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

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.

(Jeremy R.H. Tame, personal communication). Six carbohydrate-binding sites of dimer MytiLec was essential for the cytotoxicity (data not shown) similar to the property of another cytotoxic R-type lectin isolated from mushroom Clitocybe nebularis [20]. Taken together, MytiLec fits in as a new member of the R-type lectin family.
Some R-type lectins have additional domains as toxic subunits. Pierisin, isolated from Pieris rapae (cabbage butterfly), has an ADP-ribosyltransferase domain in the polypeptide and three R-type lectin domains. Pierisin induces apoptosis in HeLa cells by binding to surface Gb3 and Gb4 (GalNAcβ1-3Galα1-4Galβ1-4Glc) glycans [21]. In addition to the original MytiLec, two MytiLec variants (termed MytiLec2 and MytiLec3) containing a pore-forming aerolysin [22]-like domain in the polypeptide that creates pores into infectious organisms and kills them through initiation of innate immunity, according to the recently updated MytiBase [4].
MytiLec does not have additional functional domains or subunits beside glycan-binding domains, in contrast to other R-type lectins, although it is capable of inducing cytotoxicity. It thus occupies a unique category within the R-type lectin family. The mechanisms whereby MytiLec transmits its signals through cells to activate various signal transduction molecules for induction of cancer cell apoptosis are of great interest. We used experimental cell line, Ramos with high levels of Gb3 expression to study apoptosis-inducing molecules (mitogen-activated protein kinases (MAPK) cascade, mitochondria-controlling caspase, and death receptor signal) activated by MytiLec in Burkitt's lymphoma cells.
Mar. Drugs 2015, 13, page-page 3 (Jeremy R.H. Tame, personal communication). Six carbohydrate-binding sites of dimer MytiLec was essential for the cytotoxicity (data not shown) similar to the property of another cytotoxic R-type lectin isolated from mushroom Clitocybe nebularis [20]. Taken together, MytiLec fits in as a new member of the R-type lectin family.
Some R-type lectins have additional domains as toxic subunits. Pierisin, isolated from Pieris rapae (cabbage butterfly), has an ADP-ribosyltransferase domain in the polypeptide and three R-type lectin domains. Pierisin induces apoptosis in HeLa cells by binding to surface Gb3 and Gb4 (GalNAcβ1-3Galα1-4Galβ1-4Glc) glycans [21]. In addition to the original MytiLec, two MytiLec variants (termed MytiLec2 and MytiLec3) containing a pore-forming aerolysin [22]-like domain in the polypeptide that creates pores into infectious organisms and kills them through initiation of innate immunity, according to the recently updated MytiBase [4].
MytiLec does not have additional functional domains or subunits beside glycan-binding domains, in contrast to other R-type lectins, although it is capable of inducing cytotoxicity. It thus occupies a unique category within the R-type lectin family. The mechanisms whereby MytiLec transmits its signals through cells to activate various signal transduction molecules for induction of cancer cell apoptosis are of great interest. We used experimental cell line, Ramos with high levels of Gb3 expression to study apoptosis-inducing molecules (mitogen-activated protein kinases (MAPK) cascade, mitochondria-controlling caspase, and death receptor signal) activated by MytiLec in Burkitt's lymphoma cells.

Cytotoxic Effects of MytiLec on Burkitt's Lymphoma Cell Lines
Cytotoxic effects of MytiLec administration were evaluated by WST-8 assay rather than trypan blue assay because agglutinated cell masses were not effectively stained by trypan blue reagent. Ramos and K562 cells were cultured for 24 h, treated with MytiLec, and reduction in proportion of living cells was assayed by measuring absorbance at 450 nm. Viability was reduced in comparison with control (nontreated) cells for Ramos treated with 10 μg/mL of MytiLec, indicating a cytotoxic effect. Viability of K562 cells, which do not express Gb3, was unaffected by MytiLec treatment (Figure 2A).

Cytotoxic Effects of MytiLec on Burkitt's Lymphoma Cell Lines
Cytotoxic effects of MytiLec administration were evaluated by WST-8 assay rather than trypan blue assay because agglutinated cell masses were not effectively stained by trypan blue reagent. Ramos and K562 cells were cultured for 24 h, treated with MytiLec, and reduction in proportion of living cells was assayed by measuring absorbance at 450 nm. Viability was reduced in comparison with control (nontreated) cells for Ramos treated with 10 µg/mL of MytiLec, indicating a cytotoxic effect. Viability of K562 cells, which do not express Gb3, was unaffected by MytiLec treatment (Figure 2A). right and upper portions (respectively) of these histograms. K562 cells were unaffected by MytiLec treatment. The membrane inversion and penetration observed in MytiLec-treated Ramos cells were consistent with results of our previous study on Raji cells, another Burkittʹs lymphoma cell line [13]. These effects on the Ramos cell membrane ( Figure 2B) appeared to be associated with the cytotoxic effect of MytiLec ( Figure 3). MytiLec may increase cell fragility by suppressing biosynthesis of cell surface membrane proteins. The triggering concentration of MytiLec is lower for apoptosis (~10 μg/mL) than for necrosis (>20 μg/mL). These observations may be related to the functions of MytiLec in caspase activation and TNF-α production (Section 2.4).   consistent with results of our previous study on Raji cells, another Burkittʹs lymphoma cell line [13]. These effects on the Ramos cell membrane ( Figure 2B) appeared to be associated with the cytotoxic effect of MytiLec ( Figure 3). MytiLec may increase cell fragility by suppressing biosynthesis of cell surface membrane proteins. The triggering concentration of MytiLec is lower for apoptosis (~10 μg/mL) than for necrosis (>20 μg/mL). These observations may be related to the functions of MytiLec in caspase activation and TNF-α production (Section 2.4).  Fluorescence activated cell sorting (FACS) analysis revealed that MytiLec treatment led to deleterious biological processes such as cell membrane inversion and loss of membrane integrity. Horizontal axes in Figure 3B show binding of Fluorescein isothiocyanate (FITC)-labeled annexin V, and vertical axes show incorporation of propidium iodide. Increasing MytiLec concentration was associated with shifting of annexin V-positive and propidium iodate-positive populations into the right and upper portions (respectively) of these histograms. K562 cells were unaffected by MytiLec treatment. The membrane inversion and penetration observed in MytiLec-treated Ramos cells were consistent with results of our previous study on Raji cells, another Burkitt's lymphoma cell line [13]. These effects on the Ramos cell membrane ( Figure 2B) appeared to be associated with the cytotoxic effect of MytiLec ( Figure 3). MytiLec may increase cell fragility by suppressing biosynthesis of cell surface membrane proteins. The triggering concentration of MytiLec is lower for apoptosis (~10 µg/mL) than for necrosis (>20 µg/mL). These observations may be related to the functions of MytiLec in caspase activation and TNF-α production (Section 2.4).

Internalization of MytiLec into Burkitt's Lymphoma Cells
Internalization of fluorescein-conjugated MytiLec (20 µg/mL) by Burkitt's lymphoma cells was demonstrated by confocal microscopy. Cell surface fluorescence was observed at the beginning ( Figure 3a) and after 2 h incubation, strong intracellular fluorescence was detected due to the migration of FITC-MytiLec (Figure 3c vs. d). Such internalization was similar to that observed for TF-antigen-binding BEL lectin [24], an R-type lectin purified from mushroom. Cells with internalized MytiLec looked shrunken and irregular shaped with a characteristic rough surface (Figure 3c,d, arrows). This internalization was totally inhibited by the co-presence of D-galactose, a haptenic sugar of the lectin (Figure 3e). It can be assumed that the internalization of MytiLec activated a number of cell signaling pathways (described in following sections) whether inhibition of this internalization by the sugar gives an idea about the mechanism of Gb3-dependent signaling.

Activation of MAPK Pathways by MytiLec
MAPKs play essential roles in cell growth and differentiation, cell cycle, and cell death. We found that MytiLec activates several MAPK pathways in Burkitt's lymphoma cells.

Internalization of MytiLec into Burkitt's Lymphoma Cells
Internalization of fluorescein-conjugated MytiLec (20 μg/mL) by Burkittʹs lymphoma cells was demonstrated by confocal microscopy. Cell surface fluorescence was observed at the beginning ( Figure 3a) and after 2 h incubation, strong intracellular fluorescence was detected due to the migration of FITC-MytiLec (Figure 3c vs. d). Such internalization was similar to that observed for TF-antigen-binding BEL lectin [24], an R-type lectin purified from mushroom. Cells with internalized MytiLec looked shrunken and irregular shaped with a characteristic rough surface (Figure 3c,d, arrows). This internalization was totally inhibited by the co-presence of D-galactose, a haptenic sugar of the lectin (Figure 3e). It can be assumed that the internalization of MytiLec activated a number of cell signaling pathways (described in following sections) whether inhibition of this internalization by the sugar gives an idea about the mechanism of Gb3-dependent signaling.

Activation of MAPK Pathways by MytiLec
MAPKs play essential roles in cell growth and differentiation, cell cycle, and cell death. We found that MytiLec activates several MAPK pathways in Burkitt's lymphoma cells.

Activation of MEK-ERK Pathway
In Ramos, MytiLec activated the classical MAPK pathway of MAPK/ extracellular signal-regulated kinase (MEK)1/2 and extracellular signal-regulated kinase (ERK)1/2 signaling cascade in a dose-dependent manner, as shown by Western-blotting ( Figure 4A, P-MEK1/2 vs. MEK1/2 and P-ERK1/2 vs. ERK1). No such phosphorylation occurred in K562 (data not shown). Phosphorylation of the MEK-ERK pathway by MytiLec resulted in expression of the cyclin-dependent kinase (CDK) inhibitors p21 (Figure 4 column p21). In contrast, up-regulated levels of CDK6 and cyclinD3 were slightly reduced by MytiLec (Figure 4).   Expression of p21 (which binds to CDK and inhibits its activity) is enhanced by various external stimuli and stress factors, resulting in cell cycle arrest at the G 0/1 phase [25]. Up-regulation of p21 level by MEK has been documented using specific inhibitors [26]. The above findings indicate that binding of MytiLec and Gb3 on the cells induced MEK-ERK pathway activation and p21 expression, eventually resulting in cell cycle arrest.

Phosphorylation of Stress-Activated MAPK Pathways (JNK, p38 Kinase)
In addition to the classical MAPK pathway (MEK-ERK), MytiLec phosphorylated stress-activated MAPK pathways [c-Jun N-terminal kinase (JNK) and p38 kinase] in Ramos ( Figure 5, asterisks). Evidently, ERK mediated transduction of the MytiLec/ Gb3 binding signal to JNK and p38 kinase. The MytiLec-generated stimuli were equivalent to signals generated by oxidative stresses [27]. A recent study indicates that the MEK/ERK pathway itself can activate both JNK and p38 kinase [28]. Certain mannose-binding proteins inhibited cell proliferation through activation of these pathways [29,30]. Stress-activated kinases play roles in tumor suppression, apoptosis, termination of cell differentiation, and autophagy [31]. 6 CDK6 and cyclinD3 were shown, respectively. Cells (4 × 10 5 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.
Expression of p21 (which binds to CDK and inhibits its activity) is enhanced by various external stimuli and stress factors, resulting in cell cycle arrest at the G0/1 phase [25]. Up-regulation of p21 level by MEK has been documented using specific inhibitors [26]. The above findings indicate that binding of MytiLec and Gb3 on the cells induced MEK-ERK pathway activation and p21 expression, eventually resulting in cell cycle arrest.

Phosphorylation of Stress-Activated MAPK Pathways (JNK, p38 Kinase)
In addition to the classical MAPK pathway (MEK-ERK), MytiLec phosphorylated stress-activated MAPK pathways [c-Jun N-terminal kinase (JNK) and p38 kinase] in Ramos ( Figure  5, asterisks). Evidently, ERK mediated transduction of the MytiLec/ Gb3 binding signal to JNK and p38 kinase. The MytiLec-generated stimuli were equivalent to signals generated by oxidative stresses [27]. A recent study indicates that the MEK/ERK pathway itself can activate both JNK and p38 kinase [28]. Certain mannose-binding proteins inhibited cell proliferation through activation of these pathways [29,30]. Stress-activated kinases play roles in tumor suppression, apoptosis, termination of cell differentiation, and autophagy [31].
In conclusion, MytiLec/Gb3 binding in Ramos promoted both the classical MAPK pathway (MEK-ERK) and stress-activated MAPK pathways (JNK, P38 kinase). MytiLec-induced phosphorylation of the MEK-ERK pathway resulted in up-regulation of p21 expression that might lead to cell cycle arrest.

TNF-α Induction and Caspase Activation in Ramos Cells
MytiLec treatment of Burkitt's lymphoma Ramos cells triggered production of tumor necrosis factor (TNF)-α and activation of caspase-9 and caspase-3 ( Figure 7A). The up-regulation of TNF-α by MytiLec was inhibited by P-MEK inhibitor U0126 and by caspase-3 inhibitor Zn-DEVD-FMK ( Figure 7B). Association of TNF-α induction with caspase-3 activation was consistent with a previous observation by Burguillos et al. [33], and suggests that TNF-α expression is concurrently regulated by the MEK-ERK pathway and caspase-3 activation.

TNF-α Induction and Caspase Activation in Ramos Cells
MytiLec treatment of Burkitt's lymphoma Ramos cells triggered production of tumor necrosis factor (TNF)-α and activation of caspase-9 and caspase-3 ( Figure 7A). The up-regulation of TNF-α by MytiLec was inhibited by P-MEK inhibitor U0126 and by caspase-3 inhibitor Zn-DEVD-FMK ( Figure 7B). Association of TNF-α induction with caspase-3 activation was consistent with a previous observation by Burguillos et al. [33], and suggests that TNF-α expression is concurrently regulated by the MEK-ERK pathway and caspase-3 activation.

TNF-α Induction and Caspase Activation in Ramos Cells
MytiLec treatment of Burkitt's lymphoma Ramos cells triggered production of tumor necrosis factor (TNF)-α and activation of caspase-9 and caspase-3 ( Figure 7A). The up-regulation of TNF-α by MytiLec was inhibited by P-MEK inhibitor U0126 and by caspase-3 inhibitor Zn-DEVD-FMK ( Figure 7B). Association of TNF-α induction with caspase-3 activation was consistent with a previous observation by Burguillos et al. [33], and suggests that TNF-α expression is concurrently regulated by the MEK-ERK pathway and caspase-3 activation.   To elucidate the caspase activation pathway, U0126 was applied to Ramos prior to MytiLec administration.
Previous studies have demonstrated stimulation via phosphorylation of various signal transduction molecules by lectins isolated from lower organisms. In mouse macrophage cell lines, a recombinant B-subunit of ricin was shown to stimulate signal transduction pathways through production of inducible nitric oxide synthase, TNF-α, and interleukin-6 [34]. Human TNF-α reduced the phagocytic ability of mussel hemocytes [35]. The normal endogenous role of MytiLec in M. galloprovincialis remains unclear. Genome database analysis suggests that it may function in innate immunity [4]. Certain signal transduction molecules in M. galloprovincialis are also found in vertebrates (including humans) [5,36], suggesting similarities in the fundamental regulatory mechanisms of growth, differentiation, and cell proliferation. A role of native MytiLec in supporting innate immunity could explain its cytotoxic activity against Burkitt's lymphoma cells, since the mussel and vertebrate cells may have common surface glycans such as Gb3, as well as similar cell regulatory mechanisms. Along this line, we are attempting to identify the endogenous ligands of Gb3 and related glycans in the mussel. We previously observed binding of MytiLec to endogenous glycans in mussel tissue [13]. Surface Gb3 expression was observed on cultured cells derived from sea bass (Dicentrarchus labrax) [37].
There are difficulties in studying marine drugs based on proteins. Proteins trigger immune responses, and present logistical research problems because of their large size. However, recent studies based on each of sialic acid-binding and α-galacotside-binding lectins-coding genes from fish and sea urchin, respectively successfully recombined the genes into an adenovirus vector and applied the lectins for oncotherapy in vitro [38,39]. MytiLec, with its novel cytotoxic properties, has great potential for similar therapeutic application through Gb3-signaling.
Ponting et al. [40] reviewed studies since 1990 of β-trefoil folding, including the question of why toxins (ricin B-chain [19]), cytokines (fibroblast growth factor [41], interleukin-1 [42]), enzymes (non-catalytic domain of glycosyltransferases [43]) and protease inhibitors (Kunitz-type protease inhibitor [44]) are synchronically assigned to the same 3D structural group even though their primary structures have low similarity (<12%). The polypeptides are highly conserved, with triple-tandem repeating sequences that contain four β-sheets and one α-helix in each subdomain. It appears that β-trefoil folding is one of the most versatile templates for protein conformation, and is involved in a wide range of physiological functions in many organisms. The primary structure of MytiLec is unique in that gene coding for R-type lectins has not been reported for any other Mytilus species. We hypothesize that an ancestral gene was synchronically modified for β-trefoil folding in MytiLec in association with its role in innate immunity.
The cell regulatory properties of Gb3-binding lectins are clearly diverse and merit further study. SAL, a Gb3-specific SUEL/RBL-type lectin isolated from catfish (Silurus asotus) eggs, phosphorylated several signal transduction molecules, resulting in cell cycle delay but had no cytotoxic effect [45], unlike MytiLec. SAL was found to down-regulate the multidrug resistance (MDR) 1 P-glycoprotein (MDR1 P-gp) on Burkitt's lymphoma cells through Gb3 and did not directly influence the viability of the cells. In this study, Gb3 was found to be an effective trigger to regulate cancer cell growth through apoptosis. Therefore, along with Burkitt's lymphoma cells, a number of Gb3-expressing cancer cell lines [46] like HeLa, MCF-7 and T47D, can be good targets to study cell signaling. In particular, detailed investigation of these lectins and their properties will be useful for development of novel anti-cancer drugs and therapeutic strategies.