially higher coverage of the β-chain of LcL. A 48-h treatment with GSLL exerted antiproliferative effects on nasopharyngeal carcinoma CNE1 and CNE2 cell lines with significant inhibition at 0.125 mg/mL (P < 0.001) and 1 mg/mL (P = 0.004), respectively, and these effects were attenuated in the presence of glucose and mannose. GSLL induced apoptosis in nasopharyngeal carcinoma CNE1 cells, with detectable phosphatidylserine externalization, mitochondrial depolarization, and cell cycle arrest. Western blot analysis suggested that GSLL triggered the extrinsic apoptotic pathway involving caspase 3, 8, and 9 in CNE1 cells.
GSLL possessed some different properties from LcL (e.g., lower pI), and increased caspase 3, 8, and 9 activity in CNE1 cells.
绿色斑点小扁豆种子（兵豆）凝集素（GSLL）具有血凝活性，且拥有一些与其他小扁豆凝集素不同的特性（如分子大小、生物学活性），值得进一步研究。本研究旨在探究GSLL的基本特性（如分子大小、氨基酸序列、糖特异性）和生物学活性（如抗增殖活性）。
通过在SP-琼脂糖凝胶、Affi-凝胶蓝胶、Mono Q和Superdex 75上连续分级分离来纯化GSLL。通过SDS-PAGE、质谱分析、N端氨基酸测序和糖抑制试验来研究GSLL的生化特性。对于生物学活性，将纯化的冻干GSLL进行除菌处理，在含有胎牛血清的杜尔贝科改良伊格尔培养基中调整浓度至1至0 mg/mL（通过两倍系列稀释），在用该凝集素处理鼻咽癌CNE1和CNE2细胞系后，通过MTT法、流式细胞术和蛋白质免疫印迹法进行检测。
在非还原SDS-PAGE中，GSLL呈现为一条21 kDa的条带。它由两个分子大小分别为17 kDa和~4 kDa的亚基组成。它在与葡萄糖和甘露糖以及葡萄糖苷和甘露糖苷结合方面表现出特异性。质谱分析和N端氨基酸测序显示GSLL与兵豆凝集素（LcL）相似，尤其是LcL的β链覆盖率更高。用GSLL处理48小时对鼻咽癌CNE1和CNE2细胞系产生抗增殖作用，在0.125 mg/mL（P < 0.001）和1 mg/mL（P = 0.004）时分别有显著抑制，并且在葡萄糖和甘露糖存在的情况下这些作用减弱。GSLL诱导鼻咽癌CNE1细胞凋亡，可检测到磷脂酰丝氨酸外化、线粒体去极化和细胞周期停滞。蛋白质免疫印迹分析表明GSLL触发了CNE1细胞中涉及半胱天冬酶3、8和9的外源性凋亡途径。
GSLL具有一些与LcL不同的特性（如较低的pI），并增加了CNE1细胞中半胱天冬酶3、8和9的活性。
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