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␣1,3Gal1,4GlcNAc; 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␣1,3Gal1,4GlcNAc 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...
Ultrastructural and cytochemical investigations of ocular lens have been difficult due to limited applicability of currently available techniques for the preparation of this tissue for such studies Several modifications in currently available techniques are being made in order to preserve the fine structure of the lens. In this report we present a modified method which provides good preservation of lens fine structure and allows cytochemical localization of enzymes in this tissue. Brief freezing permits preparation of thin slices of the lens which are subsequently incubated in the appropriate media for enzyme cytochemistry at an ultrastructural level. We have used this modified method for the localization of Na-K-dependent ATPase and the results are presented in this report.
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