Analysis Of Saccharide Binding To Artocarpus integrifolia (Jackfruit) Agglutinin

Abstract

Despite the discovery of the first agglutinin in 1888, lectins remained in obscurity until the late 1940s, when it was shown that certain lectins were specific for human blood group antigens. The fact that sugars determine blood group specificity was established in the 1950s through the use of blood group specific lectins. Two discoveries in the early 1960s catalyzed rapid growth in lectin research:

The binding of certain lectins to lymphocytes, resulting in mitogenic stimulation, and Preferential agglutination of malignant cells compared to their normal counterparts.

These findings led to numerous studies in which lectins were-and continue to be-widely used to investigate cell surface carbohydrates and the changes they undergo during development, differentiation, and malignancy. Lectins have also proved to be excellent tools for the separation and structural elucidation of glycoproteins, glycopeptides, and glycolipids, and are widely used for cell fractionation. These developments have renewed interest in the isolation and characterization of lectins from various sources. Chapter I provides a comprehensive review of lectin properties, particularly plant seed lectins, their carbohydrate specificities, biological roles, and applications. The present study focuses on the lectin from Artocarpus integrifolia seed, reported to be specific for the tumor associated Thomsen–Friedenreich (T) antigen.

Since all lectin properties stem from their ability to bind specific carbohydrate structures on the cell surface, elucidating carbohydrate specificity is essential. Hemagglutination is commonly used but provides only relative affinities. Quantitative thermodynamic characterization of lectin–ligand interactions can be obtained using spectroscopic methods. Chapter II presents fluorescence spectroscopic studies on ligand specificity and binding energetics. The fluorescence intensity of N dansylgalactosamine (DnsGalN) increases by up to 100% upon binding to the A. integrifolia lectin. Binding is carbohydrate specific: addition of non fluorescent sugars restores fluorescence to the level of free DnsGalN. The association constant Ka=1.84×104 M 1K_a = 1.84 \times 10^4 \, \text{M}^{-1}Ka =1.84×104M 1 was obtained from protein dependent fluorescence changes. Temperature dependent measurements enabled determination of thermodynamic parameters. Association constants for non fluorescent sugars were obtained using competitive displacement of DnsGalN. Thermodynamic data indicate that hydroxyl groups at C 2, C 4, and C 6 of D galactose are crucial for binding, and that binding is governed primarily by enthalpic forces.

Binding of disaccharides showed exceedingly high affinity for the T antigenic disaccharide, a tumor associated antigen of non oncofetal origin. The enthalpy of association for the T antigen was greater than that of Me Gal, indicating the presence of an extended binding site. This suggests a secondary subsite, contiguous to the galactose binding region, specifically accommodating N acetylgalactosamine (GalNAc). The lectin displays remarkable selectivity, distinguishing T antigen from structurally similar disaccharides such as LacNAc, lactose, and Gal 1 3GlcNAc. Despite similar ring structures and overall topographies, significant differences in binding arise from steric hindrance at C 3 and C 4 of the reducing sugar in 1 4 and 1 3 disaccharides. In contrast, T antigen binds strongly due to favorable interactions with the C 2 acetamido group, the axial C 4 hydroxyl of GalNAc, and the non reducing galactopyranosyl residue. These findings are discussed in detail in Chapter III.

Chapter IV focuses on the kinetics of DnsGalN binding to A. integrifolia lectin using stopped flow methods. Fluorescence enhancement upon complex formation permits kinetic monitoring. Key results:

Association rate constant: kon=8.1×103 M 1s 1k_{\text{on}} = 8.1 \times 10^3 \, \text{M}^{-1}\text{s}^{-1}kon =8.1×103M 1s 1 (much slower than diffusion controlled limits)

Dissociation rate constant at 21 °C: koff=50 s 1k_{\text{off}} = 50 \, \text{s}^{-1}koff =50s 1

Both rates increase with temperature. Activation parameters derived from temperature dependent studies show that association requires an unfavourable entropy, consistent with a constrained bimolecular single step binding mechanism.

Chapter V presents NMR studies using selectively enriched ¹³C and ¹ F GalNAc. Both and anomers show slow exchange between free and bound states. Line width measurements yield association and dissociation constants, consistent with fluorescence results. The acetamido group of both anomers experiences a magnetically equivalent environment in the bound state. As with fluorescence kinetics, association is limited by unfavourable activation entropy.

Chapter VI provides a general discussion of all results presented in Chapters II–V and summarizes the conclusions drawn from this investigation.