ANTIBODIES TO LECTINS

Lectins are antigenic.While most of them elicit IgG antibodies, certain lectins such as ricin elicit IgE antibodies as well and may cause allergy in human beings.

Like for lectins, several lab and other applications have been found for lectin antibodies as well. Many workers raise antibodies in rabbits, for the lectins of their interest, for histochemical localisation of lectins, or to verify if the same lectin is present in different parts of the same organism.

The seed lectin of Ulex europaeus specifically binds to the human endothelial cells of normal and neoplastic blood and lymphatic vessels.A fluorescent labelled antibody applied to the lectin bound to vasculature, helps to distinguish vessels from the very frequent artificial cracks in the tissue.

Some lectin antibodies, raised in rabbits, are commercially available, Sigma being the major supplier. The cost of 2 ml of lyophilised antiserum is about US$ 35 to 75, depending upon the antigenic lectin.

The following are some examples of lectins for which antibodies are commercially available (the botanical source of the antigen is given along with the common name of the lectin in parenthesis):

CONJUGATED LECTINS

Conjugation is the molecular association of two different classes of compounds.Lectins are conjugated with a variety of substances to serve as tools in different lab techniques.

Conjugated lectins do not suffer from any loss of activity.Conjugation is quantified in terms of moles of the dye per mole of lectin, b) units of enzyme activity per mg of lectin, or c) mg of lectin per ml of packed gel.

MITOGENIC LECTINS

Human erythrocytes are enucleate.The differentiated leucocytes that occur in the circulatory system are nucleate, but do not divide. Some lectins trigger these cells into mitotic division. Such are mitogenic lectins. PHA, the lectin from the common red kidney bean (Phaseolus vulgaris), the first source of a mitogenic lectin, has revolutionised the study of human karyotypes.

All mitogenic lectins stimulate the T cells, except that of Phytolacca americana which stimulates both T and B cells.

The commonly known species containing mitogenic lectins and the sugar specificity of the lectins is given below. The specific fraction identified as mitogenic is indicated in parenthesis; the other fractions from the same source are not mitogenic.

Data from Hunter, 1986; Sathyananda, 1989; Anonymous, 1999

Mitogenic stimulation or mitogenecity, is the ability of a factor to induce (mitotic) division in a mature quiescent cell that does not normally divide. Prolonged mitogenic activity results in cell proliferation as in cancer.

A number of substances such as antigens, antibodies to lymphocyte surface components, bacterial toxins, calcium ionophores, oxidising agents (periodate), enzymes (galactose oxidase), etc.That some lectins also are mitogenic, is of great interest.

Since Nowell (1960) demonstrated in vitro, the dramatic induction of mitogenic activity in lymphocytes, by PHA, the lectin of Phaseolus vulgaris, several mitogenic lectins have ben discovered (Table ). Like the other activities of lectins, mitogenic stimulation also can be inhibited by a sugar specific for the lectin. All lectins do not seem to be mitogenic and some are not mitogenic in lymphocytes. Identification of appropriate cell systems will lead to the discovery of many more mitogenic lectins. PHA and con A are the best known and most widely used mitogens.

Most lectins are mitogenic only in the T (thymus dependent) lymphocytes. The pokeweed mitogen (Pa-1), however, stimulates both T cells and B cells.

Mitogenic stimulation is measured on the basis of incorporation of labelled thymidine into DNA, to obtain a measure of increase in whole cell population. The actual number of cells stimulated into division is obtained by counting the number of blast cells or by radioautography of cells pulsed with radioactive thymidine.

The use of mitogenic lectins has greatly facilitated the study of human karyotypes. Chromosomes stained using various banding techniques are used to study strucural differences betwen healthy and diseased conditions, particularly such situations as Klinefelter's, Turner's, Down's, Patau's and Edward's syndromes, and made disease diagnosis easy and more certain.

Mitogenic lectins have also been used as tools for studying the biochemical events accompanying cellular growth, differentiation and division (Ling, 1968; Lis and Sharon, 1977).

While there is great interest in the mitogenic lectins, the underlying mechanisms have been poorly understood. It is not clear yet why some lectins are mitogenic whiler others with similar properties are not. For example, the lectins of Datura stramonium, Lycopersicon esculentum (tomato) and Solanum tuberosum (potato), all of the family Solanaceae, have similar sugar specificity (oligomers of glcNAc). However, the datura lectin is mitogenic, the potato lectin is not, while the tomato lectin is actually anti-mitogenic and suppressed transformation of chicken and human lymphocytes by PHA and various stimulii (Kilpatrick et al., 1985; McCurrah and Kilpatrick, 1988).

Lectins do not seem to fall in two categories, mitogenic and non-mitogenic. Some are anti-mitogenic and some are incomplete-mitogens, providing only one of the two trans-membrane signals for proliferation. Sugar specificity is also not an indicator of mitogenicity.

Under special circumstances, a mitogenic lectin may inhibit human lymphocyte stimulation, as in the case of tuberculin-induced prolifiration inhibited by datura and wheat lectins. On the contrary, weak mitogenic activity of a lectin may be enhanced by compounds like phorbol myristate.

Mitogenic stimulation of T lymphocytes is often dependent upon other cells, such as erythrocytes and mononuclear cells. Erythrocytes stimulated the mitogenic action of the lectin of Phytolacca americana (Uiterdijk et al., 1988). Mononuclear cells enhanced the action of WGA. On the other hand the presence of monocytes enhanced the activity of Datura lectin, but the proliferation of T-cells was greatly suppressed but that of the monocytes was increased and that of T cells suppressed with WGA (McCurrah and Kilpatrick, 1988).

The concentration of the lectin appears to be one of the factors that determines proliferation (low concentration) or inhibition (high concentration), as with the lectins of Phaseolus vulgaris and Phytolacca americana ((Wiktorowicz et al., 1988). The factors that effect a switch over in this regard are the 'acute phase proteins' that accumulate in acute and chronic inflammation, trauma and cancer. Consequently, in vivo behaviour of mitogenicity can be different from in vitro responses.

Mitogenic stimulation by lectins is useful in assessing immunocompetence of patients sufferring from many diseases. Lectins can selectively stimulate T or B cells or both. Con A stimulates T cells, while pokeweed lectin stimulates only B cells.

Mitogenic lectins also induce cellular proliferation, such as in the small intestine, the result being similar to peripheral lymphocytes (Pusztai et al., 1988).

Another application of mitogenic lectins is in the preparation of chromosome maps for karyotyping, sex determination and detection of chromosomal defects (Sharon and Lis, 1989).

Lymphocyte stimulation by lectins also increases immunoglobulin production, as well as lymphokines, lymphotoxin and interferon in certain cases.

An explanation of the mechanism of lectin stimulation of lymphocytes, which seems to be a complex process, has been unsuccessfully sought after. However, two steps have been identified. The 'first signal' constitutes the binding of the lectin to lymphocyte surface sugars, and this triggers the 'second signal' transmitted into the cell, which initiates a series of biochemical eventsis leading to cell growth and proliferation. The first signal can be inhibited by the sugar specific to the mitogen.

Certain lectins require pre-treatment with neuraminidase, for becoming effective, as in the case of soybean lectin on mouse lymphocytes and peanut lectin on rat and human lymphocytes.

The divalent peanut lectin is not mitogenic on mouse lymphocytes while the monovalent form is. Contrasting valency based differences were also reported as in the case of lima bean whose tetravalent form is a much stronger mitogen than its divalent form. Mitogenic activity is not altered by valency shift from tetravalent to divalent form, but was for the monovalent form.

NON-MITOGENIC LECTINS

Some lectins or their fractions have been demonstrated to be non-mitogenic, some of which are listed below, indicating the sugar specificity of the non-mitogenic fraction:

S-1 fraction of Aloe arborescens (Liliaceae), Cicer arietinum (fetuin, Fabaceae), Euphorbia characias (Euphorbiaceae), Lycopersicon esculentum ((glcNAc)3, Solanaceae), Ononis hircina (galactose, Fabaceae), Persea americana ((Lauraceae), Solanum tuberosum 3 ((glcNAc), Solanaceae), Sophora japonica (galactose, Fabaceae), some fractions of Triticum aestivum (Poaceae), and Vicia graminea (galNAc, Fabaceae), were shown to be non-mitogenic.

STIMULATION OF PLANT CELLS

Studies on the lectin induced stimulation of plant cells are largely hindered by the cellulosic cell walls which act as a barrier to lectin mobility into the cells. In the case of tobacco pith segments and soybean roots, there was no stimulation and in the case of soybean callus cells, it was negligible. In the case of a few reports of increased mitotic activity, the presence of plant growth substances as contaminants in the lectin preparations was suspected. Nevertheless, the role of lectins in stimulating pollen germination, which is often followed by mitosis, and pollen tube growth has been well established (Kameswara Rao and Sangeeta, 1996).

LECTINS AS ENZYMES

Although some definitions of lectins describe them as non-enzymic proteins, instances of proteins with both agglutinating and enzymic activity are known (Etzler, 1985). a-galactosidase activity was demonstrated with seed lectins of mung bean, soybean and the broad bean.

Except for the mung bean lectin, no other purified lectin has shown enzymic activity. It is possible that the true lectins which agglutinate and non-enzymic are different from enzymes which agglutinate due to a slow rate of hydrolysis (Etzler, 1985). In view of some catalytic activity of toxic lectin fraction of ricin, abrin and modecin, enzyme activity other than glycosidasic (or galactosidasic), was not ruled out. But that the toxic fractions of these lectins do not agglutinate, should not be forgotten.

Soybean lectin mitogenic for neuraminidase treated lymphocytes.
ConA mitogenic as also lentil, PHA,
PHA L- leucoagglutinating, PHA R haemagglutinating
Protein A Staph.aureus binds to the Fc fragment of IgG molecules
Ricin A chain (32,000) binds to 60S ribosomal subunit.
WGA does not agglutinate normal cells unless treated with proteases; transformed cells are agglutinated.