Lectins as Carbohydrate-binding Proteins

Lectins are cell-binding and agglutinating (glyco)proteins found in a variety of living forms. They will be defined here as homogeneous carbohydrate-binding (glyco)proteins principally of plant (seed) origin but also occurring in invertebrate (and vertebrate?) forms. In recent years, lectins have been employed in studying a wide range of biological phenomena including cell differentiation and maturation , mitogenesis and spermatogenesis); cell agglutination; tumor growth and inhibition; and the nature and distribution of carbohydrates on cell surfaces. Lectin have recently been found to be valuable tools for investigating the structure Of complex carbohydrates (polysaccharides, glycoproteins, and glycolipids), for studying the molecular basis of carbohydrate—protein interactions, and for isolating and purifying polysaccharides and glycoproteins. However, in order to be truly useful as structural probes, the physical—chemical properties of lectins must be characterized and, most importantly, their carbohydrate—binding specificity must be defined. Lectins with specificity for a rather large number of sugars have been isolated and characterized. The use of carbohydrate—protein conjugates for the detection of lectins, and natural and synthetic affinity columns for the isolation of lectins will be described. The isolation and characterization of concanavalin A, the jack bean lectin, is described along with an enumeration of its carbohydrate binding specificity and the localization of its binding site by x-ray crystallography. A comparison of the carbohydrate-binding specificity of four N-acetyl-D-galac.. tosamine binding lectins from Helix pomatia, Dolichos biflorus, Phaseolus lunatus, and Glycine max is discussed. As an example of how lectins may be used in structural studies, an investigation of the interaction of several lectins with pnewnococcus S XIV capsular polysaccharide is described. The cz—D—galactopyranosyl—binding lectin from Bandeiraea simplicifolia seeds is shown to comprise a family of five isolectins, Each isolectin is a tetramer composed of two different subunits, A which binds N-acetyl..D-galactosamine and B which is specific for a—D—galactosyl units, The separation, characterization and some properties of these isolectins are presented. For almost a century we have known that many plant seed extracts clump or agglutinate animal erythrocytes. Specific proteins or glycoproteins isolated from these extracts cause the agglutination. These proteins were called plant agglutinins or phytohemagglutinins until about 25 years ago when Boyd coined the term lectin from the Latin legere to pick out or choose (1). Boyd chose this term to call attention to their serological specificity since he and Renkonen had discovered that some of these seed extracts could distinguish among human blood groups (2,3). Testing lima bean (Phaseolus lunatus) extract against a panel of blood samples, Boyd discovered that the phytohemagglutinin of lima beans was quite specific for type A erythrocytes (2). Several years ago, the lima bean lectin was purified and its specificity for type A cells was confirmed (4,5). Building upon these observations, Morgan and Watkins (6,7) followed by numerous other investigators (8,9), employed lectins as probes for studying the immunochemical, determi— nant sugars of blood group antigens of substances • All blood group substances contain the 1095 1096 IRWIN J. GOLDSTEIN, LEE A. MURPHY and SHIGEYUKI EBISU same four sugars D—galactose, L—fucose, N—acetyl-D—glucosaxnine and N-acetyl—D-galactosamine. The specific way thes sugars are linked together determines blood roup specificity. This is illustrated in Fig. 1 which shows fragments of the ABO blood group substances. The terminal nonreducing sugar of each of these structures plays a dominant Type A: a-D.-GalNAcr (l+3)-.-D-Gal-. (1÷3,4) --.D-GlcNAc. 2 1 a-L-Fuc Type B: a-D-Gal(l+3)--D-Gal(1+3,4) -13-D-.GlcNAc.... 2 1 a-L.-FucE Type 0: -D-Gal(1+3,4) ..8D-GlcNAc.,.. 2 1 a-L-Fuc Fig. 1 Sequence of sugars in oligosaccharide chains of blood group substances. role in determining immunochemical specificity. It is also chiefly these sugar residues with .which lectins interact. Thus, a-linked N-acetyl-D-galactosamine is the immunodominant sugar of type A substance. The lima bean lectin interacts with this sugar. Similarly, D—galactose-.linked ais the immunodominant sugar of blood group B substance and cx-L-fucosyl groups of 0—substance. The lectins from Lotus tetragonolobus seeds and eel serum agluti— nate type 0 erythrocytes. Morgan and Watkins discovered that this agglutination reaction between type 0 red blood cells and Lotus extracts was best inhibited by L-fucose (6, 7). This is the way they showed L-fucose to be the immunodominant sugar of Ocells. The same investigators showed N-acetyl-D-galactosamine to be the best inhibitor of the agglutination reaction between type A erythr6cytes and lima bean extracts (7). The finding that simple sugars inhibited lectin-induced hemagglutination reactions indicated that these sugars were interacting with sites on the lectin molecules. These observations indicate that lectins are a class of carbohydrate-binding proteins. Since these milestones in lectin research, substances capable of binding carbohydrates and interacting with and agglutinating animal cells have been found in many diverse sources: bacteria and molds, lichens, fish sera and roe, numerous invertebrates and even vertebrate forms (10). Approximately 25 lectins have been purified and their physical-chemical properties and carbohydrate—binding specificity studied. A group of representative lectins is presented in Table 1 along with their blood group specificity, sugar specificity and some of their molecular properties. TABLE 1. Some properties of purified lectinsa Sugar Blood Group Mol. Wt. Source Specificity Specificity Subunits Canavalia ensiformis a-D—Man 104,000 (jack bean) a—5—Glc (G1cNAc) (4) Lens culinaris a—D—Man 49,000 (lentil) a—D—Glc (G1cNAc) (2) Pisum sativum a—D-Man 55,000 (pea) a—D—Glc (G1cNAc) (4) Glycine max D-Ga1NAc A 120,000 (soy bean) — (4) Dolichos biflorus a—D—GalNAc A 113,000 (horse gram) (4) Lectins as carbohydrate—binding proteins 1097 TABLE 1. (Continued) Sugar Blood Group Mol. Wt. Source Specificity Specificity Subunits Phaseolus lunatus cx-D—GalNAc A 125,000 (lima bean) (4) 250,000 (8) Helix pomatia a-D—Ga1NAc A 100,000 (edible snail) — (6) Bandeiraeasimplicifolia i a—D—Gal B 114,000 (4) Sophora japonica a—D—Ga1NAc B,A 132,800 (Japanese pagoda tree) a-D-Gal (?) Ricinus communis 8—D—Gal 120,000 (castor bean RCA1) (4) Lotus tetragonolobus a-L-Fuc 0 120,000 (asparagus pea) (4) Anguilla anguilla cz-L-Fuc 0 123,000 (eel) (12) Triticum vulgaris GlcNAc(l4)G1cNAc 36,000 (wheat) (2) Solanum tuberosum fGlcNAc(l-4)G1cNAc) 100,000 (potato) 2 (2) Bandeiraea simlicifolia II —D—G1cNAc 113,000 cz—D-G1cNAc (4) Phaseolus vulgaris (SA)+-D—Gal.. 136,000 (SA)+8—DGa1.. (4) Streptomyces 27 S 5 L—Rha B 11,000 Limulus polyphemus Sialic acid 400,000 (horse shoe crab) (20) a. Sugar abbreviations are standard. SA, sialic acid. The physiological function of lectins is still completely unknown although there have been many speculations (Table 2). However, none of these possibilities has been convincingly demonstrated. TABLE 2. Possible functions of plant lectins Sugar transport, storage and immobilization Carbohydrases Plant antibodies Binding of nitrogen fixing bacteria Involvement in germination, differentiation, maturation, cell division Despite our ignorance: of the biological role of lectins, these carbohydrate-binding proteins have proved to be exceptionally valuable and versatile substances for examining a variety of cellular activities. Some biological properties of lectins are enumerated in Table 3. 1098 IRWIN J. GOLDSTEIN, LEE A. MURPHY and SHIGEYUKI EBISU TABLE 3. Some biological properties of lectins Agglutination of cells: Erythrocytes, lymphocytes, spermatozoa, tumor cells, microorganisms, viruses Induction of mitogenesis in lymphocytes Inhibition of enzymatic activity: : 5'-nucleosidase, 8—glucosidase Insulin—like action of fat cells Cellular poisons: Ricin, abrin Immunological activity: Immunosuppression and enhancement; histamine release The use and application of lectins especially as probes for structural studies on complex carbohydrates is shown in Table 4. TABLE 4. Some uses of lectins Blood typing; structural studies on blood group substances Detection, preliminary characterization, and structural studies on polysaccharides, glycoproteins and glycolipids Isolation and purification of carbohydratecontaining polymers Studies of carbohydrate-containing structures on cell surfaces Models for carbohydrate-protein interaction and for antibody In order for a lectin to be a useful probe in molecular biology and a structural probe in carbohydrate chemistry it is necessary to isolate it in pure form and to characterize its physical and chemical properties as completely as possible. Above all, its carbohydrate binding specificity must be studied in detail. In only one case has such a complete characterization been accomplished that of concanavalin A. First isolated by Sumner and Howell, in 1936 (11), concanavalin A (con A, from Canavalia ensiformis beans) was obtained in pure form by affinity chromatography on cross-'linked dextran gel (Sephadex) (12,13). The carbohydrate-binding specificity of this lectin has been studied in great detail (14). As illustrated in Fig. 2, the con A combining sites are most complementary to terminal, nonreducing -D-mannopyranosyl units, although 2-0-substituted a-D—mannopyranosyl residues will alsointeract with the lectin (15). We have used a series of deoxy, 0-methyl, and fluoro derivatives of D-glucose and D—mannose to identify the precise atoms of each hydroxyl group that may be involved in bind!ng to the protein (14). Our findings are summarized in Fig. 2. Con A is a metalloprotein composed of subunits, molecular weight 26,000. At pH 5-6 the lectin occurs as a diner; at pH 7 as a tetramer. The protein has been sequenced (16) and its x—ray crystallographic structure solved at a resolution of 2 A (17) and 2.4 A (18). Con A has one Mn2+ and one Ca2+ per protomer; both ions are required for carbohydrate binding activity (19). The con A carbohydrate binding site has been located in crystals of con A (20,21) after considerable controversy (22—25). Although lectins are most commonly detected by screening extracts from plant seed and animal tissue for their ability to agglutinate animal cells, more sophisticated procedures are now available. We use precipitation reactions between lectins and polysaccharides (26), and naturally occurring and synthetic carbohydrate—protein conjugates (27, 28). Recently we have developed a simple procedure for linking carbohydrates to proteins (29). The method is illustrated in Fig. 3. A peptide linkage is established between sugar acid and protein amino groups • This linkage leads to far less. nonspecific interactions. coanpared to the azophenyl linkage we have used previously. Lectins as carbohydrate—binding proteins 1099 Fig. 2 Sugar binding specificity of concanavalin A. Essential hydroxyl groups (C-3, 4 and 6) are underlined. The hydrogen and oxygen atoms thought to participate in hydrogen bonding to the protein are overscored.