Luffa acutangula agglutinin, a chito-oligo saccharide-specific lectin from the ridge gourd phloem exudate: physiochemical studies.

Abstract

The thesis entitled “Luffa acutangula Agglutinin, a Chito oligosaccharide-Specific Lectin from the Ridge Gourd Phloem Exudate: Physicochemical Studies” deals mainly with the following aspects: i) Isolation and characterization of the lectin (phloem protein 2: PP2) ii) Hemagglutination inhibition studies to determine carbohydrate specificity iii) Equilibrium dialysis to determine the stoichiometry of the lectin-saccharide interaction iv) Circular dichroism and UV difference spectroscopic studies to determine the nature and dimensions of the saccharide combining region v) Effect of saccharides and quenchers on intrinsic fluorescence of the lectin to determine the orientation of saccharides in the combining region and to investigate the microenvironment of tryptophan residues vi) Chemical modification studies to identify amino acid side chains involved in saccharide binding activity Lectins were initially discovered in plant seeds, and therefore seed lectins have been the most widely studied. Reports on their roles in plants-particularly relating to their turnover in seeds-may provide insight into their functions in development, differentiation, and root-bacterium symbiosis. Lectins, however, have also been found in other parts of the plant. Currently, major efforts are directed toward elucidating the relationship between lectins isolated from various plant tissues and their overall biological functions. In this context, the discovery of lectins in the phloem exudate of many cucurbits, including the ridge gourd (Luffa acutangula), provides an opportunity to investigate their functions owing to their unusual location and abundance. Elucidation of their molecular properties will facilitate understanding of structure-function relationships among this class of lectins. This study is a step in that direction. Chapter I is a comprehensive review of lectin properties, with emphasis on those from non leguminous plants and their applications in research and medicine. Chapter II describes the isolation, purification, and characterization of the lectin from the phloem exudate of ridge gourd (Luffa acutangula) fruits. The lectin was purified by ammonium sulfate fractionation and affinity chromatography on soybean agglutinin glycopeptide-Sepharose 6B. The purified lectin is homogeneous by PAGE at both acidic and alkaline pH, by SDS PAGE, analytical gel chromatography, and sedimentation velocity and equilibrium experiments. The presence of only one amino acid (glycine) at the N terminal end further confirms homogeneity. The relative molecular mass of the lectin is 48,000 ± 1,000 Da, determined by gel chromatography and sedimentation equilibrium, both in the presence and absence of 2 mercaptoethanol. A sedimentation coefficient of 4.06 S was obtained under both conditions. In guanidine hydrochloride, a value of 2.15 S was observed. The Stokes radius is 2.9 nm, consistent with calculated values using sedimentation and molecular mass data. SDS PAGE yielded a molecular mass of 24,000 Da, regardless of reducing agent, suggesting a non covalent dimeric lectin. The protein is not a glycoprotein. Far UV CD spectra indicate ~31% helix and predominantly non regular structure. Luffa acutangula agglutinin (LAA) does not show specificity toward human blood groups A, B, or O. It agglutinates rabbit erythrocytes, with a 3000 fold increase in affinity for trypsin treated cells. Chapter III reports hemagglutination inhibition and equilibrium dialysis studies. No monosaccharides inhibited LAA. Except for di N acetylchitobiose, disaccharides were ineffective, though N acetyllactosamine showed weak inhibition. Chito oligosaccharides (14-linked GlcNAc oligomers) were potent inhibitors, increasing in potency up to hexasaccharide. N linked glycopeptides (high mannose, hybrid, and complex types) were also potent inhibitors. Lack of binding to GlcNAc rich affinity matrices and certain oligosaccharides suggests that LAA recognizes only the core chitobiosyl sequence. Equilibrium dialysis data indicate two independent saccharide binding sites, one per monomer. Chapter IV describes thermodynamic studies using CD and UV difference spectroscopy. Chito oligosaccharide binding caused size dependent enhancement in near UV CD signals. Association constants (Ka) increased with chain length up to penta N acetylchitopentaose (values provided in the text). Thermodynamic analysis suggests an extended combining site with at least four subsites (A-D). UV difference spectra showed maxima at 288 and 292 nm, indicating tryptophan perturbation in the combining region. Chapter V examines effects of quenchers and saccharides on intrinsic fluorescence. LAA has an emission maximum at 336 nm. Binding causes ligand size dependent enhancement and blue shift. Ka and thermodynamic parameters correlate with CD results. Data indicate a tryptophan residue near subsite D. Quenching suggests tryptophans are solvent exposed, and iodide quenching decreases upon ligand binding. Chapter VI presents chemical modification studies. Modifying lysine, tyrosine, arginine, histidine, and acidic residues did not affect activity. However, modification of tryptophan residues with N bromosuccinimide (NBS) and 2 hydroxy 5 nitrobenzyl bromide (HNBBr) caused complete loss of carbohydrate binding and hemagglutinating activity. Under denaturing conditions, 6-6.8 tryptophan residues were modified; only 2 in the native state. Protection by chito oligosaccharides (except disaccharide) suggests tryptophans are located beyond subsites A and B. Fluorescence loss patterns (linear vs biphasic) support environmental changes upon ligand binding. Secondary structure remained unchanged after modification. Chapter VII provides general discussion and conclusions. Conclusion LAA is a non covalent dimeric globular protein, ~48 kDa, with two saccharide binding sites and absolute specificity for chito oligosaccharides. The combining region accommodates a tetrasaccharide. A single tryptophan residue is located in the combining region and is essential for carbohydrate binding.