About the Lectin
A human blood group A specific lectin is present in crude extracts of Vicia villosa seeds. The lectin has been purified by affinity chromatography on immobilized type A substance 1 , on immobilized αgalactose 2 , and on immobilized porcine blood group substance 3 . Lectin yields and characteristics can vary depending on the method used for isolation. As reported by Tollefsen and Kornfeld, VVA is a tetramer composed of 2 different subunits. Three related isolectins, composed of different amounts of the two subunits, have been purified. The A4 isolectin (composed of four A subunits) is responsible for the anti-A1 blood group activity. The B4 isolectin, which is composed of four B subunits, has been crystallized in the presence of carbohydrate and preliminary diffraction data obtained to 2.8Å 4 . By circular dichroism, its conformation has been shown to be primarily β-sheets, and it is exceptionally resistant to denaturation by chaotropic reagents such as 6M guanidine hydro-chloride or 7.5mM surfactant 5 . Using the fluorescent probe N-dansyl galactosamine, GalNAc-α1-OSer and GalNAc-αOMe were the best ligands, although a β-GalNAc structure is preferred in oligosaccharides 6 . The B4 isolectin does not agglutinate normal human erythrocytes at a lectin concentration below 1mg/ml, but it does agglutinate Tn erythrocytes. VVA B4 also agglutinates erythrocytes expressing the Cad determinant 7 . Carbohydrate specificity alone does not explain this affinity. The Tn antigen is a terminal O-linked αGalNAc residue, while the Cad determinant is a sialylated, O-linked polysaccharide with no terminal αGalNAc. The B4 isolectin binding site on normal type O erythrocytes can be exposed by treatment of the cells with papain 8 . VVA B4 reacts more strongly with desialylated blood group N than with M antigen 9 . The purification technique used by EY Laboratories yields a lectin that is weakly reactive with A1 erythrocytes, but which reacts strongly with neuraminidase treated cells. These data would indicate that the preparation contains little of the A4 isolectin. Purified VVA has been used extensively to separate various T cell subsets. VVA can separate human T8 + cells into two functionally different populations. T8 + cells not bound by VVA act as suppressors of immune response; T8 + cells bound by VVA act as contrasuppressors of immune response 10,11 . Contrasuppressor cells have been implicated in autoimmune diseases, including murine αTBM disease 12 and human systemic lupus erythematosus 13 . In addition, Peyer’s patch L3T4 T cells, isolated from murine intestinal mucosa, can be separated into two populations, one that secretes IL-2 and one that secretes IL-5 14 . VVA has been used to directly stain cells on human tonsil sections 15 . The lectin also specifically stains certain neuronal cells 16-18 , and after their desialylation, distinguishes α-dystroglycans of skeleton muscle from those of brain and cardiac muscle 19 . In addition to the GalNAc specific isolectins, EY Laboratories has been able to isolate a mannose specific lectin from the same seeds. When analyzed by SDS-PAGE, only low molecular weight bands of 15,000 and 18,000 Da appear. This is much different from the staining pattern obtained with the GalNAc specific preparation. This lectin has been further characterized more recently 5,20 .
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|Buffer||0.01M Phosphate – 0.15M NaCl, pH 7.2-7.4.|
|Blood Group||A1 (A4 isolectin); Tn+Cad (B4 isolectin).|
|Activity||More than 250 μg/ml is usually required to agglutinate type A1 cells. Some batches react stronger than others, requiring less than 5 μg/ml for agglutination of type A1 cells. Less than 1 μg/ml will agglutinate neuraminidase treated cells regardless of blood type.|
|Inhibitory Carbohydrate||N-acetyl‑D‑galactosamine. (Mannose for L-4603)|
|Molecular Weight||Two bands by SDS-PAGE. One band of MW=25-27,000 and another band of MW=110,120,000.|