About the Lectin
The isolectins present in the red kidney bean exhibit complex carbohydrate specificity and binding characteristics. Two crude extracts of the bean are potent blood agglutinins and possess strong mitogenic activity 1 . Various biochemical techniques have shown that different isolectins are responsible for blood agglutination and leucoagglutination 2 . Based on this finding, two of the isolectins are now referred to as PHA-E (erythroagglutinating) and PHA-L (leucoagglutinating). Initially it was assumed that PHA-L was responsible for the mitogenic activity of crude preparations, but it has since been shown that PHA-E is also a potent mitogen 3 . The reactivity of PHA-E with serum glycoproteins may explain why the isolectin originally did not exhibit mitogenic activity. Red kidney beans actually contain five isolectins. These isolectins are each tetramers composed of varying amounts of two different subunits. Purified PHA-L contains four L subunits, while PHA-E contains four E subunits. The three additional isolectins are intermediate forms composed of both L and E subunits. Ion-exchange chromatography has been used successfully to separate these five isolectinsM4. Full nucleotide sequences and deduced amino acid sequences have been determined 5 . PHA-L has been crystallized and the crystal structure resolved to 2.8 Å 6 .
PHA-P and PHA-M are both related crude preparations containing all five of the isolectins. The primary difference between these two preparations is the presence of an associated polysaccharide on PHA-M. At a pH of 1.0 the polysaccharide is cleaved from PHA-M resulting in a protein identical to PHA-P. The associated polysaccharide gives the two crude lectins different physical characteristics. PHA-M is soluble in distilled water while PHA-P is not. Dilute buffers containing 10-15mM NaCl will solubilize PHA-P. PHA-M is a weaker blood agglutinin than PHA-P 7 , requiring 10-100 times more material to exhibit the same activity.
Several methods have been developed for separating PHA-L and PHA-E from the other isolectins. These include ion-exchange chromatography, selective desorption from an affinity matrix, and electrophoresis. Each method will yield the same final products, although contamination by other isolectins will vary with the procedure used. Purified PHA-E will bind to both human erythrocytes and lymphocytes. In fact, there are 5 times more PHA-E receptors on normal human lymphocytes than there are on erythrocytes. An asparagine linked erythrocyte glycopeptide, released by trypsin and subsequently treated with borohydride and Pronase, is an inhibitor of both PHA-E induced agglutination and mitogenicity. This branched glycopeptide can be treated with neuraminidase, increasing its reactivity with PHA-E. The glycopeptide becomes inactive if it is then treated with β-galactosidase 8 . In spite of this result, PHA-E is not inhibited by β-galactose, disaccharides containing β-galactose, or any other simple sugars. Glycoproteins containing oligosaccharide chains similar to the erythrocyte glycopeptide are potent inhibitors, for example, fetuin and human IgG. The complex specificity of PHA-E indicates that the core sugar residues are important for binding. A bisecting GlcNAc unit linked to a core mannose residue of a biantennary glycoprotein is important for lectin binding 9 . PHA-E also has a separate binding site for adenine and several derivatives of adenine 10 .
Purified PHA-L exhibits almost no agglutination with human erythrocytes. Its carbohydrate binding specificity is complex, similar to that of PHA-E. There is some overlap in specificity between PHA-L and PHA-E, but both exhibit unique reactivity with several different glycoproteins. PHA-L reacts strongly with Tamm-Horsfall glycoprotein, while PHA-E will not react at all. PHA-E binds to porcine thyroglobulin, while PHA-L reacts only weakly. The binding properties of these two isolectins have been determined by their specific precipitation reactions with several glycoproteins. PHA-E forms a precipitate with a number of serum glycoproteins including fetuin. PHA-L does not precipitate fetuin, however it does bind to this glycoprotein. PHA-L is strongly inhibited by the trisaccharide Gal β(1,4)GlcNAcβ(1,2)Man, which is also a strong inhibitor of PHA-E induced agglutination. This structure is present on a number of glycoproteins, including fetuin and transferrin, however, PHA-L does not react with all of these glycoproteins. A terminal sialic acid residue is not normally considered important for binding but the presence of an α(2,6) linkage, as found in transferrin, prevents lectin binding, while an α(2,3) linkage, as found in fetuin, has no effect on lectin binding 11 . PHA-E has been used in the specific precipitation of serum glycoproteins, and PHA-L has been used successfully in anterograde transport studies 12,13 . PHA-E bound to the epidermal growth factor receptor of a human gliomal cell line so as to block the effect of EGF, whereas PHA-L binding was without effect 14 . PHA-L reacts strongly with certain carcinoma cell lines of high metastatic potential 15,16 . PHA-L has also been proposed as a potential therapeutic agent. The lectin has been used in preliminary studies indicating that it is a good biological response modifier 17 . This lectin shows the potential to inhibit graft vs. host reaction in transplantation studies. It may also promote the production of cytotoxic agents which could be useful in cancer therapy. Although PHA-L has been used alone in preliminary studies, indications are that it may even be more useful in conjunction with other therapies.
- Nowell, P. C. (1960) Cancer Res. 20: 462-466.
- Weber, T., et al. (1967) Scand. J. Haematology 4: 77.
- Kornfeld, R. D. and Kornfeld, S. (1970) J. Biol. Chem. 245: 2536.
- Leavitt, R. D., et al (1977) J. Biol. Chem. 252: 2961.
- Hoffman, L.M. and Donaldson, D.D. (1985) EMBO J. 4 : 883-889.
- Hamelryck, T.W., et al. (1996) J. Biol. Chem. 271 : 20479-20845.
- Rigas, D. A. and Osgood, E. E. (1955) J. Biol. Chem. 212 : 607-615.
- Kornfeld, R., et al. (1972) Meth. Enzymol. 28 (part B) : 344-349.
- Cummings, R. D. and Kornfeld, S. (1982) J. Biol. Chem. 257: 11230-11234.
- Maliarik, M. J. and Goldstein, I. J. (1988) J. Biol. Chem. 263: 11274-11279.
- Hammarstrom, S., et al (1982) PNAS, (USA). 79: 1611-1615.
- Glad, C. and Borrebaeck, C. A. K. (1984) J. Immunol. 133: 2126-2132.
- Gerfen, C. R. and Sawchenko, P. E. (1984) Brain Res. 290: 219-238.
- Rebbaa, A., et al. (1996) J. Neurochem. 67 : 2265-2272.
- Tanda, N., et al. (1996) Pathol. Int. 46 : 639-645.
- Schwarz, R.E., et al. (1996) Cancer Lett. 107 : 285-291.
|Buffer||0.01M Phosphate – 0.15M NaCl, pH 7.2-7.4.|
|Blood Group||For PHA-E O > A,B. PHA‑L does not react strongly with any blood type.|
|Inhibitory Carbohydrate||Not inhibited by simple sugars.|
|Molecular Weight||Aggregate MW=120,000. A single band of MW=26-30,000 by SDS-PAGE. This is the same result for both PHA-L and PHA-E.|