Biochemical characterization and cytotoxicity of polylactosamine-extended N-glycans binding isolectins from the mushroom Hericium erinaceus

The mushroom Hericium erinaceus expresses isolectins with different glycan binding specificities; of these, the ricin B-like lectin HEL1 and HEL2 (HEL2a and HEL2b) can bind fucosylated N-glycans and core 1 O-glycans, respectively. However, other lectin-like protein-coding transcripts detected in the H. erinaceus transcriptome, named HEL3, remain to be characterized. Therefore, in this study, the expression levels of all these isolectins genes were compared to characterize the molecular and biochemical properties of these carbohydrate-binding proteins. Low expression genes encoding putative cytolysin proteins, HEL3a and HEL3b, were identified. Bioinformatics analyses revealed that these proteins shared highly homologous structures and carbohydrate-binding residues with other mushroom lectins. Further, their recombinant proteins, rHEL3a and rHEL3b showed an octamer composed of identical 17 kDa subunits under non-denaturing conditions and a slightly basic isoelectric point value of approximately 8.3. The hemagglutination activity of these isolectins was strongly inhibited by glycoproteins rather than free glycans. Interestingly, glycan-binding profiles showed that rHEL3 isolectins interacted with most polylactosamine (poly-LacNAc)-extended N-glycans with relatively low binding activity. Isothermal titration calorimetry also revealed that these recombinant lectins have different binding capacities toward N-glycan-containing glycoproteins. Further, treatment with different concentrations of rHEL3 lectins showed cytotoxic effects in K562, UACC62, and CHO model cell lines, which express poly-LacNAc glycans, confirmed by inhibition of proliferation. Overall, these biochemical properties indicate that rHEL3 isolectins may be used as unique lectins for detecting poly-LacNAc-extended glycans, which are known to be over-expressed in leukemia or metastatic melanoma cells, in cancer diagnostic assays and anti-cancer therapies.