MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt's Lymphoma Cells to Trigger Apoptosis through Multiple Pathways

Abstract

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt's lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.

Keywords: Burkitt’s lymphoma cells; JNK; MEK/ERK; MytiLec; Mytilus galloprovincialis; R-type lectin; TNF-α; caspase-9/3; globotriose (Gb3); p21: p38 kinase; β-trefoil.

Figure 1

Different cell agglutination activities of MytiLec. MytiLec (0, 10, and 50 μg/mL) was applied to Ramos (5 × 10⁵ cells) and K562 (2 × 10⁵ cells) cells and observed by phase contrast microscopy.

Figure 2

Reduction of cell viability by MytiLec. (A) Determination of viability by WST-8 assay. Dotted columns: Ramos. White columns: K562. Cells (2 × 10⁵ of Ramos; 5 × 10⁵ of K562) were incubated with various MytiLec concentrations as shown. Error bars: SE (n = 3); (B) Annexin V-binding and propidium iodide (PI) incorporation in MytiLec-treated cells. Horizontal axis: binding of FITC-labeled annexin V. Phosphatidylserine externalization and PI incorporation were evaluated by FACS analysis using MEBCYTO apoptosis kit. Ramos (a,c) and K562 (b) cells were treated with MytiLec (a,b: 20 μg/mL; c: 0 μg/mL) for 30 min at 4 °C. Data shown are mean values with error bars = SD of triplicate experiments. Asterisks = significant differences (p < 0.05) between treated and control groups.

Figure 3

Internalization of FITC-conjugated MytiLec into Burkitt’s lymphoma cells. Incubation time: 0 min (a,b); and 2 h (c–f). Cells are treated with FITC-MytiLec in the presence of 25 mM D-galactose, as negative control (e,f). Cells were observed by fluorescence (a,c,e; ex 498 nm and em 522 nm) and phase-contrast (b,d,f), respectively. Arrows in c and d indicated shrunken cells. Bars indicated 50 μm.

Figure 4

Effects of MytiLec treatment on MEK, ERK, and cell cycle-related molecules in Burkitt’s lymphoma Ramos cells. (A) Phosphorylation and expression levels of MEK1/2, ERK1/2 and p21, p27, CDK6 and cyclinD3 were shown, respectively. Cells (4 × 10⁵ in each experiment) were treated with various concentrations of MytiLec as shown, and activation levels were evaluated by Western blotting of lysates. Solid and dotted lines indicated increasing and decreasing trends, respectively. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; (B) Relative densitometric quantification of P-MEK/MEK, P-ERK/ERK and p21/GAPDH. Each experiment was repeated three times.

Figure 5

Phosphorylation of JNK and p38 kinase by MytiLec in Burkitt's lymphoma Ramos. Cells (5 × 10⁵) were treated with (+) or without (−) 20 μg/mL MytiLec, and phosphorylation was evaluated by Western-blotting of cell lysates. (A) P-ERK, P-JNK, and P-p38: phosphorylated forms of ERK, JNK, and p38 kinase, respectively. Asterisks: increased phosphorylation; (B) Relative densitometric quantification of P-ERK/ERK, P-JNK/JNK and P-p38/p38. Each experiment was repeated three times.

Figure 6

Treatment with MEK inhibitor U0126 reversed up-regulation of p21 expression mediated by MytiLec-induced phosphorylation of MEK-ERK pathway. (A) Burkitt's lymphoma Ramos cells (4 × 10⁵) were treated with (+) or without (−) 20 μM U0126 for 5 h, and then with 20 μg/mL MytiLec. Expression levels of P-MEK_1/2, P-ERK_1/2, and p21 were evaluated by Western-blotting. Asterisks: disappearance of signals; (B) Relative densitometric quantification of P-MEK, P-ERK and p21 with (U0126/MytiLec) or without (MytiLec) the MEK inhibitor. Each experiment was repeated three times.

Figure 7

Activation by MytiLec of procaspase-3, procaspase-9, and TNF-α. (A) Activation of procaspase-3 in Ramos (4 × 10⁵ cells) incubated with various concentrations of MytiLec as shown, evaluated by Western blotting; (B) Up-regulation of TNF-α by MytiLec, and inhibition of caspase activation and TNF-α expression by P-MEK inhibitor U0126 and caspase-3 inhibitor Zn-DEVD-FMK (DEVD). Ramos (4 × 10⁵ cells) were treated with (+) or without (−) 10 μM U0126 or 10 μM DEVD for 2 h, and then with 20 μg/mL MytiLec. TNF-α expression and caspase-3/-9 activation were evaluated by Western blotting. Solid and dotted lines indicated increasing and decreasing trends of phosphorylation, respectively. Asterisks: disappearance of signals. Each experiment was repeated three times.