Abstract
BackgroundThe 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.MethodsGSLL 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.ResultsGSLL 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.ConclusionGSLL possessed some different properties from LcL (e.g., lower pI), and increased caspase 3, 8, and 9 activity in CNE1 cells.
Highlights
The green speckled lentil seed (Lens culinaris) lectin (GSLL) exhibits hemagglutinating activity, and possesses some properties distinct from those of other lentil lectins that deserve further investigation
Chan et al Chin Med (2015) 10:25 specific carbohydrate groups of glycoproteins on cell surfaces, allowing studies on differences in the levels of glycoproteins in different cell types [11, 12]. They are used in affinity chromatography for detection of glucoseand mannose-containing biomarkers, e.g., L. culinaris lectin (LcL) can bind to α-fetoprotein in the blood for diagnosis of hepatocellular carcinoma [13, 14]
The current protocol used for purification of GSLL from green speckled lentil seeds comprised ion exchange chromatography (SP-Sepharose: cation exchanger; Mono Q: anion exchanger), affinity chromatography (Affi-gel blue gel), and size exclusion chromatography (Superdex 75)
Summary
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. Lentil seeds contain a carbohydrate-binding protein, L. culinaris lectin (LcL) or hemagglutinin (LcH) [8]. Chan et al Chin Med (2015) 10:25 specific carbohydrate groups of glycoproteins on cell surfaces, allowing studies on differences in the levels of glycoproteins in different cell types [11, 12] They are used in affinity chromatography for detection of glucoseand mannose-containing biomarkers, e.g., LcLs can bind to α-fetoprotein in the blood for diagnosis of hepatocellular carcinoma [13, 14]. The lectin from the brown kidney bean cultivar (P. vulgaris) inhibited breast cancer MCF7 cells and nasopharyngeal carcinoma CNE1 cells more strongly than it inhibited HepG2 cells [17], and their counterpart from an Indian cultivar (P. vulgaris) was devoid of anticancer activity [18]
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