Binding of synthetic peptides by a human monoclonal IgM with an unusual combining site structure

Using X-ray crystallography, a human monoclonal IgM cryoglobulin (Mez) was found to have an unusual combining site topography. Analysis of the unliganded Fv at 2.6 A resolution revealed that the HCDR3 had partitioned the active site into two compartments [Ramsland PA et al. 2000. Mol. Immunol. 37: 295-310]. The two cavities had dimensions and chemical properties that were compatible with the binding of peptides. In this study, libraries of peptides were prepared using solid-phase synthesis. Binding of the intact Mez IgM to these peptides was tested by enzyme-linked immunoassays. Screening of 400 dipeptides revealed that binding was markedly skewed toward amino acids with aromatic side-chains (Phe and Trp), especially when located in the second position. Preferential recognition of aromatic side-chains by Mez IgM was confirmed with larger peptides of three to five residues, but C-terminal positioning was not favored in these peptides. Mez IgM also showed binding propensities for acidic residues (Asp and Glu) as well as several other side-chains with different chemical properties, including His, Pro, Asn and Gln. Mez IgM recognized sets of overlapping octapeptides representing the sequences of the constant domains of human IgG1 heavy chains. These peptides represented similar stretches of polypeptide on the three-dimensional structures of all three constant domains (CH1, CH2 and CH3). Thus, Mez IgM may recognize structurally homologous regions of immunoglobulin domains, which were conserved during the evolution of the immune system.     

Binding site mapping of a photoaffinity-labeled juvenile hormone binding protein.

The juvenile hormone binding protein (JHBP) of larval Manduca sexta was labeled by a photoaffinity analog of JH II and purified by preparative IEF and ion-exchange HPLC. The purified [3H]EHDA-labeled JHBP was selectively cleaved by CNBr and by endoproteinases Lys-C and Glu-C. The radioactive peptides were separated by tricine SDS-PAGE and sequenced after blotting to a PVDF membrane. The sequence revealed that Ala184-Asn226 contained a primary binding site of [3H]EHDA. Furthermore, peptide mapping indicated that Asp1-Glu34 also contained a second covalent attachment site of [3H]EHDA. Labeling of the N-terminal region increased when the photolysis was performed at lower temperature. Since Ala184-Asn226 is predicted to be a hydrophobic beta-sheet region, it may participate in the recognition of lipophilic backbone of JH. Five out of six cysteines are located in these two regions, consistent with a model in which the two binding regions connected by disulfide bridges provide a two-sided binding pocket for juvenile hormone.     

Binding and precipitating activities of Lotus tetragonolobus isolectins with L-fucosyl oligosaccharides. Formation of unique homogeneous cross-linked lattices observed by electron microscopy

We have recently observed that certain asparagine-linked oligosaccharides are multivalent and capable of binding and precipitating with the D-mannose-specific lectin concanavalin A [cf. Bhattacharyya, L., & Brewer, C. F. (1989) Eur. J. Biochem. 178, 721-726] and with a variety of D-galactose-specific lectins [Bhattacharyya, L., Haraldsson, M., & Brewer, C. F. (1988) Biochemistry 27, 1034-1041]. In the present study, we have examined the binding and precipitating activities of a variety of mono- and biantennary L-fucosyl oligosaccharides with three L-fucose-specific isolectins from Lotus tetragonolobus, LTL-A, LTL-B, and LTL-C. The results show that certain difucosyl biantennary oligosaccharides are capable of cross-linking and precipitating with tetrameric isolectins, LTL-A and LTL-C, but not with dimeric isolectin, LTL-B. Quantitative precipitation analyses show that biantennary oligosaccharides containing the Lewis(x) antigen (or type 2 chain of Lewis(a)), Gal beta (1-4)[Fuc alpha (1-3)]GlcNAc, at the nonreducing terminus of each arm are bivalent ligands. However, a biantennary oligosaccharide containing a Lewis(x) determinant on one arm and a type 2 chain of blood group H(O) determinant, Fuc alpha (1-2)Gal beta (1-4)GlcNAc, on the other arm and a monoantennary oligosaccharide containing two fucose residues (analogue of the Lewis(y) antigen) bind but do not precipitate with the isolectins, indicating that the positions and linkage of fucose residues are critical for cross-linking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analyses of N-linked oligosaccharides using a two-dimensional mapping technique

We propose a two-dimensional sugar map method for the simple, reproducible, and sensitive analysis of the structures of N-linked oligosaccharides. The structure of an unknown oligosaccharide can be characterized from its position on the map. The data base for the sugar map is prepared by the use of 113 kinds of standard oligosaccharides, 58 of whose structures have been confirmed by 1H NMR spectroscopy. The present method involves six steps, (i) preparation of oligosaccharides from glycopeptides by N-oligosaccharide glycopeptidase (almond) digestion, (ii) derivatization of the reducing ends of oligosaccharides with a fluorescent reagent, 2-amino-pyridine, by using sodium cyanoborohydride, (iii) separation of oligosaccharide derivatives by high-performance liquid chromatography with an ODS-silica column, (iv) analysis of the size of each separated oligosaccharide on an amide-silica column, (v) plotting of the elution position of a sample on the two-dimensional sugar map obtained for the standard oligosaccharides, and (vi) structural analysis of the oligosaccharides by a combination of sequential exoglycosidase digestion and the steps (iii-v). The present method was applied to the identification of the structures of oligosaccharides in hen ovalbumin. It was found that two unusual oligosaccharides that have not yet been reported exist in ovalbumin.     

Human alpha-N-acetylgalactosaminidase: site occupancy and structure of N-linked oligosaccharides

Human alpha-N-acetylgalactosaminidase (alpha-GalNAc; also known as alpha-galactosidase B) is the lysosomal exoglycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties in glycoconjugates. Mutagenesis studies indicated that the first five (N124, N177, N201, N359, and N385) of the six potential N-glycosylation sites were occupied. Site 3 occupancy was important for enzyme function and stability. Characterization of the N-linked oligosaccharide structures on the secreted enzyme overexpressed in Chinese hamster ovary cells revealed highly heterogeneous structures consisting of complex (approximately 53%), hybrid (approximately 12%), and high mannose-type (approximately 33%) oligosaccharides. The complex structures were mono-, bi-, 2,4-tri-, 2,6-tri-, and tetraantennary, among which the biantennary structures were most predominant (approximately 53%). Approximately 80% of the complex oligo-saccharides had a core-region fucose and 50% of the complex oligosaccharides were sialylated exclusively with alpha-2,3-linked sialic acid residues. The majority of hybrid type oligo-saccharides were GalGlcNAcMan(6)GlcNAc-Fuc(0-1)GlcNAc. Approximately 54% of the hybrid oligosaccharide were phosphorylated and one-third of these structures were further sialylated, the latter representing unique phosphorylated and sialylated structures. Of the high mannose oligosaccharides, Man(5-7)GlcNAc(2) were the predominant species (approximately 90%) and about 50% of the high mannose oligosaccharides were phosphorylated, exclusively as monoesters whose positions were determined. Comparison of the oligosaccharide structures of alpha-GalNAc and alpha-galactosidase A, an evolutionary-related and highly homologous exoglycosidase, indicated that alpha-GalNAc had more completed complex chains, presumably due to differences in enzyme structure/domains, rate of biosynthesis, and/or aggregation of the overexpressed recombinant enzymes.     

Conformational changes induced in a homogeneous anti-type III pneumococcal antibody by oligosaccharides of increasing size.

The circular polarization of luminescence (CPL) emitted by tryptophan residues was used as a sensitive probe for measuring ligand-induced structural changes in a homogeneous type III pneumococcal antibody. A series of oligosaccharide ligands of increasing size derived from type III polysaccharide by partial acid hydrolysis was assayed. Ligand-induced changes in the circular polarization of fluorescence of the antibody were observed for all antigens tested, including tetra-, hexa-, and octasaccharides and a 16-residue oligomer, the largest changes being recorded upon interaction with the intact soluble type III pneumococcal (SIII) polysaccharide. When Fab' or F(ab')2 fragments were used instead of the antibody IgG for binding of SIII polysaccharide, the extent of conformational changes was decreased. This suggests interactions between Fab and Fc portions in the IgG molecule and subsequent conformational changes in Fc part upon antigen binding. Reduction of interchain disulfide bonds abolished the additional spectral changes attributed to the Fc part but not the changes observed in the Fab part, thus suggesting that the presence of the interchain disulfide bond in the hinge region is required for maximal CPL changes to occur. Small monovalent ligands, i.e., the tetra-, hexa-, and octasaccharides, were capable of inducing CPL changes in the Fab part of the antibody molecule as well as CPL changes attributed to the Fc portion. A multivalent ligand containing about 16 sugar residues appears to be the minimal antigenic size required for triggering conformational changes attributed to the Fc part, similar to those seen in the interaction with the whole polysaccharide antigen.     

Binding of quercetin with human serum albumin: a critical spectroscopic study

Flavonols are plant pigments that are ubiquitous in nature. Quercetin (3,3',4',5,7-pentahydroxyflavone) and other related plant flavonols have come into recent prominence because of their usefulness as anticancer, antitumor, anti-AIDS, and other important therapeutic activities of significant potency and low systemic toxicity. Quercetin is intrinsically weakly fluorescent in aqueous solution, showing an emission maximum at approximately 538 nm. Upon binding to human serum albumin (HSA), quercetin undergoes dramatic enhancement in its fluorescence emission intensity, along with the appearance of dual emission behavior, consisting of normal and excited-state proton transfer (ESPT) fluorescence. In addition, the occurrence of a third emitting species has been noted for the first time. This is attributed to a electronic ground-state complex formed in the protein environment. High values of the fluorescence anisotropy (r) are obtained in the presence of HSA for the ESPT tautomer (r = 0.18), as well as the complex species (r = 0.37) of quercetin, indicating that the precursor ground-state molecules for both these emitting species of quercetin molecules are located in the motionally constrained sites of HSA. The steady-state emission data suggest that quercetin binds to two distinct sites in HSA from which the emissions from the normal tautomer and complex species take place. The preliminary results of studies on emission decay kinetics are also reported herein. Studies by far-UV circular dichroism spectroscopy reveal that binding of quercetin induces no significant perturbation in the secondary structure of HSA.     

Binding matrix: a novel approach for binding site recognition

Recognition of protein-DNA binding sites in genomic sequences is a crucial step for discovering biological functions of genomic sequences. Explosive growth in availability of sequence information has resulted in a demand for binding site detection methods with high specificity. The motivation of the work presented here is to address this demand by a systematic approach based on Maximum Likelihood Estimation. A general framework is developed in which a large class of binding site detection methods can be described in a uniform and consistent way. Protein-DNA binding is determined by binding energy, which is an approximately linear function within the space of sequence words. All matrix based binding word detectors can be regarded as different linear classifiers which attempt to estimate the linear separation implied by the binding energy function. The standard approaches of consensus sequences and profile matrices are described using this framework. A maximum likelihood approach for determining this linear separation leads to a novel matrix type, called the binding matrix. The binding matrix is the most specific matrix based classifier which is consistent with the input set of known binding words. It achieves significant improvements in specificity compared to other matrices. This is demonstrated using 95 sets of experimentally determined binding words provided by the TRANSFAC database.     

Two-dimensional mapping by the high-performance liquid chromatography of oligosaccharides released from glycosphingolipids by endoglycoceramidase

A two-dimensional sugar mapping method has been developed by which sensitive, reproducible, and simple analysis can be carried out on the structures and compositions of oligosaccharides released from glycosphingolipids by endoglycoceramidase. The oligosaccharides were labeled quantitatively with an ultraviolet-absorbing compound, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-oligosaccharides were separated first on an amide-silica column and then on a C4-silica column by high-performance liquid chromatography. The acidic ABEE-oligosaccharides were eluted as a group at the start of the chromatography while the neutral ABEE-oligosaccharides were separated according to size and structure on an amide-silica column using an eluent without salt. The acidic oligosaccharides were separated according to size and structure when rechromatographed on the same column using an eluent containing KH2PO4. NeuAc-containing ABEE-oligosaccharides were extensively separated from the corresponding NeuGc derivatives. The ABEE-oligosaccharides separated on an amide-silica column were then chromatographed on a column of C4-silica on which lactotriose and neolacto-series oligosaccharides were clearly shown to be separated from the others. On the basis of the retention times of the individual ABEE-oligosaccharides on two separate columns, 9 neutral and 15 acidic oligosaccharides derived from glycosphingolipid standards were two-dimensionally mapped without overlapping. The gangliosides of a human chondrosarcoma tissue and glycosphingolipids of tumor tissue of FBJ virus-transformed murine osteosarcoma cells were analyzed by this method in conjunction with exoglycosidase treatment. At least 11 species of glycosphingolipids were identified in both cases.     

Binding of milk oligosaccharides by several enterotoxigenic Escherichia coli strains isolated from calves

Milk oligosaccharides have been proposed to play an important role in newborn defense, blocking bacterial adhesion to the intestinal mucosa and preventing infections. Some studies have been performed on human milk oligosaccharides. Here we checked whether bovine milk oligosaccharides would achieve the same protective action against the most common calf enteric pathogens. Seven enterotoxigenic Escherichia coli strains, isolated from diarrheic calves, were selected. All strains managed to agglutinate horse erythrocytes, and we therefore used the inhibition of hemagglutination in the presence of oligosaccharides as an indicator of the union between oligosaccharide and bacterial adhesins. Oligosaccharides from different stages of bovine lactation and standard oligosaccharides were assayed. Midlactation milk, in particular that corresponding to the transition period, proved to be the most efficient at inhibiting hemagglutination. The standard oligosaccharides used pointed to the preference of several strains (K99-, F41-, and F17-fimbriated) for 2,6-linked sialic acid. By contrast, B23 fimbriae exhibited higher affinity for 2,3-sialylated isomers and B64 seemed to require N-acetylglucosamine for binding.Our results suggest a general trend for milk oligosaccharides. Probably they participate in the protection of newborn mammals from pathogens.     

Binding of berberine to bovine serum albumin: spectroscopic approach

Fluorescence spectroscopy in combination with UV–vis absorption spectroscopy was employed to investigate the binding of an important traditional medicinal herb berberine to bovine serum albumin (BSA) under the physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of BSA by berberine is a result of the formation of berberine–BSA complex. Fluorescence quenching constants were determined using the Stern–Volmer equation and Scatchard equation to provide a measure of the binding affinity between berberine and BSA. The results of thermodynamic parameters ΔG, ΔH, ΔS at different temperatures indicate that the electrostatic interactions play a major role for berberine–BSA association. Site marker competitive experiments indicated that the binding of berberine to BSA primarily took place in site II. Furthermore, the Effect of supramolecules to berberine–BSA system, and the distance r between donor (BSA) and acceptor (berberine) was obtained according to fluorescence resonance energy transfer (FRET).     

Binding and catalytic contributions to site recognition by flp recombinase

Flp catalyzes site-specific recombination in a highly sequence-specific manner despite making few direct contacts to the bases within its binding site. Sequence discrimination could take place in the binding and/or the catalytic steps. In this study, we independently measure the binding affinity and initial cleavage rate of Flp recombinase with approximately 20 designed alternate target DNA sequences. Our results show that Flp specificity is largely, although not entirely, imparted at the binding step and is the result of a combination of direct and indirect readout. The Flp binding site includes an A/T-rich region that displays a characteristically narrow minor groove. We find that many A --> T changes are tolerated at the binding step, whereas C or G substitutions tend to decrease binding affinity. The effects of the latter can be alleviated by replacing guanine with inosine, which removes the N2 amino group that protrudes into the minor groove. Some A --> T changes reduce binding affinity, due to clashing with nearby residues, reinforcing that specificity requires avoiding negative contacts as well as creating positive ones. A tracts, which can lead to unusually rigid DNA structure, are tolerated during the binding step when placed within the region where the minor groove is already narrow. However, most A tracts slow catalysis more than C or G substitutions. Understanding what kind of sequence variation is tolerated in the binding and catalytic steps helps us understand how the target DNA is recognized by Flp and will be useful in guiding the design of Flp variants with altered specificities.     

A spectroscopic and thermodynamic study of Taxol nucleic acid complexes

The interactions of natural and synthetic polynucleotide double strands with the antitumor agent paclitaxel and the oncological product "Taxol for Injection Concentrate" (abbreviated as taxol) were examined in diluted aqueous solutions by thermal denaturation profiles (Tm), CD spectra and UV-absorption measurements. Furthermore, DNA-paclitaxel and -taxol complexes in condensed nucleic acid solutions were studied by differential scanning calorimetry. As polynucleotides alternating and homologous poly[d(AT)] and poly[d(GC)] and calf thymus DNA were used. The results point to stabilizing interactions of paclitaxel to AT nucleotides, whereas in the presence of GC base pairings no interaction took place. Thereby the interaction to homologous (dA).(dT)-tracts seems to be preferred.     

Binding of lipophilic cations to the liposomal membrane: thermodynamic analysis

Lipophilic ions are widely used as probes for measuring membrane potentials. Since binding of the probes to the membrane interferes with the accurate estimation of the membrane potential, it is necessary to clarify the characteristics of probe binding to membranes. The present paper deals with the binding of lipophilic cations to liposomes. The results can be summarized as follows: (1) The binding of triphenylmethylphosphonium, its homologues and tetraphenylphosphonium to liposomes of dipalmitoylphosphatidylcholine followed the Langmuir adsorption isotherm. (2) Spin-labeled lipophilic cations were synthesized and the binding to liposomes of egg phosphatidylcholine was examined. The binding also followed the Langmuir adsorption isotherm. The dissociation constant (the concentration giving half-maximal binding), K, was independent of the temperature, indicating that the binding is entropy-driven. (3) The binding was influenced by the fluidity of the membrane. Except in the case of triphenylmethylphosphonium (TPMP+), K and A (maximum amounts of binding) increased above the transition temperature. In other words, above the phase transition temperature the binding affinity is decreased, while maximum amounts of binding are increased for all phosphoniums used except TPMP+.     

Binding properties of palmatine to DNA: spectroscopic and molecular modeling investigations

Palmatine, an isoquinoline alkaloid, is an important medicinal herbal extract with diverse pharmacological and biological properties. In this work, spectroscopic and molecular modeling approaches were employed to reveal the interaction between palmatine and DNA isolated from herring sperm. The absorption spectra and iodide quenching results indicated that groove binding was the main binding mode of palmatine to DNA. Fluorescence studies indicated that the binding constant (K) of palmatine and DNA was ~ 10⁴ L·mol^?1. The associated thermodynamic parameters, ΔG, ΔH, and ΔS, indicated that hydrogen bonds and van der Waals forces played major roles in the interaction. The effects of chemical denaturant, thermal denaturation and pH on the interaction were investigated and provided further support for the groove binding mode. In addition to experimental approaches, molecular modeling was conducted to verify binding pattern of palmatine–DNA. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of base stacking on the relative thermodynamic stability of oligonucleotide complexes: a spectroscopic study

Three-strand oligonucleotide complexes are employed to assess the effect of base stacking and base pair mismatch on the relative thermodynamic stabilities of oligonucleotide duplexes. The melting behavior of three-strand oligonucleotide complexes incorporating nicks and gaps as well as internal single base mismatches is monitored using temperature-dependent optical absorption spectroscopy. A sequential three-state equilibrium model is used to analyze the measured melting profiles and evaluate thermodynamic parameters associated with dissociation of the complexes. The free-energy of stabilization of a nick complex compared to a gap complex due to base stacking is determined to be -1.9 kcal/mol. The influence of a mispaired base in these systems is shown to destabilize a nick complex by 3.1 kcal/mol and a gap complex by 2.8 kcal/mol, respectively.