The Mushroom Marasmius oreades Lectin Is a Blood Group Type B Agglutinin That Recognizes the Galα1,3Gal and Galα1,3Galβ1,4GlcNAc Porcine Xenotransplantation Epitopes with High Affinity

Abstract

A blood group B-specific lectin from the mushroom Marasmius oreades (MOA) was investigated with respect to its molecular structure and carbohydrate binding properties. SDS-PAGE mass spectrometric analysis showed it to consist of an intact (H; 33 kDa) and truncated (L; 23 kDa) subunit in addition to a small polypeptide (P; 10 kDa). Isolation in the presence of EDTA produced only the H subunits, indicating that the latter two are formed by metalloprotease cleavage of the intact H subunit. Tryptic digestion of the H, L, and P polypeptide chains followed by mass spectral analysis supports this view. The lectin strongly precipitated blood group type B substance, was nonreactive with type A substance, and reacted weakly with type H substance. Carbohydrate binding studies reveal a high affinity for Gal␣1,3Gal (but not for the isomeric ␣1,2-, ␣1,4-, and ␣1,6-disaccharides); Gal␣1,3Gal␤1,4GlcNAc; and the type B branched trisaccharide. MOA also reacts strongly with murine laminin from the Engelbreth-Holm-Swarm sarcoma and bovine thyroglobulin, both of which contain multiple Gal␣1,3Gal␤1,4GlcNAc end groups. This linear B trisaccharide is a component of porcine tissues and organs, preventing their transplantation into humans. MOA also shares carbohydrate recognition of this trisaccharide with toxin A elaborated by Clostridium difficile.Blood group-specific lectins have served as serological reagents for typing blood for over 65 years (1)(2)(3). Among the useful reagents are Dolichos biflorus lectin for human type A 1 erythrocytes (4, 5), the B 4 Griffonia simplicifolia isolectin for type B erythrocytes (6), and the Ulex europeus I (7, 8) and eel (Anguilla anguilla) serum agglutinins for type O(H) erythrocytes (1, 2). Of all the blood group-specific lectins studied, group B-specific lectins are probably fewest in number.Presently, the fruiting bodies of mushrooms are being studied as sources for lectins with novel carbohydrate binding activity. Several surveys have been published that classify these proteins based on their ability to agglutinate human (and rabbit) erythrocytes according to blood type (9 -12).Crude extracts of the mushroom Marasmius oreades (Tricholomataceae) were first reported to exhibit human type B erythrocyte agglutinating activity in 1951 (9, 13). A lectin from this source was isolated by affinity chromatography on a matrix of ␣-galactosyl-polyacrylamide gel by Horejsi and Kocourek (14). It was reported to agglutinate type B erythrocytes six times more avidly than type A erythrocytes and was stated to be a heterodimeric protein of 50 kDa, with subunits of 33 and 23 kDa. No mono-or oligosaccharides (25 mM) tested were found to inhibit agglutination of human type B erythrocytes.We have undertaken a more detailed investigation of this potentially important B-specific lectin with interesting results. Quantitative precipitation studies using a highly purified preparation with A, B, and O(H) blood group substances show absolute specificity for B versus A substance, with slight Obinding activity. Binding stud...

The Pathogenic Fungus Paracoccidioides brasiliensis Exports Extracellular Vesicles Containing Highly Immunogenic α-Galactosyl Epitopes

“…These results suggested the involvement of terminal ␣-galactosyl epitopes in the reaction and were corroborated by intense reactivity in ELISA with MOA lectin (Fig. 4, right), which specifically binds to terminal nonreducing Gal␣1,3Gal moieties (41). The reactivity with MOA lectin decreased about 75% upon treatment of P. brasiliensis vesicles from both Pb3 and Pb18 with ␣-galactosidase (Fig.…”

Entomotoxic and nematotoxic lectins and protease inhibitors from fungal fruiting bodies

“…Entomotoxic b (Cordara et al 2011;Grahn et al 2007;Grahn et al 2009;Rempel et al 2002;Winter et al 2002;Wohlschlager et al 2011) na Entomotoxic (Brzin et al 2000;Renko et al 2010;Sabotič et al 2007a;Sabotič et al 2006;Sabotič et al 2011;Šmid et al 2015 (91 % sequence similarity) and are approximately 32 % identical (43 % similar) to CGL2. Galectins CGL1 and CGL2 are highly abundant in young fruiting bodies but hardly produced in the vegetative mycelium of C. cinerea (Boulianne et al 2000;Plaza et al 2014).…”

Nematotoxicity of Marasmius oreades Agglutinin (MOA) Depends on Glycolipid Binding and Cysteine Protease Activity

“…The dimeric lectin consists of 293-residue protomers and has been shown to specifically recognize Gal␣1,3-containing structures, exhibiting the highest affinity for the branched blood group B trisaccharide Gal␣1,3(Fuc␣1,2)Gal (6). Crystal structures of MOA in complex with the xenotransplantation epitope and the blood group B trisaccharide revealed an N-terminal ricin B-type lectin domain with three canonical carbohydrate-binding sites and a C-terminal domain that is involved in dimerization.…”

“…Protein Expression, Purification, and Biotinylation-MOA and MOA(C215A) were expressed recombinantly in Escherichia coli and purified as described (6,11). Detailed information can be found under supplemental "Materials and Methods."…”