Analysis Of the Saccharide Specificity Of an Acidic Lectin from Winged Bean (Psophocarpus Tetragonolobus)

Abstract

Discovered as early as 1888, lectins did not receive much attention until the late 1940s, when it was shown that certain lectins were specific for human blood group antigens. Sugars were demonstrated as the determinants of blood group specificity in the 1950s using lectins. Two discoveries in the early 1960s led to a phenomenal upsurge in the application of lectins to study cell biology and carbohydrate chemistry: (i) binding of certain lectins to sugars on lymphocyte cell surfaces induced mitogenic stimulation, (ii) lectins preferentially agglutinated malignant cells as compared to their normal counterparts. Later, numerous studies followed in which lectins have been-and continue to be-widely used to study cell surface carbohydrates and their architecture during development, differentiation, and malignancy. Their usefulness in isolating glycoproteins and glycopeptides, as well as in fractionating cells, is well established. Lectins are now routinely employed in the structural elucidation of carbohydrate moieties of glycoconjugates. Moreover, recent discoveries about their ubiquitous presence in all forms of life and strong conservation of their amino acid sequences among lectins belonging to a given family indicate that they likely subserve important physiological functions in nature. These developments have provided strong impetus for the isolation and characterization of lectins from various sources. Chapter I is a comprehensive review of the properties of plant lectins-particularly those from the family Leguminosae, which constitute the most extensively studied group with regard to their saccharide specificities, nature of binding sites, modes of interaction with sugars and cell surface receptors, and their applications in biology and medicine. Two reports on the isolation of a lectin from winged bean (Psophocarpus tetragonolobus) have appeared earlier. These studies differed considerably with respect to the nature of the lectin, its carbohydrate specificity, and its erythroagglutinating activity. Puppke (1) reported a lectin of Mr 46,000 (subunit 29,000) which agglutinated only trypsinized and desialylated human erythrocytes; D galactose was a better inhibitor than GalNAc. Appukuttan and Basu (2), however, reported a lectin of Mr 41,000 (subunit 35,000) which agglutinated untreated human erythrocytes; GalNAc was a more effective inhibitor than D galactose. Later, Kortt (3,4) demonstrated the presence of two groups of lectins in winged bean seeds which had almost identical molecular weights but differed in isoelectric points, agglutinating activity, and carbohydrate specificities. One of these lectins with pI > 9.5, called Winged Bean Basic Lectin (WBA I), bound GalNAc better than galactose, agglutinated trypsinized rabbit erythrocytes, and failed to agglutinate human O group erythrocytes. WBA I has been studied in detail in this laboratory (5). The second lectin, with pI 5.5, is the focus of this thesis entitled: “Analysis of the Saccharide Specificity of an Acidic Lectin from Winged Bean (Psophocarpus tetragonolobus)”. This thesis deals with the following aspects: 1. Characterization of cell surface receptors for the winged bean acidic lectin (WBA II) using human erythrocytes. 2. Analysis of thermodynamic and kinetic parameters involved in WBA II-erythrocyte receptor interaction. 3. Thermodynamic analysis of WBA II interaction with various mono and disaccharides. 4. Identification of essential amino acid residues involved in the binding site of WBA II. Additionally, two appendices are included: a) Chemical modification studies on Abrus agglutinin. b) Studies on the tryptophan residues of Abrus agglutinin. Since lectin binding to cell surface receptors precedes their biological activities, identifying the receptors is crucial. Although hemagglutination inhibition provides preliminary carbohydrate binding information, it yields limited insight into receptor structures. Receptors can be identified by isolating detergent solubilized membrane glycoproteins using affinity chromatography on lectin matrices or by competitive binding with lectins of known specificities. The latter approach was used in Chapter II to identify WBA II receptors on human red blood cells. Two distinct receptor types that differed significantly in binding strengths were identified. Competitive binding with blood group specific lectins and various saccharides revealed that WBA II binds to H and T antigenic determinants on human erythrocytes. Chapter III deals with the thermodynamic and kinetic analysis of WBA II binding to erythrocytes. The interaction is characterized by positive entropy and negative enthalpy, which differs from typical lectin-simple sugar interactions where both S and H are negative. Parameters obtained from equilibrium and kinetic studies indicate that both ionic and hydrogen bonding interactions contribute. The data also suggest a conformational change in the lectin and/or receptor during binding. Kinetic data support a two step binding mechanism, with an initial diffusion controlled step followed by mutual fitting between lectin and receptor. Quantitative determination of saccharide specificity through hemagglutination is limited, especially due to the presence of two receptor types on cells. Therefore, association constants for WBA II-simple sugar interactions were obtained by fluorescence spectroscopy using fluorescent sugars. Chapter IV describes the saccharide specificity of WBA II, mapping the binding site and determining forces that stabilize WBA II-sugar interactions. MeGalNAc and T antigen were found to be the most potent mono and disaccharide inhibitors. Hydrogen bonding and van der Waals forces are major contributors. Temperature dependent binding studies indicate an extended binding site: a primary galactose binding site interacting with the 2-, 4-, and 6 hydroxyl groups, adjacent to a site accommodating a GalNAc residue. The acetamido group of GalNAc and its glycosidic linkage are crucial; (13) linkages bind better than (14). Because sugar specificity alone does not fully define the binding site, essential amino acids were identified through chemical modification (Chapter V). Modification of two amino groups per WBA II molecule abolished saccharide binding and agglutinating activity. Since WBA II is a dimer with two binding sites, one essential amino group per site is implicated. To further understand binding, studies on another legume lectin-Abrus agglutinin from Abrus precatorius-are presented in Appendices I and II. Appendix I reports chemical modification studies showing that one tryptophan residue per binding site is essential. Appendix II details stopped flow kinetic studies of tryptophan oxidation, revealing two phases of tryptophan reorganization upon lactose binding. Quenching studies using acrylamide, succinimide, and cesium indicate heterogeneous tryptophan exposure and a hydrophobic microenvironment with nearby acidic residues.