Since the 1880’s, it has been known that extracts from certain plants could agglutinate red blood cells. In the 1940’s, agglutinins were discovered which could “select” types of cells based on their blood group activities. Although “lectin” was originally coined to define agglutinins which could discriminate among types of red blood cells, today the term is used more generally and includes sugar-binding proteins from many sources regardless of their ability to agglutinate cells. Lectins have been found in plants, viruses, microorganisms and animals, but despite their ubiquity, their function in nature is unclear. Although lectins share the common property of binding to defined sugar structures, their roles in various organisms are not likely to be the same.
Most lectins studied to date are multimeric, consisting of non-covalently associated subunits. A lectin may contain two or more of the same subunit, such as Con A, or different subunits, such as Phaseolus vulgaris agglutinin. It is this multimeric structure which gives lectins their ability to agglutinate cells or form precipitates with glycoconjugates in a manner similar to antigen-antibody interactions. Although most lectins can agglutinate some cell type, cellular agglutination is not a prerequisite. Some lectins can bind to cells and not cause agglutination, such as succinylated Con A, or the lectin may not bind to cells at all. The latter property may be a consequence of the structure of the lectin or the absence of a suitable receptor oligosaccharide on the cell surface. Since agglutination of cells is the assay most generally employed to measure lectins, many non-agglutinating lectins may exist in nature but have not been easily detected.
In spite of our ignorance about the function of lectins in nature, this unique group of proteins has provided researchers with powerful tools to explore a myriad of biological structures and processes. Because of the specificity that each lectin has toward a particular carbohydrate structure, even oligosaccharides with identical sugar compositions can be distinguished or separated. Some lectins will bind only to structures with mannose or glucose residues, while others may recognize only galactose residues. Some lectins require that the particular sugar be in a terminal non-reducing position in the oligosaccharide, while others can bind to sugars within the oligosaccharide chain. Some lectins do not discriminate between a and b anomers, while others require not only the correct anomeric structure but a specific sequence of sugars for binding. The affinity between a lectin and its receptor may vary a great deal due to small changes in the carbohydrate structure of the receptor. All of these properties which are peculiar to lectins enable the researcher to discriminate between structures, to isolate one glycoconjugate, cell or virus from a mixture or to study one process among several. Since virtually all biological membranes and cell walls contain glycoconjugates, all living organisms can be studied with lectins.
Another property of some lectins is an ability to induce mitosis in cells which are normally not dividing. This property has been exploited extensively in an attempt to understand the process of lymphocyte blastogenesis and the biochemical and structural alterations associated with mitogenesis. It is not clear why some lectins are mitogenic since the structures to which mitogenic lectins bind are not necessarily the same, and not all lectins with similar binding specificities are mitogenic. It is likely that binding to the cell surface alone is not sufficient to cause mitosis but that other interactions on the cell surface are equally important.
Additional Information:
Purification of Lectins
Lectins have been purified by “conventional” procedures including salt-induced crystallization, ethanol precipitation, ion exchange chromatography and gel filtration, or by affinity chromatography. The former methods rely on the physicochemical properties of the proteins for separation while affinity chromatography depends on the specific interaction between the lectin and a carbohydrate structure attached to an inert matrix. Although affinity chroma-tography is a highly specific procedure, some carbohydrate matrices used for affinity chromatography can adsorb not only the lectin but also glycosidases capable of hydrolyzing the sugar structures to which the lectin may bind. Contamination by glycosidases could greatly affect the activity of a lectin preparation. To isolate lectins of the highest purity and to avoid possible contamination by glycosidases, we employ combinations of both “conventional” procedures and affinity chromatography for all of our lectins.
Lectins have been purified by “conventional” procedures including salt-induced crystallization, ethanol precipitation, ion exchange chromatography and gel filtration, or by affinity chromatography. The former methods rely on the physicochemical properties of the proteins for separation while affinity chromatography depends on the specific interaction between the lectin and a carbohydrate structure attached to an inert matrix. Although affinity chroma-tography is a highly specific procedure, some carbohydrate matrices used for affinity chromatography can adsorb not only the lectin but also glycosidases capable of hydrolyzing the sugar structures to which the lectin may bind. Contamination by glycosidases could greatly affect the activity of a lectin preparation. To isolate lectins of the highest purity and to avoid possible contamination by glycosidases, we employ combinations of both “conventional” procedures and affinity chromatography for all of our lectins.
Each step of the purification is monitored for lectin activity by measuring agglutination of specifically prepared red blood cells. Agglutination of cells is a complex process likely to require binding of the lectin to the cell surface, lateral movement and clustering of lectin receptors, changes in the cytoskeletal network, intercellular associations, biochemical alterations, redistribution of cell surface charges and other changes which ultimately result in multicellular aggregates. Because cellular agglutination is such a complex process, it is difficult to measure quantitatively. For example, the degrees of purity or lectin activities are often assessed by comparing the titers of lectins versus a particular red blood cell. The results of these comparisons may be misleading. Unless performed side by side, under identical conditions, assays measuring agglutination titers may not be valid. The age of the cells, whether they have been trypsinized, fixed or neuraminidase-treated, the animal species or individual from which the cells were taken, the buffer used, metal ions or other solutes present during agglutination, the type of assay performed (test tube, microtiter well, etc.), the temperature, the length of time used and the interpretation regarding “end point of the titer,” all have a significant effect on the titers obtained. Additional criteria should also be used to assess purity including the patterns on high resolution polyacrylamide electrophoretic gels, immuno-precipitation with antisera and the ability of the lectin to bind to a specific affinity column.
This last test is of prime importance since it determines whether the carbohydrate binding characteristics of the final product have survived the procedures utilized in the purification of the lectin.
Because any one of these criteria is not sufficient to adequately assess the purity and activity of lectins, we use all four procedures on our highly purified lectins. This rigorous quality control provides the assurance that our customers have the best lectins available.
Description of Agarose Bound Lectins
The relatively new approach of lectin affinity chromatography has become a standard and widely used technique for the isolation of soluble glycoproteins, hormones, antigens and polysaccharides, as well as detergent-solubilized membrane-bound glycoconjugates and cell surface receptors. An excellent review article on this topic has been published by Lotan and Nicolson (Biochim. Biophys. Acta, 559, 329-376, 1979).
Lectin affinity chromatography combines simplicity with potentially high resolution. During the purification of glycoconjugates, the immobilized lectin is allowed to bind to the glycoconjugate, and the unbound residual material can be readily removed by subsequent washing. The bound glycoconjugates are displaced from the immobilized lectins by the addition of a solution of a sugar known to inhibit binding of the particular lectin.
A continually increasing number of glycoproteins have been purified by using immobilized lectins. Among these are hormones, blood group substances, viral glycoproteins, histocompatibility antigens, interferons, enzymes, lymphocyte markers, serum proteins, and oncofetal antigens.
Our immobilized lectins are prepared from affinity-purified lectins. Heat stable, cross-linked 4% agarose beads with a molecular weight exclusion limit of about 2x107 are used as the solid-phase matrix to which the lectins are covalently bound. The attachment of the lectins to the solid phase is carefully controlled in order to preserve the activity of the lectins as well as to minimize conformational changes of the bound lectins which might result in nonspecific ionic or hydrophobic interactions.
The technique we have developed to couple lectins to agarose provides a very hydrophilic spacer arm between the protein and the matrix. This ensures maximum expression of the carbohydrate binding activity of the lectin. The linkage is very stable over a range of pH values and, unlike cyanogen bromide linkages, proteins are not leached off the gel by Tris or other routinely used buffers. In addition, residual charges generated during cyanogen bromide conjugation which can produce nonspecific binding are not present on the gel following our coupling procedure.
Our agarose bound lectins are supplied at a constant concentration of lectin per ml of gel. The concentration at which each lectin is prepared is selected in order to achieve the highest glycoconjugate binding capacity per mg of lectin present in the gel.
Before each lot is made available, its binding capacity is tested using glycoproteins or derivatives of glycoproteins known to bind to the particular lectin being evaluated. This provides a guideline for the user and assures the quality of our agarose bound lectins.
Description of Fluorescent Lectins
The number and distribution of charged groups on protein surfaces, the hydrophilic or hydrophobic nature of protein domains, the size of the protein or its carbohydrate content, the method employed to label the protein, as well as many other factors have an effect on the final product of fluorescent conjugation. Some lectins can tolerate a large number of attached fluorochromes and still remain fully active, soluble, and retain low nonspecific binding properties, while others cannot. By using the highest quality fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate, our affinity-purified lectins and special conjugation procedures, we are able to produce reagents with maximum fluorescence. Each of our fluorochrome-labeled lectins has an appropriate number of fluorochromes bound which provide the optimum staining characteristics for that particular lectin. These conjugates are supplied essentially free of unconjugated fluorochromes and inactive lectin.
Accompanying each fluorescent lectin is an analysis data sheet summarizing the results of our quality control tests and providing pertinent information on the product. All of these reagents are supplied as solutions perserved with sodium azide.
Description of Peroxidase Conjugated Lectins
Peroxidase conjugated lectins generally tend to produce high nonspecific background staining when used with cells or tissues and are not recommended for routine histochemical use. However, we offer two peroxidase conjugates for other specific applications. These reagents are prepared from highly purified succinylated Con A or wheat germ agglutinin and conjugated with an ultrapure grade of horseradish peroxidase. These products can be used to follow transport pathways when the use of antibodies to the lectin and VECTASTAIN® ABC reagents is precluded.
Specificity Guide for Lectins
* This table shows the primary sugar specificity of each lectin. Most lectins have additional structural requirements for binding, so this table should only be used as a quick reference guide.
† Lectins in each box are listed in alphabetical order. Please refer to text and cited references for other properties and recommended inhibiting/eluting sugars.
General Lectin References
Literally thousands of articles on lectins have been published during the past forty years examining hundreds of different aspects and uses of lectins. In this catalog, only a few of the uses and properties of each lectin are described. However, both general and specific information can be found in the excellent review articles listed below:
(1) W.C. Boyd, “Introduction to Immunochemical Specificity,” Wiley-Interscience, New York, 1962, pp. 1-158.
(2) G.C. Toms and A. Western, in “Chemotaxonomy of the Leguminosae,” J.B. Harborne, D. Boulter, and B.L. Turnes, eds., Academic Press, London and New York, 1971, pp. 367-482.
(3) N. Sharon and H. Lis, Science, 177, 949-959 (1972).
(4) H. Lis and N. Sharon, Ann. Rev. Biochem., 42, 541-574 (1973).
(5) G.L. Nicolson, Int. Rev. Cytol., 39, 89-190 (1974).
(6) D.F.H. Wallach, “Membrane Molecular Biology of Neoplastic Cells,” Elsevier Scientific Publ. Co., Amsterdam, Oxford and New York, 1975, pp. 435-481.
(7) I.E. Liener, Ann. Rev. Plant Physiol., 27, 291-319 (1976).
(8) I.J. Goldstein and C.E. Hayes, Adv. Carb. Chem. Biochem., 35, 127-340 (1978).
(9) G.L. Nicolson, “Cancer Markers-Diagnostic & Developmental Significance,” Stewart Sell, ed., Humana Press, Clifton, N.J., 1980, pp. 403-443.
(10) N. Sharon, “Advances in Immunology,” F.J. Dixon and H.G. Kunkel, eds., Academic Press, London and New York, 1983, pp. 213-298.
(11) “The Lectins. Properties, Functions, and Applications in Biology and Medicine,” I.E. Liener, N. Sharon, and I.J. Goldstein, eds., Academic Press, Inc., Orlando, 1986.
Lectin Perfusion References (Tomato Lectin)
Debbage, P.L., et al., Lectin intravital perfusion studies in tumor-bearing mice: Micrometer-resolution, wide-area mapping of microvascular labeling, distinguishing efficiently and inefficiently perfused microregions in the tumor. J. Histochem Cytochem, 1998. 46(5): p. 627-639. (General lectin perfusion reference).
Thurston, G., et al., Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice. J. Clin Invest, 1998. 101 (7): p. 1401-1413.
Hashizume, H., et al., Openings between defective endothelial cells explain tumor vessel leakiness. Amer J Pathol, 2000. 156(4): p. 1363-1380.
Debbage, P.L., et al., Intravital lectin perfusion analysis of vascular permeability in human micro- and macro- blood vessels. Histochemistry Cell Biol, 2001. 116(4): p. 349-359.
Lee, J.C., et al., Interleukin-12 inhibits angiogenesis and growth of transplanted but not in situ mouse mammary tumor virus-induced mammary carcinomas. Cancer Res, 2002. 62(3): p. 747-755.
Akerman, M.E., et al., Nanocrystal targeting in vivo. Proc Nat Acad Sci Usa, 2002. 99(20): p. 12617-12621.
Gee, M.S., et al., Tumor vessel development and maturation impose limits on the effectiveness of anti-vascular therapy. Amer J Pathol, 2003. 162(1): p. 183-193.
Jilani, S.M., et al., Selective binding of lectins to embryonic chicken vasculature. J Histochem Cytochem, 2003. 51(5): p. 597-604.
Krasnici, S., et al., Effect of the surface charge of liposomes on their uptake by angiogenic tumor vessels. Int J Cancer, 2003. 105(4): p. 561-567.
Huang, J.Z. et al., Regression of established tumors and metastases by potent vascular endothelial growth factor blockade. Proc Nat Acad Sci Usa, 2003. 100(13): p. 7785-7790.
Inai, T., et al., Inhibition of vascular endothelial growth factor (VEGF) signaling in cancer causes loss of endothelial fenestrations, regression of tumor vessels, and appearance of basement membrane ghosts. Amer J Pathol, 2004. 165(1): p. 35-52.
