key: cord-0034613-bexp1ubn
authors: nan
title: Detection, Occurence And Isolation
date: 2007
journal: Lectins
DOI: 10.1007/978-1-4020-6953-6_3
sha: 2e23077bf9317636061999c82a5e1c199e9b454b
doc_id: 34613
cord_uid: bexp1ubn

Lectins are ubiquitous in nature, and are found in all classes of organism. They are easy to detect and often to isolate. In addition, many are available from commercial suppliers. They are now obtainable also by recombinant techniques. The classic, and still simplest, way to detect the presence of a lectin in a biological material is to prepare an extract from the material and examine its ability to agglutinate erythrocytes (Fig. 3.1) (Rüdiger, 1993). A more refined screening procedure is based on the ability of these proteins to precipitate polysaccharides (Goldstein, 1976) (Fig. 3.2) or glycoproteins. If a positive result is obtained, it is essential to show that the agglutination or precipitation is specifically inhibited by mono- or oligosaccharides, i.e., it is sugar specific (Fig. 3.1). Hemagglutination is commonly assayed by the serial dilution technique using erythrocytes from humans or rabbits. Occasionally erythrocytes that have been treated with trypsin or sialidase are employed, since such cells are often more sensitive to agglutination than the untreated cells (Fig. 3.3). Hemagglutination also serves to monitor and quantify the activity of lectins in the course of purification. Because of the wide use of the agglutination reaction, it deserves some comments (Lis & Sharon, 1986). For agglutination to occur, the lectin must bind to the cells and form cross-bridges between them. There is however no simple relation between the amount of lectin bound and agglutination. Cases are even known where considerable amounts of lectin are bound to cells, without causing agglutination. This is because agglutination is affected by many factors, among them accessibility of receptor sites, membrane fluidity and metabolic state of the cells. It is also influenced by external conditions of the assay, such as temperature, cell concentration, mixing and so on. The relative contribution of the different factors depends on both the lectin and the cells examined. When agglutination does occur and it is inhibited by monoor oligosaccharides, it serves as an indication that carbohydrate structures for which the lectin is specific are present on the surface of the cell. Additional information on the nature of the receptors may be obtained with erythrocytes pretreated with enzymes, in particular glycosidases, or with sugar-modifying reagents, such as periodate. Agglutination with lectins is also of use in following changes on cell surfaces during physiological and pathological processes.

which the lectin is specific are present on the surface of the cell. Additional information on the nature of the receptors may be obtained with erythrocytes pretreated with enzymes, in particular glycosidases, or with sugar-modifying reagents, such as periodate. Agglutination with lectins is also of use in following changes on cell surfaces during physiological and pathological processes.

Currently, a number of other methods for lectin detection is available. Thus, microarrays of different carbohydrates coupled to wells of a microtiter plate have been developed, with a range and complexity such as found in naturally occurring glycans (see fig. 4 .1) (Bryan et al., 2002; Gargir, 2001) . Such glycochips should greatly facilitate the screening for lectins in biological materials, as well as the definition of their specificity. By a completely different method, lectins can be detected in situ, in tissue sections and on cells, by staining with suitably derivatized glycoproteins or Fig. 3 .1 Hemagglutination and its inhibition as a means to demonstrate the presence of lectins in seed extracts. Ground seeds (or wheat germ) were extracted with ten times their weight of phosphate buffered saline, pH 7.4. In the case of soybeans, the oil was removed prior to extraction. Each well of the microtiter plate contained 50 µl of seed extract, 50 µl of a 4% suspension of rabbit erythrocytes and 50 µl of 0.2 M sugar solution in phosphate-buffered saline, pH 7.4. The agglutinated erythrocytes form a carpet that covers the whole well; where no agglutination occurs, the cells form a button at the bottom of the well. Picture taken after 2 hours at room temperature.

between an anti-Type SIII polysaccharide antibody and the polysaccharide. Courtesy Dr.Irwin J. Goldstein, University of Michigan, Ann Arbor. neoglycoproteins (Gabius, H. J. et al., 1994) . A third method is based on sequence similarities of newly discovered proteins to known lectins, by homology searches in data bases at the protein or cDNA level. It has had a pronounced impact in the field of animal lectins, where it led to the identification of many new proteins of this class. It also resulted in the discovery of large numbers of diverse lectin-like proteins, not all of which however possess carbohydrate-binding activity (Drickamer & Dodd, 1999) .

Lectins occur in all classes and families of organisms examined, although not necessarily in every genus or species {For an extensive survey of the early literature on the subject, see (Gold, 1975) . Their tissue and cellular distribution is variable, and it may be affected by miscellaneous factors, such as developmental stage, age and pathological conditions. In the following we focus on the distribution of lectins in nature, touching only briefly on their specificity, which is the subject of Chapter 4.

Lectins have been detected in over a thousand species of plants and several hundreds have been isolated (Goldstein & Poretz, 1986; Rüdiger, 1988; Van Damme et al., 1998c) . Some of the better characterized plant lectins and their specificity are listed in Table 3 .1. The majority of these have been obtained from the seeds, especially those of the dicotyledonous legumes, where they accumulate during maturation and disappear upon germination. They may constitute as much as 10% of the total seed protein, although the quantities isolated are usually lower, between 0.1-1%. Their location within the seeds differs among various plant families (Rüdiger, 1998) . In seeds of the legumes, most of the lectin is localized in the cotyledons in protein bodies, subcellular organelles related to lysosomes, (Fig. 3 .4) where it may be in complex with endogenous proteins, named "lectin binders" (Rüdiger, 1998) . In those of the Euphorbiaceae (e.g. castor bean), the endosperm is the major site where the lectins occur and here, too, they are confined mainly to protein bodies. In rhizomes of the hedge bindweed (Calystegia sepium) the lectin is in the cytoplasm (Peumans et al., 2000a) whereas in cereals, it is all in the seed embryo.

Besides seeds, lectins have been found in all kinds of vegetative tissue (Table 3 .1). The level of the lectins in these tissues is variable, and exhibits seasonal changes. It is usually lower than in seeds, but can be as high as 30% of the total tissue protein, e.g., the bulb lectins of garlic and ransom, or as low as 0.01% (e.g. in leaves of the leek) (Peumans et al., 2000a) .

Most plant tissues contain a single lectin, although occasionally two (or more) lectins that differ in their sugar specificities and other properties are found in the same tissue (Peumans, W. J. et al., 2000a lectins occur in the seeds of gorse, jackfruit and Vicia cracca, while in seeds of Griffonia simplicifolia three different lectins (in addition to a number of isolectins, see below) are present. Lectins with dissimilar specificities are also found in the bark of the elderberry and the Japanese pagoda tree. In the same plant, lectins are not necessarily confined to a single tissue. Cases are known when lectins found in vegetative parts of the plant are identical with those in the seeds (e.g., the three lectins of Griffonia simplicifolia seeds are also present in leaves of the same plant) but this is not always so. For example, in Dolichos biflorus, the most extensively studied plant with respect to the distribution of lectins in various tissues, the leaves contain a lectin (DB58) homologous to that of the seed lectin (DBL), but with some differences in its fine specificity (Etzler, 1997) . In addition, a root lectin (LNP) has been found in the same plant that is distinct from the seed lectin both in amino acid composition, molecular weight, isoelectric point and specificity (Etzler et al., 1999) . Single lectins, in particular from legumes, occur occasionally as a mixture of isoforms referred to as isolectins. Typically, isolectins have a similar molecular structure, although they may differ slightly in their specificity or some physical property such as electric charge. They can, therefore, be separated by ion exchange chromatography, as found for WGA ( Fig. 3 .5), or by affinity chromatography on immobilized sugars. Some isolectins originate from distinct genes, as is the case of those of WGA (cf. Chapter 7). Others result from the differential posttranslational modifications of a single lectin gene product (Young et al., 1995b) or of the different assembly of closely related subunits. The two winged bean seed isolectins WBA-I and WBA-II differ mainly in their isoelectric points, one being acidic and the other basic, whereas the Maackia amurensis seed isolectins differ in their cell specificity, one (MAH) being a hemagglutinin and the other (MAL) having leukoagglutinating activity (i.e. the ability to agglutinate preferentially white blood cells such as lymphocytes) (Van Damme et al., 1998b) . GSL-I consists of a family of five isolectins, each a tetramer of one or two types of subunit, A and B (Fig. 3 .6) that are very similar in molecular size and amino acid composition, but differ in specificity (see 4.2.1.b). The PHA isolectins also represent a family of five tetrameric proteins with varying proportions of two classes of subunit, E and L. They differ in carbohydrate and cell specificity, as well as in biological properties: E 4 (E-PHA) is a potent hemagglutinin, L 4 (L-PHA) has leukoagglutinating activity and is a potent mitogen; the intermediate forms (E 1 L 3 , E 2 L 2 or E 3 L) possess lower levels of the above activities. Similar mixtures of isoforms are found in the seeds of Vicia villosa and in the bark of Maackia amurensis, Robinia pseudoacacia and Sophora japonica (Van Damme et al., 1998c) . In Datura stramonium seeds three isolectins are present, two of which are homodimers composed of either A or B subunits, whereas the third is a heterodimer comprised of both subunits. The two types of lectin (toxin and agglutinin) found, among others, in the seeds of Ricinus communis and of Abrus precatorius may be considered as a special case of isolectins.

Hemagglutinating activity has been detected in many species of marine algae, particularly red ones (Hori et al., 2000; Rogers, D. J. & Fish, 1991) . However, only a few algal lectins have been purified and characterized, such as the N-acetylgalactosamine-specific lectin from the green alga Codium fragile subspecies tomentosoides (Wu, A. M. et al., 1997) , the galactosespecific one from the red alga Ptilota filicina (Sampaio et al., 1998) . 

A A A A A A A A B B B B B B B B B

The first lectins to be purified from these sources were from the fruiting bodies of the meadow mushroom, Agaricus campestris, and the common (commercial) mushroom, Agaricus bisporus (Goldstein & Poretz, 1986) . By now, many other fungal lectins became known (Guillot, J. & Konska, 1997) . Lectins have also been found in phytopathogenic fungi, such as Botrytis cinerea (Kellens et al., 1992) , Pleurotus ostreatus (Chattopadhyay et al., 1999; Wang, H. et al., 2000) , Rhizoctonia solani (Candy et al., 2001) , in different members of the Sclerotiniaceae (Goldstein, 1990; Inbar, J. & Chet, 1994) and in the nematode-trapping fungus Arthrobotrys oligospora (Rosen, S. et al., 1996) . Recently, a lectin with unique carbohydrate-binding properties, including blood group-B-specificity, and high affinity for Galα3Gal and Galα3Galβ4GlcNAc, but no reactivity with methyl αgalactoside, has been isolated from the mushroom Marasmius oreades (Winter et al., 2002) . A galectin has been isolated from the fruiting bodies of Coprinus cinereus, the first of this lectin family found outside the animal kingdom (Cooper et al., 1997) . It is possible that when additional galactosespecific fungal lectins are sequenced, at least some of them, too, will turn out to be galectins. The above and other fungal lectins are listed in Table 3 .2. 

Lectins have ben isolated from a few yeast species, namely a galactosespecific one from a fatty acid auxotroph of Saccharomyces cerevisiae (Kundu et al., 1987) and two from the culture medium of Kluyveromyces bulgaricus, one specific for galactose and the other for N-acetylglucosamine (al-Mahmood et al., 1991) .

Until the late 1980's, the major source of animal lectins was invertebrates. During the last decade, numerous lectins have been isolated from higher animals, and their number is fast growing. Unlike plant lectins, which can be grouped in families along taxonomic lines, animal lectins often exhibit structural similarities even when derived from diverse phyla. These lectins are therefore classified largely on the basis of shared sequence characteristics of their carbohydrate recognition domains (CRDs) (Drickamer, 1988; Dodd & Drickamer, 2001) as discussed in detail in section 5.2. According to a recent count, at least 12 structural families of animal lectins are known to exist (Kilpatrick, 2002a) , the major ones of which are the C-type lectins (a superfamily), galectins and siglecs. However, not all animal lectins fall into any of the known families.

The most widely occurring family of animal lectins is that of the galectins, (originally S-lectins), so called because they are galactose-specific (Table  3 .3). Twelve mammalian galectins have been described, as well as many additional ones from other species, including birds, lower vertebrates, worms and sponges (Leffler, 2001; Rabinovich et al., 2002; Vasta et al., 1997) . They occur in nearly all cell types, both inside and outside cells, but each galectin tends to be enriched in a few cell types. Thus, galectin-4 and 6 are present almost exclusively in epithelial cells of the gastrointestinal tract, galectin-5 is expressed in erythrocytes and galectin-7 in keratinocytes. The intracellular galectins are located in the cytosol as well as in the nucleus; the extracellular ones are either attached to the cell surface or present in the intercellular space between closely packed cells. They are also found in connective tissues, where they are usually not free but bound to N-acetyllactosamine-containing carbohydrate units of glycoproteins. C-Type lectins (so called because they require Ca 2+ for binding of sugars) have been identified in a wide range of animals (Drickamer, 1999) . They consist of three major classes -endocytic lectins, collectins and selectinsand a minor one -lecticans, and are confined to particular species, organs, or tissues (Table 3 .3). Many are associated both with the plasma membrane and with intracellular membranes. The prototype endocytic lectin is the galactosespecific mammalian hepatic asialoglycoprotein receptor (ASOGR), or hepatic binding protein (HBP), originally isolated from rabbit liver (Ashwell & Harford, 1982; Ashwell & Morell, 1974) ; similar lectins are also present in liver of man, rat and other mammals. In birds, the corresponding lectin is specific for N-acetylgalactosamine and not for galactose. The prototype of the avian lectins is that of chicken, known as chicken hepatic lectin (CHL). In all cases examined, the HBPs are located exclusively on parenchymal hepatocytes. Other endocytic C-type lectins are the fucose-and the galactosespecific receptor found on liver macrophages (Kupffer cells), and the mannose receptor of macrophages (MMR) and of hepatic endothelial cells. The collectins, so called because of their collagenous domains, are present predominantly in mammals and are the only family of C-type lectins that are soluble and not membrane bound. Examples are the serum and liver mannose binding lectins (MBLs), bovine serum conglutinin, and the pulmonary surfactant proteins A and D (SP-A and SP-D, respectively), components of the surfactant that line alveoli in the lung.

The selectins (E-, L-and P-) are membrane lectins found on vascular endothelium, and on leukocytes. L-Selectin is present on essentially all blood 

Ig gene rearrangement, and just before the mature, virgin, immunocompetent B cells migrate out of the bone marrow (Kansas, 1996) . E-Selectin is confined to endothelial cells, its expression being stimulated at the level of transcription, principally in response to inflammatory stimuli such as interleukin-1 (IL-1) or tumor necrosis factor-α (TNF-α). Cytokine induction of the E-selectin gene requires activation and nuclear translocation of the transcription factor NF-κB, that controls many genes involved in immune and inflammatory responses. Within 6 to 9 hours after induction, transcription of the E-selectin gene is sharply down regulated. In addition, the lectin is rapidly internalized and degraded in the lysosomes. These are the major factors ensuring that the expression of E-selectin on cytokinesimulated cells is transient.

In addition to C-type lectins proper, domains homologous to the C-type CRD, referred to as C-type lectin-like domains or CTLDs, have been identified in a variety of proteins that do not appear to have carbohydratebinding activity (Drickamer & Dodd, 1999) . Many such CTLDs are found, for instance, in surface receptors of natural killer (NK) cells (Kogelberg & Feizi, 2001; Weis, W. I., Taylor, M.E. & Drickamer, 1998) , e.g., the murine Ly-49 family and the NKR-P1 series that is polymorphic in rodents, but has only one known homologue in humans, and in anti-freeze proteins from arctic fish (Davies & Sykes, 1997) .

In contrast to the large family of C-type lectins that is highly diverse, both with respect to structure and to carbohydrate specificity, the P-type lectin a For references see Kilpatrick, D.C., 2000, unless Tsuji et al., 2001; (9) Bookbinder et al., 1995; (10) Töpfer-Petersen et al., 1998; (11) Chang et al., 2001; b Found in all kinds of organism, from nematodes to mammals; c Neu5Ac-OS, Neu5Ac-terminated oligosaccharides; d ER, endoplasmic reticulum; e DC-SIGN, dentritic cellspecific ICAM-3-grabbing nonintegrin; f Man-OS, Man-terminated oligoccharides; g DC-SIGNR, DC-SIGN-related protein; h EDEM, ER degradation-enhancing α-mannosidase-like protein; i ERGIC, endoplasmic reticulum Golgi intermediate compartment; j found also in lower animals, e.g. ascidians (Kenjo et al., 2001) ; k VIP, vesicular integral membrane protein. family consists of only two homologous members. It owes its name to the fact that both are specific for mannose-6-phosphate (Man6P). These lectins, known as Man6P-receptors, are widespread and occur on most cell types. The eleven siglecs (sialic acid-binding, Ig-like lectins) identified to date constitute a distinct subset of the Ig superfamily (see 5.2.5). Apart from siglec-4a and -4b, that are expressed exclusively in the nervous system, on oligodentrocytes and Schwann cells, respectively, all other members of this family are confined to discrete subpopulations of leukocytes (Fig. 3.7) . Fig. 3 .7 Expression pattern of siglecs within the hematopoietic system. Apart from CD33 and CD22, little is known about the expression patterns of siglecs on stem cells and progenitors. To date, CD33 and siglec-7 are the only CD33-related siglecs reported to be expressed on monocyte-derived dendritic cells. Sialoadhesin, CD33 and siglec-5 are expressed by subsets of tissue macrophages, but nothing is known about the expression of other CD33-related siglecs on macrophages. NK, natural killer. Reproduced from Crocker & Varki, 2001b; copyright 2001, with permission from Elsevier Science. The recently discovered siglec-11 was not found on peripheral blood leukocytes but was present on macrophages (Angata et al., 2002) . The pentraxins, so named for the arrangement of their subunits into discs with cyclic pentameric symmetry, are serum proteins. The intracellular animal lectins include calnexin, calreticulin and EDEM present in the endoplasmic reticulum, ERGIC-53 in the ER-Golgi intermediate compartment and VIP36 in the Golgi. The newly discovered lectins DC-SIGN and dectin are constituents of the membranes of dendritic cells and of macrophages, respectively. Nuclei also contain lectins, as indicated by the specific binding of neoglycoproteins to these organelles, but only one such lectin (galectin-3, previously known as CBP35) has been isolated and characterized (Gaudin et al., 1995; Wang, L. et al., 1995) .

Some other proteins, such as the different interleukins and tumor necrosis factor exhibit carbohydrate binding activities (Cebo et al., 2002) .

Practically all classes and subclasses of invertebrate examined have lectins (Table 3 .4). These include crabs, snails, worms (helminths) (Greenhalgh et al., 1999; Hirabayashi et al., 1998 ), insects (Ingram & Molyneux, 1991 Kubo et al., 2001) , mollusks and sponges (Müller et al., 1997) . The lectins are present mainly in the hemolymph and sexual organs, e.g. albumin glands and eggs, and occur also on membranes of hemocytes, cells that function in innate immunity (Vasta, 1992) . Perhaps the best known invertebrate lectins are from the garden snail, Helix pomatia, from the body wall of the slug, Limax flavus, and from the serum of the horseshoe crab, Limulus polyphemus. These and other well-characterized invertebrate lectins are listed in Table 3 .4. More exhaustive listings are found in (Kilpatrick, 2000) . Many invertebrates contain multiple lectins, several of which have been purified and characterized. Examples are the four and three lectins, respectively, of the cockroaches Periplaneta americana and Blaberus discoidalis, the two of sea urchin, and the three of sea cucumber (Cucumaria echinata) (Kilpatrick, 2000) . A bewildering array of lectins/isolectins has been detected in the Japanese horseshoe crab, some of which are antigenically distinct and exhibit different species specificity for erythrocytes.

Certain invertebrate lectins show significant sequence similarity to vertebrate lectins (Vasta, 1992) . Examples are the lectins of the larva of Sarcophaga peregrina and of Anthocidaris crasipina that are similar to Ctype lectins, of Geodia cynodium similar to galectins, and of the horseshoe crab and Didenum candidum that show sequence similarity to pentraxins. No less than 125 proteins containing CTLDs have been discovered in the genome of Caenorhabditis elegans (Drickamer & Dodd, 1999) . Since the amino acid sequences of most invertebrate lectins are however not known, the vast majority of these proteins cannot yet be classified into families based on structural similarities.

Among the protozoan lectins (Ward, 1997) the primary examples are the two surface proteins of the pathogenic ameba (Entamoeba histolytica), specific for N-acetylglucosamine (Mirelman and Ravdin, 1986) and for Gal/ GalNAc, respectively (Petri & Schnaar, 1995) . Two lectins, one specific for N-acetylneuraminic acid, the other for N-acetylglucosamine, were isolated from merozoites of the human malarial parasite Plasmodium falciparum, and a For references see (Kilpatrick, D.C., 2000) , unless otherwise stated; b (Cole & Zipser, 1994) ; c (Hirabayashi, J. et al., 1998) ; d Neu5Ac-OS, Neu5Ac-terminated oligosacharides; e the hemolymph contains a mixture of lectins, two of which have been isolated; f (Lü et al., 1999) ; g four lectins with different specificities have been isolated from the hemolymph; two of them (regenectin and the 26kDa lectin) appear transiently in leg homogenates during regeneration; h contains at least five different lectins; i contains four lectins; j detectable only during budding; k the two galactose-specific lectins isolated from this organism were shown to be galectins (Hirabayashi, J. et al., 2001) ; l contains 5 lectins, one of which is specific for hexuronic acids. , 1997) . A lectins with the latter specificity has been purified from trypomastigotes of Trypanosoma cruzi, the parasite causing the Chagas disease (Ward, 1997) . N-Acetylneuraminic acid-specific lectins were obtained from culture supernatants of the protozoan Trichomonas mobilensis (Babál et al., 1994) and of the cattle parasite Trichomonas foetus (Babál et al., 1999) .

Many bacterial species and genera express lectins, frequently of more than one type and with distinct specificities (Ofek & Doyle, 1994; Sharon & Lis, 1997) (Table 3 .6). It is not known however whether individual cells coexpress multiple lectins or if each lectin is confined to a distinct cell population. In Gram negative bacteria (such as E. coli, K. pneumoniae and Salmonellae spp.) the lectins often are in the form of submicroscopic hair-like appendages, known as fimbriae (or pili), that protrude from the surface of the cells (Fig. 3.8) . During the fimbriated phase, a typical Gram negative bacterium carries 200-500 peritrichously arranged fimbriae.

Fimbrial surface lectins are also produced by Gram positive bacteria, among them the oral Actinomyces naeslundii and Actinomyces viscosus. Non-fimbrial lectins associated with the bacterial surface have been purified from Rhizobium lupinii, and from Agrobacterium tumefaciens, also a member of the Rhizobia family. In rare cases the lectins are predominantly intracellular. Two such lectins, PA-IL and PA-IIL, have been isolated from Pseudomonas aeruginosa (Gilboa-Garber et al., 1997) .

Viruses contain sugar-specific surface proteins or glycoproteins that act as hemagglutinins and are therefore classified as lectins (Table 3 .5) (Sharon & Lis, 1997) . Much information is available on the influenza and polyoma (Fry et al., 1999) , HIV (Haidar et al., 1992) and herpes simplex (Spillmann, 2001) . , 1999; ; Dormitzer et al., 2002a; (8) Freund et al., 1991; (9) Fry et al., 1999; (10) Haidar et al., 1992; Mbemba et al., 1994; Rider, 1997. 

Purified lectins are essential in order to establish their molecular properties and is 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 (section 3.3.2).

Isolation of a lectin begins commonly with extraction of the tissue or organ in which it is present. This is quite simple in the case of plants, especially their seeds (Fig. 3.9 ) (Goldstein & Poretz, 1986; Rüdiger, 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 Fig. 3.9 Scheme for lectin purification. reproduced with permission from Rüdiger, 1993; copyright 1993 Springer Verlag.. 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 (Fig. 3.10) .

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 (Table 3 .7). These include polysaccharides such as Sephadex, a polymer of glucose employed for the purification of concanavalin A and pea lectin; Fig. 3 .10 Isolation of Erythrina corallodendron lectin from an ammonium sulfate fraction of a seed protein preparation by affinity chromatography on a column of galactose-derivatized Sepharose 4B. The first peak is of inactive protein eluted with phosphate buffered saline, and the second peak is the pure lectin, eluted with galactose. O--O, protein; •--•, hemagglutinating activity. Inset, analysis by polyacrylamide gel electrophoresis of: I, crude protein preparation applied to column; II, first peak; III, second peak. M, molecular weight markers. 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 for the purification of the lectins from peanut, eel electric organ or calf heart muscle. 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 Sepharosebound asialoglycophorin for the purification of the blood type N-specific lectin from Vicia graminea. Often, a number of techniques are available for the purification of the same lectin, as illustrated in Table 3 .8 for PNA.

An alternative approach for the preparation of lectins has been made possible by the advent of recombinant DNA technology. It is based on the isolation of the cDNA or genomic DNA of the lectin, its insertion into a suitable vector and expression in an appropriate host cell. Isolation of the cDNA requires knowledge of at least part of the primary sequence of the a Modified from Lis & Sharon, 1981a. Compilations of methods for the affinity chromatography of lectins are found in Methods in Enzymology, volumes 28, 34, 50, 83, 138, 179 and 230; b immobilized polysaccharides serve both as supports and as ligands for lectins with suitable specificities; c columns of immobilized glycoproteins, preferably desialylated, can be used for the purification of lectins with different sugar specificities, or those that do not interact with simple sugars. In the latter case elution is done with solutions of low or high pH. Desorption of Ca 2+requiring lectins can be achieved by the addition of EDTA; d the specificity of the lectins isolated depends on the carbohydrate used as ligand. 

peanut (Sharma & Surolia, 1994) and Griffonia simplicifolia (Zhu et al., 1996) have been expressed in E. coli. Expression of plant lectins was also achieved in other systems, e.g. WGA in Saccharomyces cerevisiae

Galβ3GalNAc GP Roussel & Lamblin, 1996; Sharon, N. & Ofek, 2000 . b Ruiz-Palacios et al., 2003