A lectin from the mussel Mytilus galloprovincialis has a highly novel primary structure and induces glycan-mediated cytotoxicity of globotriaosylceramide-expressing lymphoma cells

Abstract

A novel lectin structure was found for a 17-kDa α-D-galactose-binding lectin (termed "MytiLec") isolated from the Mediterranean mussel, Mytilus galloprovincialis. The complete primary structure of the lectin was determined by Edman degradation and mass spectrometric analysis. MytiLec was found to consist of 149 amino acids with a total molecular mass of 16,812.59 Da by Fourier transform-ion cyclotron resonance mass spectrometry, in good agreement with the calculated value of 16,823.22 Da. MytiLec had an N terminus of acetylthreonine and a primary structure that was highly novel in comparison with those of all known lectins in the structure database. The polypeptide structure consisted of three tandem-repeat domains of ∼50 amino acids each having 45-52% homology with each other. Frontal affinity chromatography technology indicated that MytiLec bound specifically to globotriose (Gb3; Galα1-4Galβ1-4Glc), the epitope of globotriaosylceramide. MytiLec showed a dose-dependent cytotoxic effect on human Burkitt lymphoma Raji cells (which have high surface expression of Gb3) but had no such effect on erythroleukemia K562 cells (which do not express Gb3). The cytotoxic effect of MytiLec was specifically blocked by the co-presence of an α-galactoside. MytiLec treatment of Raji cells caused increased binding of anti-annexin V antibody and incorporation of propidium iodide, which are indicators of cell membrane inversion and perforation. MytiLec is the first reported lectin having a primary structure with the highly novel triple tandem-repeat domain and showing transduction of apoptotic signaling against Burkitt lymphoma cells by interaction with a glycosphingolipid-enriched microdomain containing Gb3.

FIGURE 1.

Purification of MytiLec. A, extract from mussel (M. galloprovincialis) by TBS containing 100 mm d-Gal was extensively dialyzed and applied to a melibiose-conjugated agarose column (1 × 5 cm) equilibrated with TBS. MytiLec bound to the column was eluted with TBS containing 200 mm melibiose (arrow). B, SDS-PAGE pattern under reducing (R) and nonreducing (NR) conditions. Numbers at right indicate the molecular masses of marker proteins as follows: phosphorylase b (97 kDa), BSA (66 kDa), Ovalbumin (44 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20 kDa), and lysozyme (14 kDa). M, molecular marker; C, crude extract; L, lectin.

FIGURE 2.

Estimation of the molecular mass of MytiLec by gel permeation chromatography. A Superdex 75 column (1.0 × 30 cm) was equilibrated with TBS containing 50 mm melibiose. Molecular standards are shown in relation to elution position. Marker proteins: BSA (66 kDa) (1); ovalbumin (44 kDa) (2); carbonic anhydrase (29 kDa) (3), and lysozyme (14 kDa) (4).

FIGURE 3.

Elution (HPLC) profiles of peptides generated by enzymatic digestion and chemical cleavage of MytiLec. A, Achromobacter protease I digest on an Aquapore RP-300 column. B, CNBr cleavage on a Superspher Select B column. C, Asp-N digest on a Mightysil RP-18 column. Peptides were eluted by a gradient of acetonitrile into dilute aqueous TFA.

FIGURE 4.

Proven sequence of MytiLec. Sequences determined by Edman degradation of specific peptides (indicated by italic type) are shown by one-letter code below the summary sequence (top line). Molecular mass values obtained from MALDI-TOF and FT-ICR MS are indicated in parentheses. Uppercase letters, peptide sequences proven by Edman degradation. Lowercase letters, sequences tentatively identified or deduced from MS. m′, homoserine lactone. Dashes, sequences not identified by the protein sequencer.

FIGURE 5.

Amino acid sequence homology of the internal tandem-repeat domains of MytiLec. A, boxes indicate the common (homologous) amino acid residues within the repeat domains. Common residues at bottom summarizes the common residues in the three domains. Uppercase letters, residues identical in all three domains. Lowercase letters, residues identical in two of the domains. B, residue numbers of the polypeptide are shown. Acidic and basic amino acids are indicated as Acidic a.a. and Basic a.a., respectively.

FIGURE 6.

Glycan-binding profile of MytiLec obtained by FACT analysis. The numbers on the vertical axis correspond to the oligosaccharide numbers in the list of PA-glycans shown in Table 5 and used in the text. Horizontal axis, difference in relative intensity between the elution front volume of each PA-oligosaccharide (V) and PA-rhamnose (Vo). Error bars, S.E. (n = 3).

FIGURE 7.

Kinetic analysis by SPR. MytiLec was applied to a CM5 sensor chip coupled with Galα1–4Galβ1–4Glc-HSA at 6.8 ng/mm². MytiLec was applied to a asialofetuin-conjugated sensor chip at 20 μl/min for 2.5 min. MytiLec concentrations (red lines from top to bottom): 800 (6), 400 (5), 200 (4), 100 (3), 50 (2), and 0 (1) nm. The chip was washed with TBS for 2.5 min. Vertical axis, resonance units (indicating association and dissociation of the analyte). Kinetics were analyzed using the BIAevaluation software program, version 3.0 (GE Healthcare).

FIGURE 8.

Glycan-dependent reduction of viability of Burkitt lymphoma Raji cells by MytiLec. Cell viability and numbers of living cells were determined using trypan blue exclusion assay (A and C) and WST-8 (B and D), respectively. A and B, Raji cells were treated with various concentrations (0–50 μg/ml) of MytiLec. Black bars (or circles) and gray bars (or circles) indicate the viability (or absorbance) of Raji cells and erythroleukemia K562 cells (negative control), respectively. C and D, blocking by various added saccharides (100 mm each) of the reduction of cell viability caused by MytiLec. Suc, sucrose; Mel, melibiose; Lac, lactose. Black bars (or circles) and shaded bars (or circles) indicate the viability (or absorbance) of Raji cells and Raji cells added with saccharides, respectively. Error bars, S.E. (n = 3).

FIGURE 9.

Detection of annexin V and incorporation of propidium iodide (PI) in MytiLec-treated Raji cells as analyzed by FACSCalibur. Horizontal axes, binding of FITC-labeled anti-annexin V antibody. Phosphatidylserine externalization and propidium iodide incorporation were detected using the MEBCYTO apoptosis kit by FACScan. A, Raji and K562 cells were treated with various concentrations (0–50 μg/ml) of MytiLec (MytL) as indicated for 30 min at 4 °C. B, blocking of the cytotoxic activity of MytiLec (10 μg/ml) by saccharide (100 mm) addition to Raji cells. Cont, PBS; Mel, melibiose; Suc, sucrose.