**9.1 Composition**

156 Antimicrobial Agents

Fig. 4. The β-trefoil folds illustrated by the first domain of the subunit ricin (in complex with lactose), the first domain of amaranthin (in complex with the T-antigen) In each case, for clarity, only a single β-trefoil domain is shown in an identical orientation, although each protein is a multidomain as well as a multimeric protein. Bound carbohydrate is shown in

Lectins comprise a structurally very diverse class of proteins characterized by their ability to bind carbohydrates with considerable specificity. Although lectins bind monosaccharides rather weakly, they employ common strategies for enhancing both the affinity and specificity of their interactions for more complex carbohydrate ligands. Members of the legume lectin family show considerable sequence and structural homology, but differences in their carbohydrate-binding specificity. The legume lectin monomer has a molecular weight of 25,000 and is composed primarily of a six- and a seven-stranded antiparallel-sheet. Concanavalin A (Con A), *Lathyrus ochrus* isolectin I (LOL I), and pea lectin all show mannose and glucose-binding specificity, and the X-ray crystal structures of their carbohydrate complexes show a monosaccharide binding- site geometry very similar to that of the LOL I-

The first lectin structures to be determined derived from two phylogenetically conserved families, the *Leguminoseae* (Sharon and Lis 2001; and Young and Oomen, 1992) and the *Gramineae* (Raikhel et al., 1993). Over the past ten years, major advances in X-ray crystallographic technology and the relative ease of isolation and crystallization of plant lectins have led to a rapid increase in crystal structures. The leguminous lectins clearly have dominated the field with some ten structures known today. Four of these, peanut lectin, soybean lectin, lentil lectin and hemagglutinin L, have been determined within the past

Lectins of the *Amaryllidaceae, Orchidaceae, Alliaceae, Araceae* and *Liliaceae* families are in the class of the mannosespecific *Liliatae* and constitute the third major structurally characterized plant lectin superfamily (Van Damme et al., 1994). They are nonseed lectins of multigene families isolated from plant bulbs (Van Damme et al., 1994) and function either as dimers or tetramers, as do the legume lectins. Their strict and exclusive specificity solely for mannose has imparted some unusual biological properties *in vitro* to this lectin family including their antiviral properties against retroviruses (e.g. HIV) (Hammaar et al., 2006). Snowdrop lectin (GNA) is a tetrameric lectin (Mr =50,000 Da) and is the first member of the *Amaryllidaceae* 

three years. These lectins display diverse sugar-binding specificitiesistructures.

green ball-and-stick (Loris, 2002).

α-methyl-o-mannopyranoside complex.

family crystallographically investigated.

There are no structural features common to all lectins. Many of these proteins are relatively rich in aspartic acid, serine and threonine, which comprise as much as 30% of their amino acid content and are low in sulfur-containing amino acids. Such a pattern of amino acids is characteristic of plant proteins. In contrast, lectins such as those from wheat germ, potato and pokeweed are rich in cysteine with 20, 11.5 and 18% of the total amino acid residues respectively, most or all of which are in the form of cysteine. The high content of disulfide bonds in wheat germ agglutinin endows the protein with stability to heat (Aub et al., 1963), to proteolytic enzymes and to denaturing agents such as detergents, urea, alkali and acids (Nagata and Burger, 1972; and Rice and Etzler, 1974). The potato and *Datura stramonium*  lectins are rich in hydroxyproline (Lamport, 1969). A few lectins, such as Concanavalin A, wheat germ and peanut agglutinins are devoid of covalently bound sugars. Most lectins, however, are glycoproteins with carbohydrate contents that can be as high as 50%, e.g., potato lectin. The table shown below (Table 1) is on the sugar contents of certain important glycoprotein lectins. The sugar constituents in animal glycoproteins are the same as those found in other plant glycoproteins, with the exception of L-arabinose.


Table 1. Well-characterized glycoprotein lectins

The molecular weight of lectins in plants ranges from 36,000 Da for wheat germ agglutinin (Nagata and Burger, 1972; and Rice and Etzler, 1974) to 265,000 for lima bean lectin (Galbraith and Goldstein, 1970). The lower limit of MW of animal lectins is found to be 14 kDa (Lis and Sharon, 1998). Some lectins exhibit a pronounced tendency to aggregate. Thus, the MW of Concanavalin A at pH below 6 is 51,000 Da and at physiological pH it is 12,000 Da (Mc Cubbin and Kay, 1971; and Wang et al., 1971). Upon storage at room temperature, soybean agglutinin and peanut agglutinin also possibly undergo irreversible self-association to high molecular weight aggregates (Lotan et al., 1975). The subunits are identical in most

Antimicrobial Activity of Lectins from Plants 159

ions can be achieved under acidic conditions. The Mn2+ in lectins can be replaced by a variety of transition-metal ions without loss of biological activity as demonstrated for Concanavalin A (Agrawal and Goldstein, 1968; and Shoham et al., 1973). Ca2+ in Concanavalin A could be replaced by Cd2+, but not by Ba2+ (Shoham et al., 1973). The metal ions confer a high degree of structural stability to Concanavalin A, protecting the lectin against heat inactivation and hydrolysis by proteolytic enzymes (Thomasson and Doyle, 1975). Ni2+ alone protects Concanavalin A against proteolysis at pH 7.0 but not at pH 8.2. Some lectins require metal ions for the saccharide-binding activity (Sumner and Howell, 1936). Extensive studies by NMR have revealed a complicated set of interlocking equilibrium involving the apoprotein and various complexes with metal ions and the

Purified lectins are essential for establish their molecular properties and are highly desirable for their many applications. In the past, lectins have been obtained solely from native sources, but they can now be produced also by recombinant techniques. Isolation of a lectin begins commonly with extraction of the tissue or organ in which it is present. This is simple in the case of plants, especially their seeds (Goldstein and Poretz, 1986; and Rudiger, 1993). The seeds are ground and the meal obtained is extracted with a neutral buffer. Often it is advisable to pre-extract the dry meal with an organic solvent, such as petroleum ether, to remove colored materials derived from the seed coat and lipids that may be present in large amounts. Animal tissues are either homogenized directly in the extraction buffer or the tissue is extracted first with acetone to remove water and lipids. The extraction buffer should preferably contain protease inhibitors to prevent degradation of the lectin during purification, and, in the case of membrane bound lectins, a detergent as well. Preliminary fractionation of the crude extract (e.g., by ammonium sulfate precipitation) is often done to obtain a protein fraction devoid of other constituents (e.g., polysaccharides in the case of plants). Final purification is achieved by affinity chromatography on a suitable adsorbent. A wide variety of affinity adsorbents, to suit any taste or purse, have been described in the literature and many of them can be purchased ready-made These include polysaccharides such as Sephadex, a polymer of glucose employed for the purification of Concanavalin A and pea lectin agarose (or Sepharose), a polymer of galactose, for the purification of the lectins from castor bean; acid-treated Sepharose for the purification of SBA; and chitin, a polymer of *N-*acetylglucosamine, for the purification of WGA. In the absence of readily available polysaccharides, use can be made of adsorbents consisting of carbohydrates or glycoproteins as such, or in the form of a synthetic derivative, that are covalently attached to an insoluble carrier. For instance, lactose coupled to Sepharose is the reagent of choice the purification of the lectins from peanut, eel electric organ or calf heartmuscle. *N-*acetylglucosamine bound to the same support serves for the purification of potato lectin and WGA, whereas immobilized porcine AH blood type substance is employed for the purification of the blood type A specific DBL and HPA. When working with lectins of an uncommon specificity, adsorbents have to be tailor made, as for example Sepharose bound asialoglycophorin for the purification of the

The lectin was purified from crude extract of mixer solution, commonly use chromatography technique such as, affinity chromatography, ion exchange

saccharides (Brewer et al., 1983).

**10. Isolate and purification of lectin** 

blood type *N*-specific for lectin from *Vicia graminea*.

lectins. But lectins comprising of non-identical subunits are known as seen in soyabean agglutinin (Lotan et al., 1975) and the lectin from *Dolichos biflorus* (Carter and Etzler, 1975) which are tetramers, consisting of two types of subunits (Wright et al., 1996). A different type of subunit heterogeneity was first demonstrated in Concanavalin A (Abe et al., 1971; and Wang et al., 1971). The anti-B lectin from *Bandeiraea simplicifolia* consists of a family of five closely related proteins, each of which is a tetramer of one or two types of subunits. One of the subunits is specific for *N*-acetyl galactosamine, whereas the specificity of the other is confined to α-galactose (Goldstein and Hayes, 1978). The structure of *Bandeiraea simplicifolia*  isolectins is analogous to that of PHA isolectins. They have five tetrameric proteins comprising of varying proportions of two classes of subunits (Miller et al., 1973; Rasanen et al., 1973; and Leavitt et al., 1977). These subunits show difference in properties. It is assumed that it is due to their difference in the primary structure of subunits (Miller et al., 1973).
