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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.

An invertebrate sialyltransferase, cST3Gal-I, identified from the sea squirt Ciona savignyi, was functionally characterized in vitro using recombinant enzyme expressed in yeast strains. cST3Gal-I was localized to the Golgi membrane when expressed in Saccharomyces cerevisiae. Enzymatic characterization for substrate specificity and kinetic property indicate that cST3Gal-I prefers O-glycans, rather than N-glycan, of asialoglycoproteins as substrates. Interestingly, C. savignyi sialyltransferase exhibited effectively Neu5Ac transfer to core 1 O-glycan, Gal β(1,3)GalNAc, compared to orthologous human glycosyltransferase. Further, it is shown that cST3Gal-I catalyzes the formation of α(2,3)-linkage, through lectin blot analysis with Maackia amurensis lectin and by linkage-specific sialidase treatments. The putative active sites of cST3Gal-I for putative acid/base catalysts and sialic acid acceptor/donor substrate bindings were also identical to the counterpart residues of a mammalian enzyme, porcine ST3Gal-I, as predicted through homologous structure modeling. These results could imply that an ancestral tunicate ST3Gal-I in C. savignyi would prefer O-glycan onto glycoproteins as its sialic acid acceptor than vertebrate enzymes.

Mucin-type O-glycans are involved in biological functions on the cell surface as well as the glycoproteins and can also be used as specific carbohydrate biomarkers of many diseases. In this study, I purified a novel core 1 O-linked glycan specific lectin, Hericium erinaceus lecin (HeL), from the fruiting body of the mushroom Hericium erinaceus, which is known as the natural source for a sialic acid-binding lectin. Upon optimization of the purification conditions, a sequence of ion exchange, affinity, ion exchange, and size-exclusion chromatography resulted in the highest yield and best quality of lectin without protease activity. The resulting purified HeL is an apparent hexameric protein with a subunit molecular weight of 15kDa, and a pI of 4.3. In hemagglutination inhibition assay, the purified lectin was only inhibited by glycoproteins containing mucin-type O-glycans and reacted weakly with Galβ(1,3)GalNAc. Glycan array analyses showed that HeL specifically interacts with core 1 O-linked glycans as well as extended O-glycan structures containing sialylation or fucosylation. The glycan binding specificity of HeL is comparable to that of peanut agglutinin for detection of a broader range of extended core 1 O-glycan structures. Taken together, these results provide an efficient and optimized procedure for the purification of HeL from the fruiting body of the mushroom Hericium erinaceus. Moreover, HeL represents a powerful tool for analyzing core 1 and extended core 1 O- glycan structures in diagnosis assays.

The mushroom Hericium erinaceus contains isolectins, including the ricin B-like lectin HEL1 and the core 1 O-glycan-binding lectin HEL2. Recombinant HEL2 reportedly binds O-linked glycans, but recombinant HEL1 (rHEL1) has not been characterized. HEL1 and Stereum hirsutum lectin (SHL1) orthologues, which contain the typical (QxW)3 ricin-B like motif, were evaluated. Interestingly, under non-denaturing conditions, recombinant SHL1 (rSHL1) existed as a trimer and exhibited agglutination activity, whereas rHEL1 existed as a monomer with no agglutination activity. The hemagglutination activity of rSHL1 was inhibited by N-linked glycoprotein transferrin. A glycan-array analysis revealed that the two recombinant lectins had different binding intensities toward fucosylated N-glycans harboring fucose-α(1,2) galactose or fucose-α(1,4) N-acetylglucosamine. Isothermal calorimetry showed that compared with rHEL1, rSHL1 interacted more strongly with transferrin, a fucosylated glycoprotein, than with other fucosylated disaccharide glycoconjugates. Finally, rSHL1 and rHEL1 were comparable in their ability to detect highly fucosylated N-glycans within glycoproteins on the surface of SW1116 human colorectal carcinoma cells. Therefore, these ricin B-like lectins might enable detection of highly fucosylated glycoepitopes on cancer cells for diagnostic applications.

In Hericium erinaceus mushroom fruiting body, two different lectin groups, HEL1 and HEL2, were identified by using peptide mass fingerprinting based on customized protein sequence databases derived from RNA-Seq data. The HEL2 group included four isoforms designated HEL2a–d. Codon-optimized genes encoding HEL1, HEL2a, and HEL2b were expressed in Escherichia coli to produce fully active soluble proteins designated rHEL1, rHEL2a, and rHEL2b. Interestingly, these lectins showed different molecular weights: approximately 15 kDa for rHEL1 and approximately 120 kDa for rHEL2a and rHEL2b under non-denaturing conditions. rHEL2a and rHEL2b exhibited agglutination activities, but rHEL1 did not show any agglutination activity toward animal erythrocytes. The hemagglutination activity of rHEL2 lectins was strongly inhibited by glycoproteins containing mucin-type O-glycans. Glycan array analysis and isothermal titration calorimetry revealed that rHEL2 isolectins interacted strongly with O-glycans harboring the core 1 O-glycan motif, Galβ(1,3)GalNAc. Moreover, the glycan binding specificities of rHEL2 isolectins were comparable to that of peanut agglutinin in their ability to recognize O-glycans attached to leukosialin as tumor-associated carbohydrate antigens on the surface of K562 human leukemia cells. These results indicate that rHEL2 isolectins could be used as a powerful tool for analyzing mucin-type O-glycans expressed on the surface of cancer cells.