Sneha Sudha Komath

Beyond carbohydrate binding: new directions in plant lectin research

Although for a long time carbohydrate binding property has been used as the defining feature of lectins, studies carried out mostly during the last two decades or so demonstrate that many plant lectins exhibit specific interactions with small molecules that are predominantly hydrophobic in nature. Such interactions, in most cases, appear to be at specific sites that do not interfere with the ability of the lectins to recognise and bind carbohydrates. Further, several of these ligands have binding affinities comparable to those for the binding of specific carbohydrates to the lectins. Given the ability of lectins to specifically recognise the glycocode (carbohydrate code) on different cell surfaces and distinguish between diseased and normal tissues, these additional sites may be viewed as potential drug carrying sites that could be exploited for targeted delivery to sites of choice. Porphyrin-lectin complexes are especially suited for such targeting since porphyrins are already under investigation in photodynamic therapy for cancer. This review will provide an update on the interactions of plant lectins with non-carbohydrate ligands, with particular emphasis on porphyrin ligands. The implications and potential applications of such studies will also be discussed.

Efflux Pumps in Drug Resistance of Candida

The incidences of human pathogenic yeast Candida albicans and its related species acquiring resistance to antifungals have increased considerably, which poses serious problems towards its successful chemotherapy. The resistance of these pathogenic fungi is not restricted to the commonly used triazole compounds but is even encountered, though not often, with polyene derivatives as well. The efflux pump proteins belonging to ABC (ATP Binding Cassette) and MFS (Major Facilitators) super family are the most prominent contributors of multidrug resistance (MDR) in yeasts. The abundance of the drug transporters and their wider specificity suggest that these transporters may not be exclusively drug exporters in yeasts and may have other cellular functions. In this article we focus on some of the recent advances on the structure and function, evolution and transcriptional control of drug efflux proteins of Candida. A short discussion on the physiological relevance of drug transporters is also included.

Binding of Porphyrins by the Tumor-Specific Lectin, Jacalin (Jack Fruit (Artocarpus integrifolia) Agglutinin)

Jacalin (Artocarpus integrifolia agglutinin) specifically recognizes thetumor-associated T-antigenic disaccharide structure,Galβ13GalNAc. Porphyrins and their derivatives are currently used asphotosensitizers in photodynamic therapy to treat malignant tumors. In thisstudy, the interaction of several free base porphyrins and their metalderivatives with jacalin is investigated by absorption and fluorescencespectroscopy. Each lectin subunit was found to bind one porphyrin moleculeand the association constants were estimated to be in the range of2.4×103M−1 to 1.3×105M−1 at room temperaturefor the interaction of different porphyrins with jacalin. These values arein the same range as those obtained for the interaction of monosaccharidesto jacalin. Both free lectin and lectin saturated with the specificsaccharide were found to bind different porphyrins with comparable bindingstrength indicating that porphyrin binding takes place at a site differentfrom the sugar binding site. Further, both anionic and cationic porphyrinswere found to interact with the lectin with comparable affinity, clearlyindicating that the charge on the porphyrin does not play any role in thebinding process and that most likely the interaction is mediated byhydrophobic forces. These results suggest that jacalin and other lectins maypotentially be useful for targeted delivery of porphyrins to tumor tissuesin photodynamic therapy.

Fluorescence and absorption spectroscopic studies on the interaction of porphyrins with snake gourd (Trichosanthes anguina) seed lectin.

The interaction of several free-base porphyrins and their corresponding copper(II) and zinc(II) derivatives with the galactose-specific lectin from snake gourd (Trichosanthes anguina) seeds has been investigated by absorption and fluorescence spectroscopic techniques. The lectin dimer contains two apparently equivalent binding sites for the porphyrins. Association constants obtained for the interaction of various porphyrins with the lectin are in the range 1.7 x 10(4)-6.2 x 10(5) M(-1), with the metalloporphyrins being seen to have higher affinity for the lectin compared with the free-base analogues. Both positively charged and negatively charged porphyrins bind to snake gourd seed lectin (SGSL) with comparable affinities, suggesting that binding occurs primarily via hydrophobic interactions. Further, binding of porphyrins is found to be largely unaffected by the presence of the sugar ligand, lactose, indicating that the binding sites for the carbohydrate and porphyrin are different. This study thus suggests that the lectin may serve as a receptor for some endogenous non-carbohydrate, hydrophobic ligand in vivo, in addition to the saccharide ligands. It also opens up the possibility of employing the T. anguina lectin in applications such as photodynamic therapy, which involve the use of porphyrins.

Fluorescence quenching, time-resolved fluorescence and chemical modification studies on the tryptophan residues of snake gourd (Trichosanthes anguina) seed lectin

Abstract Fluorescence quenching and time-resolved fluorescence studies have been performed on the galactose-specific lectin purified from snake gourd (Trichosanthes anguina) seeds, in order to investigate the tryptophan accessibility and environment in the native protein and in the presence of bound ligand. Estimation of the tryptophan content by N-bromosuccinimide modification in the presence of 8 M urea yields four residues per dimeric molecule. The emission spectrum of native lectin in the absence as well as in the presence of 50 mM methyl-α- d -galatopyranoside (MeαGal) shows a maximum around 331 nm, which shifts to 361.8 nm upon reduction of the disulfide bonds and denaturation with 8 M urea, indicating that all four tryptophan residues in the native state of this protein are in a hydrophobic environment. The extent of quenching that is observed is highest with acrylamide, intermediate with succinimide, and low with Cs+ and I−, further supporting the idea that the tryptophan residues are predominantly buried in the hydrophobic core of the protein. The presence of MeαGal (50 mM) affects the quenching only marginally. Time-resolved fluorescence measurements yield bi-exponential decay curves with lifetimes of 1.45 and 4.99 ns in the absence of sugar, and 1.36 and 4.8 ns in its presence. These results suggest that the tryptophan residues are not directly involved in the saccharide binding activity of the T. anguina lectin. Of the four quenchers employed in this study, the cationic quencher, Cs+, is found to be a very sensitive probe for the tryptophan environment of this lectin and may be useful in investigating the environment of partially buried tryptophan residues and unfolding processes in other proteins as well.

Differential scanning calorimetric studies on the thermotropic phase transitions of dry and hydrated forms of N-acylethanolamines of even chainlengths.

N-acylethanolamines (NAEs) have attracted the attention of researchers in the last two decades due to their occurrence in biological membranes under conditions of stress as well as under normal conditions. Differential scanning calorimetric studies have been carried out on dry and hydrated samples of a homologous series of N-acylethanolamines containing saturated acyl chains of even number of carbon atoms (n = 8-20). In both cases a major sharp endothermic transition was observed which occurs at the melting point for the dry NAEs whereas for the hydrated samples it occurs at considerably lower temperatures. The enthalpies and entropies corresponding to this transition could be fitted, in each case, to a straight line suggesting that the transition enthalpy and transition entropy consist of a fixed component from the polar head group and the terminal methyl group, whereas the contribution of the methylene groups, (CH2)n, is linearly proportional to the number of carbon atoms in it. The contributions of each methylene unit to the transition enthalpy and transition entropy of NAEs were found to be deltaH(inc) = 0.82 (+/-0.02) and 0.96 (+/-0.06) kcal mol(-1), and deltaS(inc) = 2.01 (+/- 0.06) and 2.37 (+/-0.17) cal mol(-1) K(-1), respectively, for the dry and hydrated samples of NAEs, whereas the end contributions arising from the head group and the terminal methyl group were determined to be deltaH(o) = -0.10 (+/-0.26) and -0.52 (+/-0.82) kcal mol(-1) and deltaS(o) = 2.12 (+/-0.71) and 3.1 (+/-2.3) cal mol(-1) K(-1), respectively, for the dry and hydrated samples of NAEs. These results are relevant to an understanding of the thermodynamics of the phase properties of NAEs in membranes.

Purification in high yield and characterisation of the galactose‐specific lectin from the seeds of snake gourd (Trichosanthes anguina)

The galactose‐specific lectin present in the seeds of snake gourd (Trichosanthes anguina) was purified in high yield by affinity chromatography on cross‐linked guar gum. The purified snake gourd seed lectin (SGSL) yielded a single symmetrical peak on gel filtration with an Mr of 62 kDa and gave a single band in PAGE under non‐denaturing conditions. In SDS‐PAGE, SGSL gave a single band of Mr 53 kDa in the absence of β‐mercaptoethanol, and two bands of Mr 32 and 23 kDa in its presence, indicating that the lectin is a heterodimer in which the subunits are linked by a disulphide bridge. The lectin gave a single precipitin line in immunodiffusion experiments with antiserum raised against the purified SGSL. No cross‐reactivity was found between SGSL and antiserum raised against the Momordica charantia lectin and vice versa, suggesting that the two lectins are antigenically dissimilar. Haemagglutination‐inhibition data show that MeβD‐Gal is the best monosaccharide inhibitor of SGSL and indicate that an equatorial hydroxyl at C‐2 and axial hydroxyl at C‐4 in the pyranose form are important binding loci for the lectin.

Purification in high yield and characterisation of a new galactose-specific lectin from the seeds of Trichosanthes cucumerina

Abstract Ten Cucurbitaceae species have been investigated for the presence of seed lectins of which only two species, Trichosanthes cucumerina and T. palmata, have displayed agglutination activity which was inhibited by galactose. The lectin from T. cucumerina seeds has been purified in high yield (∼350 mg lectin/100 g deshelled seeds) by affinity chromatography on cross-linked guar gum. The purified T. cucumerina seed lectin (TCSL) moved as a single symmetrical peak on gel filtration on Sephadex G-150 with an apparent molecular weight of 62 (±5) kDa and gave a single band on PAGE under non-denaturing conditions. In SDS-PAGE, TCSL gave a single band at 69 kDa in the absence of 2-mercaptoethanol, whereas in the presence of 2-mercaptoethanol two bands corresponding to 41 and 22 kDa were observed, suggesting that the lectin is made up of two non-identical subunits that are linked by one or more disulphide bridges. TCSL is a glycoprotein with about 3.0% covalently bound neutral sugar. The lectin cross-reacted with rabbit antiserum raised against the Trichosanthes anguina (snake gourd) seed lectin (SGSL), yielding a single precipitin line and SGSL cross-reacted with the anti-TCSL antiserum raised in rabbits, indicating that the two lectins are antigenically very similar. This was further confirmed by Western blot analysis where the two subunits of TCSL were found to react with both anti-TCSL and anti-SGSL antisera and vice versa. On the other hand, TCSL did not cross-react with the antiserum raised against Momordica charantia lectin and vice versa, suggesting that these two cucurbit seed lectins are antigenically dissimilar. Haemagglutination-inhibition data show that TCSL is specific for the β-anomer of galactose with MeβGal and lactose being the best mono- and disaccharide inhibitors, respectively.

Insight into Pleiotropic Drug Resistance ATP-binding Cassette Pump Drug Transport through Mutagenesis of Cdr1p Transmembrane Domains

Background: The Candida albicans efflux pump Cdr1p causes clinically significant antifungal resistance. Results: Biochemical mapping of Cdr1p transmembrane domain mutants reveals residues affecting drug transport. Conclusion: Functional characterization and homology modeling provide insight into the drug binding cavity and substrate promiscuity of Cdr1p. Significance: A platform is provided for systematic Cdr1p structure/function analysis and the rational design of transport modulators/inhibitors. The fungal ATP-binding cassette (ABC) transporter Cdr1 protein (Cdr1p), responsible for clinically significant drug resistance, is composed of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). We have probed the nature of the drug binding pocket by performing systematic mutagenesis of the primary sequences of the 12 transmembrane segments (TMSs) found in the TMDs. All mutated proteins were expressed equally well and localized properly at the plasma membrane in the heterologous host Saccharomyces cerevisiae, but some variants differed significantly in efflux activity, substrate specificity, and coupled ATPase activity. Replacement of the majority of the amino acid residues with alanine or glycine yielded neutral mutations, but about 42% of the variants lost resistance to drug efflux substrates completely or selectively. A predicted three-dimensional homology model shows that all the TMSs, apart from TMS4 and TMS10, interact directly with the drug-binding cavity in both the open and closed Cdr1p conformations. However, TMS4 and TMS10 mutations can also induce total or selective drug susceptibility. Functional data and homology modeling assisted identification of critical amino acids within a drug-binding cavity that, upon mutation, abolished resistance to all drugs tested singly or in combinations. The open and closed Cdr1p models enabled the identification of amino acid residues that bordered a drug-binding cavity dominated by hydrophobic residues. The disposition of TMD residues with differential effects on drug binding and transport are consistent with a large polyspecific drug binding pocket in this yeast multidrug transporter.

Molecular theory of ultrafast solvation in liquid acetonitrile

A microscopic calculation of solvation dynamics of an immobile solute ion in liquid acetonitrile is presented. The dynamical information necessary for this calculation is obtained from the Kerr and dielectric relaxation of the neat liquid. The calculated solvation time correlation function is in excellent agreement with both the experimental and the simulated results.