Saccharide Binding Research Articles

The Amino-terminal Heptad Repeats of the Coiled-coil Neck Domain of Pulmonary Surfactant Protein D Are Necessary for the Assembly of Trimeric Subunits and Dodecamers

Pulmonary surfactant protein D (SP-D), a lung host defense protein, is assembled as multimers of trimeric subunits. Trimerization of SP-D monomers is required for high affinity saccharide binding, and the oligomerization of trimers is required for many of its functions. A peptide containing the alpha-helical neck region can spontaneously trimerize in vitro. However, it is not known whether this sequence is necessary for the complete cellular assembly of disulfide-cross-linked, trimeric subunits and dodecamers. For the present studies, we synthesized mutant cDNAs with deletions or site-directed substitutions in the neck domain of rat SP-D, and examined the assembly of the newly synthesized proteins after transfection of CHO-K1 cells. The neck domain contains three "classical" heptad repeat motifs with leucine residues at the "d position," and a distinctive C-terminal repeat previously suggested to drive trimeric chain association. Deletion of the highly conserved core of the latter repeat (FSRYLKK) did not interfere with the secretion of dodecamers with lectin activity. By contrast, deletion of the entire neck domain or deletion of one or two amino-terminal repeats resulted in defective molecular assembly. The secreted proteins eluted in the position of monomers by gel filtration under nondenaturing conditions. In addition, the neck + carbohydrate recognition domain of SP-D was necessary and sufficient for the trimerization of a heterologous collagen sequence located amino-terminal to the trimeric coiled-coil. These studies provide strong evidence that the amino-terminal heptad repeats of the neck domain are necessary for the intracellular, trimeric association of SP-D monomers and for the assembly and secretion of functional dodecamers.

Modular fluorescence sensors for saccharides

Modular and modular polymer supported fluorescence photoinduced electron transfer (PET) sensors 2 and 3 with two boronic acid receptor units, a pyren-1-yl fluorophore, and hexamethylene linker show selective saccharide binding in aqueous methanolic solution at pH 8.21.

Thermodynamic analysis of saccharide binding to snake gourd (Trichosanthes anguina) seed lectin

The interaction of different saccharides with the snake gourd (Trichosanthes anguina) seed lectin (SGSL) was investigated by fluorescence spectroscopy. Binding of 4-methylumbelliferyl-beta-D-galactopyranoside (MeUmb beta Gal) to SGSL resulted in a significant increase in the fluorescence emission intensity of the sugar at 376 nm, and this change was used to estimate the association constants for the binding interaction. Interestingly, the increase in emission intensity changed with a change in temperature, increasing from 19.2% at 20 degrees C to 80.2% at 40 degrees C. At 20 degrees C the association constant, K(a), for the MeUmb beta Gal-SGSL interaction was found by fluorescence titration to be 5.8 x 10(4) M(-1). From the temperature dependence of the association constants, the changes in enthalpy (Delta H) and entropy (Delta S) associated with binding of MeUmb beta Gal to SGSL were estimated to be -80.85 kJ.mol(-1) and -184.0 J.mol(-1).K(-1), respectively. Binding of unlabeled sugars was investigated by monitoring the decrease in fluorescence intensity when they were added to a mixture of SGSL and MeUmb beta Gal. The Ka values for different sugars were determined at several temperatures, and Delta H and Delta S were determined from the van't Hoff plots. Enthalpy-entropy compensation was noticed in all cases. The results indicate that saccharide binding to SGSL is enthalpy-driven and the negative contribution from entropy is, in general, quite high.

Diaza-18-crown-6-Based Saccharide Receptor Bearing Two Boronic Acids. Possible Communication between Bound Saccharides and Metal Cations

A novel diaza-18-crown-6-based saccharide receptor (4) bearing two boronic acid groups has been developed. In 4 boronic acid groups in the side arms interact as Lewis acids with amines and boron atoms are changed to sp3 hybridization through the B···N interaction. Because of this hybridization, the boronic acid groups can bind saccharides even in a neutral pH region. Judging from the distance between two boronic acid groups in 4 assuming a syn conformation, one can expect that 4 forms 1:1 cyclic complexes with monosaccharides. These are confirmed by circular dichroism spectroscopy of 4 in the presence of glucose and allose. UV photometric titration studies showed that the boronic acid groups and metal cation bound to the crown cavity competitively interact as Lewis acids with the crown amines. As a result, saccharides and metal cations “communicate” with each other in an allosteric manner. It was shown that added metal cations can affect the saccharide binding ability of 4 through the competitive interaction with the crown amines and the conformational change of the crown ether ring.

Detection of total sugar concentration using photoinduced electron transfer materials: development of operationally stable, reusable optical sensors

The work presented here describes the synthesis and testing of a number of related boronic-acid containing photoinduced electron transfer (PET) materials for use in saccharide measurement. These chemicals consist of a fluorophore and boronic acid attached to an amine and show increased fluorescence on saccharide binding to the boronic acid. When the boronic acid complexes with the saccharide the boron atom becomes more acidic leading to an enhanced Lewis acid–Lewis base interaction with the amine nitrogen. This reduces the interaction of the nitrogen lone pair with the fluorophore suppressing the PET process and increasing fluorescence. The work described here investigates the effect of spacer length between two boronic acids on the same molecule, on the selectivity and sensitivity of saccharide binding. The stability of these compounds against stress from high temperatures and ionic strength has also been investigated. A second type of chemical, with a vinyl group has been synthesised and tested for saccharide measurement abilities. This chemical may be crosslinked into a polymer and enable its incorporation onto a polymer-coated optical fibre for the potential production of reusable saccharide sensors.

Crystal structure of YbaK protein from Haemophilus influenzae (HI1434) at 1.8 A resolution: functional implications.

Structural genomics of proteins of unknown function most straightforwardly assists with assignment of biochemical activity when the new structure resembles that of proteins whose functions are known. When a new fold is revealed, the universe of known folds is enriched, and once the function is determined by other means, novel structure-function relationships are established. The previously unannotated protein HI1434 from H. influenzae provides a hybrid example of these two paradigms. It is a member of a microbial protein family, labeled in SwissProt as YbaK and ebsC. The crystal structure at 1.8 A resolution reported here reveals a fold that is only remotely related to the C-lectin fold, in particular to endostatin, and thus is not sufficiently similar to imply that YbaK proteins are saccharide binding proteins. However, a crevice that may accommodate a small ligand is evident. The putative binding site contains only one invariant residue, Lys46, which carries a functional group that could play a role in catalysis, indicating that YbaK is probably not an enzyme. Detailed sequence analysis, including a number of newly sequenced microbial organisms, highlights sequence homology to an insertion domain in prolyl-tRNA synthetases (proRS) from prokaryote, a domain whose function is unknown. A HI1434-based model of the insertion domain shows that it should also contain the putative binding site. Being part of a tRNA synthetases, the insertion domain is likely to be involved in oligonucleotide binding, with possible roles in recognition/discrimination or editing of prolyl-tRNA. By analogy, YbaK may also play a role in nucleotide or oligonucleotide binding, the nature of which is yet to be determined.

Why has porcine VEG protein unusually high stability and suppressed binding ability?

Von Ebner gland protein (VEGP) and odorant-binding protein (OBP) were purified from porcine lingual epithelium and nasal mucosa, respectively. Both VEGP and OBP preparations were homogeneous as indicated by SDS–PAGE, isoelectric focusing, gel-filtration and electrospray mass spectrometry. However, high-sensitivity differential scanning calorimetry (HS-DSC) yielded multiphasic denaturation thermograms for both proteins indicating their conformational heterogeneity. The unfolding transition of VEGP is observed at extremely high temperatures (about 110°C), which is unexpected for a protein with significant structural homology to OBP and other lipocalins. Isothermal titration calorimetry (ITC) did not detect the binding of either aspartame or denatonium saccharide to VEGP nor did it detect binding of 2-isobutyl-3-methoxypyrazine (IBMP) to OBP. Extraction of OBP with mixed organic solvents eliminated the conformational heterogeneity and the protein showed a reversible two-state transition in HS-DSC thereafter. ITC also showed that the extracted OBP was able to bind IBMP. These results imply that tightly bound endogenous ligands increase the thermal stability of OBP and block the binding of other ligands. In contrast to OBP, the extraction of VEGP with organic solvents failed to promote binding or to establish thermal homogeneity, most likely because of the irreversible denaturation of VEGP. Thus, the elucidation of the functional behaviour of VEGP is closely related to the exhaustive purging of its endogenous ligands which otherwise very efficiently mask ligand binding sites of this protein.

A C-Terminal Carbohydrate-Binding Domain in the Endothelial Cell Regulatory Protein, Pigpen: New Function for an EWS Family Member

The potential for encoding information in carbohydrate (CHO) structures has long been recognized. Selective CHO-binding proteins known as lectins and the biological events they mediate are well known. However, many lectins were originally discovered for biological activities other than saccharide binding, and only subsequently was it realized that one or more of their key functions were mediated by specific CHO recognition. Our previous observations suggested that the nuclear protein pigpen had an affinity for CHO structures. This would represent a new attribute for proteins of the EWS (Ewing's sarcoma) family, of which pigpen is a member. In this study we demonstrate that a CHO-binding domain resides in the C-terminus of the molecule and can be preferentially inhibited by saccharides, most notably N-acetyl-d-galactosamine (GalNAc) and the GalNAc-containing polysaccharide, chondroitin sulfate. Ligand blotting experiments were subsequently performed with fractionated, [3H]galactose-labeled cells to demonstrate the presence of chondroitin sulfate-inhibitable endogenous CHO ligands for pigpen in endothelial nuclei. Finally, microinjection of polysaccharide competitor into the nucleus of cultured endothelial cells resulted in a loss of pigpen focal accumulations, suggesting that the CHO-binding activity may be instrumental in subcellular localization of the protein. In summary, our results show ligand preference and domain specificity for pigpen's CHO affinity and provide initial evidence for physiological ligands and function. They may also shed new light on the mechanisms of oncogenic transformation involving EWS proteins.

キクイモ塊茎由来レクチンの清酒酵母に対する凝集特性

Lectin isolated from the Helianthustuberosus tuber (tHTA) is a mannose/glucose-binding lectin that has a very similar saccharide binding specificity to lectin isolated from callus (cHTA). In this study, we showed that tHTA had a very similar yeast agglutinating-activity to cHTA at a suitable pH and yeast density. However, we found that tHTA could agglutinate all the sake yeast strains tested and its ability was not effected by trypsintreatment of yeast, on the other hand cHTA could agglutinate some sake yeast strains that had been treated with trypsin that were known to adhere to the surface of air bubbles.

Thermodynamic Studies of Saccharide Binding to Artocarpin, a B-Cell Mitogen, Reveals the Extended Nature of Its Interaction with Mannotriose [3,6-Di-O-(α-d-mannopyranosyl)-d-mannose

The thermodynamics of binding of various saccharides to artocarpin, from Artocarpus integrifolia seeds, a homotetrameric lectin (M(r) 65, 000) with one binding site per subunit, was determined by isothermal titration calorimetry measurements at 280 and 293 K. The binding enthalpies, DeltaH(b), are the same at both temperatures, and the values range from -10.94 to -47.11 kJ mol(-1). The affinities of artocarpin as obtained from isothermal titration calorimetry are in reasonable agreement with the results obtained by enzyme-linked lectin absorbent essay, which is based on the minimum amount of ligand required to inhibit horseradish peroxidase binding to artocarpin in enzyme-linked lectin absorbent essay (Misquith, S., Rani, P. G., and Surolia, A. (1994) J. Biol. Chem. 269, 30393-30401). The interactions are mainly enthalpically driven and exhibit enthalpy-entropy compensation. The order of binding affinity of artocarpin is as follows: mannotriose>Manalpha3Man>GlcNAc(2)Man(3)>MealphaMan>Man>M analpha6Man> Manalpha2Man>MealphaGlc>Glc, i.e. 7>4>2>1.4>1>0.4>0.3>0.24>0.11. The DeltaH for the interaction of Manalpha3Man, Manalpha6Man, and MealphaMan are similar and 20 kJ mol(-1) lower than that of mannotriose. This indicates that, while Manalpha3Man and Manalpha6Man interact with the lectin exclusively through their nonreducing end monosaccharide with the subsites specific for the alpha1,3 and alpha1,6 arms, the mannotriose interacts with the lectin simultaneously through all three of its mannopyranosyl residues. This study thus underscores the distinction in the recognition of this common oligosaccharide motif in comparison with that displayed by other lectins with related specificity.

Interaction between chitosomes and concanavalin A

Abstract Chitosomes, small secretory vesicles with S-value of ca 100 that act as conveyors of proteolytically-activatable chitin synthase (ChS) to its site of action at the cell surface of fungi, display an unusually high affinity for concanavalin A (ConA), if present in a compacted milieu. The outstandingly strong bonding between the lectin and 100 S-ChS established under this condition in all probability results from a superposition of four types of interaction: ‘classical’ lectin recognition, hydrophobic complexation, extended lectin-receptor bridging, and lectin-mediated liposome/liposome aggregation. The following three-step-procedure for the purification of ChS, together with the result of lipo-selective affinity chromatography (AC) of the enzyme on heparin, allowed the identification of a single 60-kDa polypeptide as a UDPGlcNAc transferase with defining enzymic properties of ChS: (i) isolation of gradient-purified chitosomes according to a standard procedure; (ii) ConA-AC of chitosomes to remove non-binding as well as other contaminating proteins that interact with the lectin only at its saccharide binding site; (iii) selective desorption of ChS with digitonin, methyl mannoside or NaCl following dilution of the chitosome-loaded ConA-gel. The results support the notion of a glycoconjugate nature of ChS, provide a means of obtaining homogeneous preparations of ChS for structural and mechanistic analyses as well as for biotechnological applications, throw light on some hitherto unexplained findings encountered using the Scarborough method to label the plasma membrane with ConA, and weaken the experimental basis for the firmly entrenched tenet of the plasma membrane as the only locus operandi of ChS.

X-ray structure of Novamyl, the five-domain "maltogenic" alpha-amylase from Bacillus stearothermophilus: maltose and acarbose complexes at 1.7A resolution.

The three-dimensional structure of the Bacillus stearothermophilus "maltogenic" alpha-amylase, Novamyl, has been determined by X-ray crystallography at a resolution of 1.7 A. Unlike conventional alpha-amylases from glycoside hydrolase family 13, Novamyl exhibits the five-domain structure more usually associated with cyclodextrin glycosyltransferase. Complexes of the enzyme with both maltose and the inhibitor acarbose have been characterized. In the maltose complex, two molecules of maltose are found in the -1 to -2 and +2 to +3 subsites of the active site, with two more on the C and E domains. The C-domain maltose occupies a position identical to one previously observed in the Bacillus circulans CGTase structure [Lawson, C. L., et al. (1994) J. Mol. Biol. 236, 590-600], suggesting that the C-domain plays a genuine biological role in saccharide binding. In the acarbose-maltose complex, the tetrasaccharide inhibitor acarbose is found as an extended hexasaccharide species, bound in the -3 to +3 subsites. The transition state mimicking pseudosaccharide is bound in the -1 subsite of the enzyme in a 2H3 half-chair conformation, as expected. The active site of Novamyl lies in an open gully, fully consistent with its ability to perform internal cleavage via an endo as opposed to an exo activity.

Evolution of chitin-binding proteins in invertebrates.

Analysis of a group of invertebrate proteins, including chitinases and peritrophic matrix proteins, reveals the presence of chitin-binding domains that share significant amino acid sequence similarity. The data suggest that these domains evolved from a common ancestor which may be a protein containing a single chitin-binding domain. The duplication and transposition of this chitin-binding domain may have contributed to the functional diversification of chitin-binding proteins. Sequence comparisons indicated that invertebrate and plant chitin binding domains do not share significant amino acid sequence similarity, suggesting that they are not coancestral. However, both the invertebrate and the plant chitin-binding domains are cysteine-rich and have several highly conserved aromatic residues. In plants, cysteines have been elucidated in maintaining protein folding and aromatic amino acids in interacting with saccharides [Wright HT, Sanddrasegaram G, Wright CS (1991) J Mol Evol 33:283-294]. It is likely that these residues perform similar functions in invertebrates. We propose that the invertebrate and the plant chitin-binding domains share similar mechanisms for folding and saccharide binding and that they evolved by convergent evolution. Furthermore, we propose that the disulfide bonds and aromatic residues are hallmarks for saccharide-binding proteins.

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

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.

FT infrared and Raman investigation of saccharide–phosphatidylcholine interactions using novel structure probes

Conformational consequences of adduct formation between saccharides (trehalose, glucose, raffinose) and sorbitol with dipalmitoylphosphatidylcholine (DPPC) in multibilayers are revealed by relative intensity changes of the band components corresponding to the ν asN(CH 3) 3 and ν sC–N(CH 3) 3 stretching modes of the choline chain terminal and those of the ν CO band. The conformational sensitivity of those modes was demonstrated previously (J. Grdadolnik et al., Chem. Phys. Lipids 65 (1993) 121) and used to demonstrate the effects of stepwise hydration of phosphatidylcholines. The latter are compared with the effects of saccharide binding and found to be qualitatively similar, but not identical. The same is true of the low frequency shifts of the ν asPO 2 − vibration: the shifts due to saccharide binding correspond to the binding of six to seven water molecules per phosphate which is about 20 cm −1 less than the shift caused by full hydration. A particularly interesting finding concerns the appearance of two bands in the ν asPO 2 − region of the DPPC-saccharide adducts. The relative intensities of the two bands (1243 and 1223 cm −1) change on additional hydration; it is the one at 1223 cm −1 that prevails at high hydration levels. Major changes in saccharide conformation are not detectable but minor differences between the DPPC bound and crystal spectra are observed.

Heteroditopic ruthenium(II) bipyridyl receptor with adjacent saccharide and phosphate binding sites

A luminescent ruthenium(II) bipyridyl receptor with pendant boronic acids is synthesized and shown to bind phosphorylated sugars in water. The association constant for fructose-6-phosphate is log K a = 3.1 M −1 at 298 °K. The receptor simultaneously binds saccharide phophate sitive cooperativity.

Fluorimetric studies on saccharide binding to the basic lectin from Artocarpus hirsuta.

The binding of Artocarpus hirsuta lectin to galactose and its derivatives was examined by fluorescence spectroscopy. The intrinsic fluorescence intensity of the lectin was enhanced by 55% upon binding to methyl alpha-galactose without any change in the emission maximum (333 nm). 4-Methyl umbellifery alpha-galactopyranoside showed 100% quenching of its fluorescence intensity upon binding to the lectin without any shift in the emission maximum (373 nm). The association constant for the binding of the above sugars to the lectin decreases with increasing temperature. Methyl group in the alpha anomeric position of galactose enhanced the binding while that in the beta position reduced the binding to the lectin. Solute quenching studies of the lectin using acrylamide, potassium iodide and cesium chloride indicated that the tryptophan residues were fully accessible to the neutral quencher, while only partly accessible to the ionic quenchers.

The mast cell function-associated antigen exhibits saccharide binding capacity.

The mast cell function-associated antigen (MAFA) is a membrane glycoprotein first identified on rat mucosal type mast cells (line RBL-2H3) and known to inhibit the Fc epsilon RI-mediated secretory response. In its extracellular domain, an amino acid stretch homologous to the carbohydrate binding domain of calcium-dependent animal lectins has been found. To investigate its carbohydrate binding capacity, the MAFA has been expressed in the Spodoptera frugiperda insect cell line (Sf9) using the baculovirus expression system. Analysis by flow cytometry and surface labeling with 125I showed that the recombinant MAFA (rMAFA) was expressed as a monomeric and disulfide-linked homodimeric glycoprotein in the membrane of the insect cells, and both forms exhibited the same epitopes as the protein isolated from RBL-2H3 cells. Immunoaffinity-purified rMAFA was then employed for studies of its saccharide binding capacity by using different neoglycans and glycoproteins. The rMAFA was found to bind specifically terminal mannose residues in a Ca(2+)-dependent manner. These results support the notion that the extracellular domain of the MAFA is indeed able to bind ligands, which may be modulatory for the mast cell response.

A Covalent Enzyme-Substrate Adduct in a Mutant Hen Egg White Lysozyme (D52E)

A mutant hen egg white lysozyme, D52E, was designed to correspond to the structure of the mutant T4 lysozyme T26E (Kuroki, R., Weaver, L. H., and Matthews B. W. (1993) Science 262, 2030-2033) to investigate the role of the catalytic residue on the alpha-side of the saccharide in these enzymes. The D52E mutant forms a covalent enzyme-substrate adduct, which was detected by electron ion spray mass spectrometry. X-ray crystallographic analysis showed that the covalent adduct was formed between Glu-52 and the C-1 carbon of the N-acetylglucosamine residue in subsite D of the saccharide binding site. It suggests that the catalytic mechanism of D52E mutant lysozyme proceeds through a covalent enzyme-substrate intermediate indicating a different catalytic mechanism from the wild type hen egg white lysozyme. It was confirmed that the substitution of Asp-52 with Glu is structurally and functionally equivalent to the substitution of Thr-26 with Glu in T4 lysozyme. Although the position of the catalytic residue on the beta-side of the saccharide is quite conserved among hen egg white lysozyme, goose egg white lysozyme, and T4 phage lysozyme, the adaptability of the side chain on the alpha-side of the saccharide is considered to be responsible for the functional variation in their glycosidase and transglycosidase activities.

Energy profile of maltooligosaccharide permeation through maltoporin as derived from the structure and from a statistical analysis of saccharide-protein interactions.

The crystal structure of the maltodextrin-specific porin from Salmonella typhimurium ligated with a maltotrioside at the pore eyelet is known at 2.4 A resolution. The three glucose units assume a conformation close to the natural amylose helix. The pore eyelet fits exactly the cross-section of a maltooligosaccharide chain and thus functions as a constraining orifice. The oligomer permeates the membrane by screwing along the amylose helix through this orifice. Because each glucose glides along the given helix, its interactions can be sampled at any point along the pathway. The interactions are mostly hydrogen bonds, but also contacts to aromatic rings at one side of the pore. We have derived the energy profile of a gliding maltooligosaccharide by following formation and breakage of hydrogen bonds and by assessing the saccharide-aromatics interactions from a statistical analysis of saccharide binding sites in proteins. The resulting profile indicates smooth permeation despite extensive hydrogen bonding at the orifice.