A GM1b/asialo‐GM1 oligosaccharide‐binding R‐type lectin from purplish bifurcate mussels Mytilisepta virgata and its effect on MAP kinases

Abstract

A 15‐kDa lectin, termed SeviL, was isolated from Mytilisepta virgata (purplish bifurcate mussel). SeviL forms a noncovalent dimer that binds strongly to ganglio‐series GM1b oligosaccharide (Neu5Acɑ2‐3Galβ1‐3GalNAcβ1‐4Galβ1‐4Glc) and its precursor, asialo‐GM1 (Galβ1‐3GalNAcβ1‐4Galβ1‐4Glc). SeviL also interacts weakly with the glycan moiety of SSEA‐4 hexaose (Neu5Acα2‐3Galβ1‐3GalNAcβ1‐3Galα1‐4Galβ1‐4Glc). A partial protein sequence of the lectin was determined by mass spectrometry, and the complete sequence was identified from transcriptomic analysis. SeviL, consisting of 129 amino acids, was classified as an R(icin B)‐type lectin, based on the presence of the QxW motif characteristic of this fold. SeviL mRNA is highly expressed in gills and, in particular, mantle rim tissues. Orthologue sequences were identified in other species of the family Mytilidae, including Mytilus galloprovincialis, from which lectin MytiLec‐1 was isolated and characterized in our previous studies. Thus, mytilid species contain lectins belonging to at least two distinct families (R‐type lectins and mytilectins) that have a common β‐trefoil fold structure but differing glycan‐binding specificities. SeviL displayed notable cytotoxic (apoptotic) effects against various cultured cell lines (human breast, ovarian, and colonic cancer; dog kidney) that possess asialo‐GM1 oligosaccharide at the cell surface. This cytotoxic effect was inhibited by the presence of anti‐asialo‐GM1 oligosaccharide antibodies. With HeLa ovarian cancer cells, SeviL showed dose‐ and time‐dependent activation of kinase MKK3/6, p38 MAPK, and caspase‐3/9. The transduction pathways activated by SeviL via the glycosphingolipid oligosaccharide were triggered apoptosis. Database Nucleotide sequence data have been deposited in the GenBank database under accession numbers , , , , , , , , , , and .

The structure of SeviL, a GM1b/asialo-GM1 binding R-type lectin from the mussel Mytilisepta virgata

“…Hemagglutination is also lost in a SeviL mutant that retains sugar binding, but forms only monomers. The dimeric form therefore appears to be required for at least some of the biological effects of the native protein, which include stimulation of apoptotic pathways in cells that SeviL can bind 16 . The models described in this paper form the basis of further work to develop a new biosensor for asialo-GM1, a useful addition to present lectin arrays, as well as understand the biological effects of SeviL on cells that display its target saccharide.…”

“…The protein was found to cause hemagglutination of human red cells in the presence of calcium, and to bind the sugar chains of the gangliosides GM1b and asialo-GM1. Glycan array profiling of SeviL against a wide variety of oligosaccharides, 52 in all, showed no other significant interaction, apart from weak binding to SSEA-4, Neu5Acα (2-3)Galβ(1-3)GalNAcβ(1-3)Galα(1-4)Galβ(1-4)Glc 16 . The first three monosaccharide residues of GM1b and SSEA-4 are identical.…”

“…Hemagglutination activity. SeviL is able to hemagglutinate red cells in the presence of calcium 16 . Mutants of SeviL, designed on the basis of the model described here, that are unable to bind the saccharide or form the dimer do not hemagglutinate (Fig.…”

“…The mytilectin subfamily shows unique sequence patterns compared to other lectins with the trefoil fold, and is of interest for its unusual specificity towards -linked substrates. The mytilectins were the only -trefoil lectins known in the mytilid mussels, until the recent discovery in the purplish bifurcate mussel Mytilisepta virgata of a new protein called SeviL, which was purified from homogenised tissues using immobilised lactose 16 . The protein was found to cause hemagglutination of human red cells in the presence of calcium, and to bind the sugar chains of the gangliosides GM1b and asialo-GM1.…”

“…The natural function of SeviL remains unknown, but if SeviL is added to mammalian carcinoma cells expressing asialo-GM1 then it activates apoptotic pathways, causing cell death 16 . The 129 residue sequence of SeviL is unrelated to the 149 residue MytiLec, but shows three weakly-conserved tandem repeats of about 40 residues, each with a tryptophan residue at an equivalent position.…”