The polyfunctional properties of carbohydrate-binding proteins, lectins, allow their application to various biological and medical studies as speciﬁc agglutinins in blood typing; in synthesis of afﬁne stationary phases for fractionation and analysis of polysaccharides, glycolipids, or glycoproteins; and in cytochemical and histochemical studies of glycoconjugates of tissues and cell surfaces. The hemagglutination reaction, based on speciﬁc lectin binding to carbohydrates on erythrocyte surface, is the most common and appropriate test system for the determination of lectin activity and carbohydrate speciﬁcity. Treatment with trypsin interferes the pattern of erythrocyte binding by lectins and, therefore, the analytical characteristics of the test-system used for disclosing their carbohydrate-binding properties. Erythrocytes obtained from different organisms differ in carbohydrate determinants, the trypsin-treatment being capable of enzymatic constructing the optimal receptor sites on their surface.
Then we turn our attention to the results that our group has obtained over the last ten years.
Trypsin-stimulated agglutination of animal (rabbit, horse, ram, bovine) erythrocytes was observed to be appropriate semi-qualitative analytical method for studying the lectin activity and carbohydrate specificity of the agglutinins of basidial mushrooms at the dikaryotic mycelium step. The difference in taxonomic characteristics and in the medium composition on growing the fungal cultures – representatives of xylotrophic basidiomycetes – influenced the hemagglutinating activity to a much lesser extent, as compared with the aggregate state of nutrient medium and the type of erythrocytes in hemagglutination reaction.
Hemagglutinating activity of Lentinula edodes intracellular lectins at different morphogenesis steps was explored depending upon the type of erythrocytes, the growth medium, and the grade of proteins purity. The lectin activity under a solid-phase cultivation was appeared to depend on the type of erythrocytes used in a hemagglutination reaction, including the trypsin-treatment of erythrocytes, to a great extent. The sensitivity of the lectins towards trypsin-treated rabbit erythrocytes was no less than a hundredfold higher than towards any other erythrocyte type studied.
The culture of L. edodes F-249 occured to synthesize two extracellular lectins. We have isolated those from fungal culture liquid and purified to electrophoretic homogeneity. The lectins differ in physicochemical properties, composition, carbohydrate specificity, and ability for agglutination of erythrocytes of various types. The purified extracellular lectins of L. edodes are highly selective at recognition of definite structures on the surface of trypsinized rabbit erythrocytes and do not react with the erythrocytes of other animals and humans.
At the same time, a preferable agglutination of human trypsinized erythrocytes compared to untreated ones was not observed under any experimental conditions when exploring the blood-group specificity of the lectins of basidial xylotrophs from different systematic groups. The extent of manifestation of the hemagglutinating activity in respect to human erythrocytes had the peculiarities general for all the cultures along with somewhat differences caused obviously by the individual characteristics of each basidiomycete. The maximal values of hemagglutination titers for all the fungi were on the agar media. Judging from the results of solid-phase cultivation, the agglutinins of Armillariella mellea 1346 should be considered to be Н(О) blood-group specific.
Trypsin-treated erythrocytes appeared to be worse test-system for agglutinating ability of mushroom mycelial hemagglutinin, a dimeric membrane-bound 68 kDa-lectin, which we isolated from the surface of dikaryotic mycelium of xylotrophic basidiomycete Grifola frondosa. The lectin exhibited high afﬁnity to native rabbit erythrocytes and to human erythrocytes of the O blood group, but not to trypsin-treated ones. Only 13.9 µg/ml of the homogeneous preparation of a polysaccharide, a linear D-rhamnan with the definite structure of the repeated component, blocked hemagglutination completely. The lectins specific toward glycoconjugate, but not to any monosaccharide, presumably oppose a trypsin-assisted revision of the erythrocytes surface. Enzymatically lost complex carbohydrate-containing molecules – more or less perfect sites of endolectins binding with erythrocytes – results in poor agglutination and, may be, inadequate conclusions on lectins properties.