Lectin purification from carp roe (Cyprinus carpio L.), investigation of its carbohydrate specificity and application in histochemistry.

A method of lectin purification from carp roe (CCRA) was elaborated, which includes affinity chromatography on cross-linked ovomucoid and copolymers of polyvinyl alcohol and blood group-specific substances. That allowed obtaining lectin with electrophoretic purity and yielding of ˜ 42 mg÷kg roe. Electrophoresis in 15% polyacrylamide gel in the presence of ß-mercaptoethanol showed one band with molecular mass ˜ 15 kDa, whereas in the absence of ß-mercaptoethanol, CCRA exposed band with molecular mass ˜ 60 kDa. The resulting lectin was thermostable, withstanding heating to 75°C for 15 minutes, without noticeable loss of hemagglutinating activity. Gel column chromatography on Toyopearl HW-55 determined the lectin molecular weight of 120±3 kDa. For the lectin activity, divalent metal ions (Ca2+ and Mg2+) were not necessary. CCRA showed the best agglutination titer with pigeon erythrocytes, weaker - with rabbit and dog erythrocytes, and significantly weaker - with human and rat erythrocytes. CCRA lectin was specific to N-acetyl-D-galactosamine and D-galactose group carbohydrates. The best lectin activity inhibition possessed alkaline phosphatase of calf intestine and fetuin. CCRA exposed highest affinity to complex oligosaccharide similar to the receptor of Phaseolus vulgaris erythroagglutinin (PHA-E). A comparative study on the histochemical specificity of CCRA and PHA-E using specimens of normal tissues, and that of colon neoplasia, showed similar, yet not identical binding properties. CCRA lectin rather differentially labeled adenoma and adenocarcinoma of colon, which suggests its prospective applicability in diagnostic histopathology.

Lectin purification from fruiting bodies of brown roll-rim fungus, Paxillus involutus (Fr.) Fr., and its application in histochemistry.

A lectin (agglutinin) from fresh fruit bodies of the brown roll-rim fungus [Paxillus involutus (Fr.) Fr.] has been purified with output approx. 60 mg÷kg of raw material. Method of purification included the sedimentation of viscous polysaccharide by ethanol, removal of ethanol by dialysis, ion-exchange chromatography on DEAE-Toyopearl and affinity chromatography on Sepharose 6B column with immobilized mannose-specific Polygonatum multiflorum lectin. The obtained lectin preparation (abbreviated PIFA) is a glycoprotein with 6.5±1% carbohydrates, molecular mass of 64 kDa, consisting of four identical subunits. Lectin interacted only with N-acetyl-lactosamine and glycoproteins that contained Galß1-4GlcNAc disaccharide moieties; agglutinated erythrocytes of dog, sheep and horse, but not of humans. The specificity of PIFA binding to tissue samples of the rat has been investigated. Lectin selectively reacted with gastric parietal cells, submandibular salivary gland duct cells. In the kidney, PIFA labeled epithelial cells of renal tubules, collecting ducts, nuclei of podocytes and mesangiocytes. It was also revealed selective lectin binding to Purkinje cells of cerebellum. Brush border of absorptive cells in small intestine was also strongly reactive, while goblet cells both in small and large intestine were completely negative. Considering similarities in carbohydrate specificity of Paxillus involutus (PIFA) and Ricinus communis agglutinin (RCA-120), histochemical reactivity of these two lectins was compared. It was similar, yet not identical: differences included absence of PIFA binding to the brush border of renal tubules, higher interaction with absorptive cells of the small intestine, lower background staining of cerebellar cortex and renal corpuscles. A conclusion was made that due to the unique carbohydrate specificity PIFA lectin can cover prospective position in experimental histochemistry and diagnostic histopathology comparable to PNA (Peanut agglutinin) and SNA (Sambucus nigra agglutinin).