Switchable binding affinity of mannose tethered to collagen peptide by temperature-dependent triple-helix formation.

Novel glycopeptides were created with a view to regulate the bindings of carbohydrates to lectins as a means of controlling biological function. We synthesized glycopeptides containing mannose (Man) tethered to a collagen peptide moiety (MPOG10: -(Pro-Hyp-Gly)10- or MGPP10: -(Gly-Pro-Pro)10-). Circular dichroism spectra showed formation of a triple helical structure for MPOG10, and the melting temperature indicates that MPOG10 forms a more stable triple helical structure than MGPP10 in phosphate buffered saline (PBS). At 25 °C, fluorescence polarization (FP) values of MPOG10 and MGPP10 increased following the addition of concanavalin A (ConA), and the addition of α-methyl-mannose (MeMan) to a mixed solution of each glycopeptide with ConA resulted in a decrease in FP values. These results confirm that the previous increase in FP values observed was caused by ConA binding to Man on MPOG10 or MGPP10. The binding affinity of MPOG10 was higher than that of MGPP10, and the dissociation constant of MPOG10 to ConA was 1.9 × 10(-5) (mol/L). The observed binding of MPOG10 to ConA at 25 °C was reduced at higher temperature (50 °C). Therefore, the enhanced binding affinity of MPOG10 to ConA could be accounted for by formation of a clustered Man moiety triggered by the formation of a more stable triple helical structure of MPOG10 compared with MGPP10.

Carbohydrate immobilized on a dendrimer-coated colloidal gold surface for fabrication of a lectin-sensing device based on localized surface plasmon resonance spectroscopy.

Carbohydrate-mediated functions in biological systems have generated considerable interest in recent years. We have developed a device bearing immobilized carbohydrates on a colloidal gold surface and applied this device to the detection of carbohydrate-binding molecules by using localized surface plasmon resonance (LSPR) spectroscopy. The sensing device was constructed by using cyanuric chloride as an amine-linker between an amino residue of a polyamidoamine (PAMAM) dendrimer-coated colloidal gold surface and the amino residue of a 12-aminododecyl glycoside. After optimizing the construction of the device, we characterized its LSPR-based sensing capability. Binding specificity with lectins and linear range responses were obtained with the device. Our LSPR-based sensing device thus provides a label-free, low-cost detection method for use as a laboratory research tool or in medical glycan arrays.

Immobilization of carbohydrate clusters on a quartz crystal microbalance sensor surface.

The immobilization of carbohydrates on gold surfaces is a prerequisite technology for carbohydrate-related studies, including those of carbohydrate-biomolecule interactions. Glycolipid domains in cell membranes, such as lipid rafts, are thought to play an important role in cell biology through their carbohydrate portions. To understand the recognition of glycolipid domains such as receptors for bacterial toxins and viruses, we immobilized clusters of carbohydrates on a gold surface by using polyamidoamine (PAMAM) dendrimers as a scaffold. The PAMAM dendrimers were adsorbed on the gold-coated surface of a quartz crystal microbalance (QCM) sensor and were observed by means of QCM with dissipation (QCM-D). After adsorption of the PAMAM dendrimers, lysoganglioside-GM(1) and 12-aminododecyl-N-acetylglucosaminide (GlcNAc-C12-NH(2)) were immobilized on the amino groups of PAMAM dendrimers by means of an NH(2) cross-linker. Immobilization of the carbohydrates was confirmed by observation of their specific interaction with anti-ganglioside GM(1) antibody or wheat germ agglutinin (WGA). Surfaces with different GlcNAc-C12-NH(2) cluster sizes and densities were prepared by varying the size of the PAMAM dendrimers or the concentration of GlcNAc-C12-NH(2) immobilized on the dendrimers, respectively. Analysis of the binding between the GlcNAc-C12-NH(2)-immobilized surface and WGA revealed that the size of the PAMAM dendrimers influenced the GlcNAc-C12-NH(2)-WGA interaction, with larger dendrimers resulting in higher WGA binding constants.

Development of lipid A-imprinted polymer hydrogels that selectively recognize lipopolysaccharides.

To remove lipopolysaccharide (LPS) from pure water, we developed polymer hydrogels that selectively recognize LPS. A molecular imprinting technique was used to prepare the polymer hydrogels. We prepared the polymer hydrogels with LPS-binding sites by using acryloyllysine and acryloylphenylalanine as functional monomers and used lipid A as a template because it is the biologically active part of LPS and contains two phosphate groups. Co-existence of n-octane during the polymerization process was highly effective in promoting the formation of LPS-accessible sites on the surface of the hydrogels. Both an electrostatic and a hydrophobic interaction between the lipid A portion of LPS and the recognition site of the imprinted hydrogel are necessary for LPS recognition. The adsorption isotherm of LPS to the lipid A-imprinted hydrogels was Langmuir-type; the saturated adsorption capacity and the adsorption constant, calculated by applying an equation for Langmuir-type adsorption isotherms, were 1.0 × 10(-11)mol/cm(2) and 2.5 × 10(5)M(-1), respectively. The imprinted hydrogels selectively recognized toxic LPS in a competition experiment in which two other kinds of LPS with similar chemical structures to that of the LPS of E. coli (toxic LPS) were adsorbed to the lipid A-imprinted hydrogels.

Detection of mutant uromodulin in transgenic mouse harboring a mutant human UMOD gene.

Uromodulin is the most abundant protein secreted in urine, and the mutated form of the uromodulin gene is associated with uromodulin-associated kidney disease (UAKD). Although uromodulin accumulates in the kidney of UAKD patients, it is unclear whether this is the wildtype or mutant form. In this study, we established a liquid chromatography (LC)-mass spectrometry/mass spectrometry (MS/MS)-based method for the detection of uromodulin mutants, using the C148W mutant as a target molecule. Membrane and cytosolic fractions of kidney samples from transgenic (Tg) mice expressing the C148W uromodulin mutant were shown to contain human uromodulin by western blotting, and mutant uromodulin with the C148W mutant sequence was observed by proteomic and selected reaction monitoring analyses. Our LC-MS/MS-based method is therefore useful for detection of mutant uromodulin that is undetectable by western blotting alone.

Fluorescence emission and polarization analyses for evaluating binding of ruthenium metalloglycoclusters to lectins and tetanus toxin C-fragment.

We develop a fluorescent ruthenium metalloglycocluster for use as a powerful molecular probe in evaluating the binding between carbohydrates and lectins by fluorescence emission (FE) and fluorescence polarization (FP) analyses. Changes in the FE and FP of these metalloglycoclusters are measured following the addition of lectin [peanut agglutinin (PNA), Ricinus communis agglutinin 120, Concanavalin A (ConA), or wheat germ agglutinin] or tetanus toxin c-fragment (TCF). After the addition of PNA, the FE spectrum of [Ru(bpy-2Gal)(3)] shows a new emission peak and the FP value of [Ru(bpy-2Gal)(3)] increases. Similarly, the FE spectrum of [Ru(bpy-2Glc)(3)] shows a new emission peak and the FP value increases on addition of ConA. Because other combinations of metalloglycoclusters and lectins show little change, specific binding of galactose to PNA and that of glucose to ConA are confirmed by the FE and FP measurements. Resulting dissociation constants (K(d)) prove that the metalloglycoclusters with highly clustered carbohydrates show higher affinity for the respective lectins than those with less clustered carbohydrates. Furthermore, specific binding of [Ru(bpy-2Gal)(3)] to TCF was confirmed by the FP measurement.

Super-hydrophilic silicone hydrogels with interpenetrating poly(2-methacryloyloxyethyl phosphorylcholine) networks.

We synthesized silicone hydrogels from 2-methacryloyloxyethyl phosphorylcholine (MPC) and bis(trimethylsilyloxy)methylsilylpropyl glycerol methacrylate (SiMA) using two methods: random copolymerization with a small amount of cross-linker (P(SiMA-co-MPC)) and construction of an interpenetration network (IPN) structure composed of cross-linked poly(MPC)(PMPC) chains and cross-linked poly(SiMA)(PSiMA) chains (PSiMA-ipn-PMPC). The polymerization was carried out by photoreaction. The surface hydrophilicity and water absorbability of P(SiMA-co-MPC) increased with an increase in the MPC unit composition. On the other hand, in the case of PSiMA-ipn-PMPC, a super-hydrophilic surface was obtained by the surface enrichment of MPC units. The optical and mechanical properties of PSiMA-ipn-PMPC are suitable for use as a material for preparing contact lenses. In addition, the oxygen permeability of PSiMA-ipn-PMPC remains high because of the PSiMA chains. The MPC units at the surface of the hydrogels reduce protein adsorption effectively. From these results for PSiMA-ipn-PMPC, we confirmed that it has the potential for application to silicone hydrogel contact lenses.

Fluorescence emission and polarization for analyzing binding of ruthenium metalloglycocluster to lectin and tetanus toxin C-fragment.

We have designed and synthesized ruthenium complexes bearing clustered galactose Ru(bpy-2Gal)(3) and glucose Ru(bpy-2Glc)(3). Changes in fluorescence emission (FE) and fluorescence polarization (FP) of the metalloglycoclusters were measured by adding each lectin (peanut agglutinin (PNA), Ricinus communis agglutinin 120 (RCA), concanavalin A (ConA), or wheat germ agglutinin (WGA)) or tetanus toxin c-fragment (TCF). Following the addition of PNA and ConA, the FE spectra of Ru(bpy-2Gal)(3) and Ru(bpy-2Glc)(3) showed new emission peaks, respectively. In addition, Ru(bpy-2Gal)(3) and Ru(bpy-2Glc)(3) exclusively increased the FP values by addition of PNA and ConA. Since other combinations of the metalloglycoclusters and lectin caused little change, specific bindings of galactose to PNA and glucose to ConA were confirmed by the FE and FP measurement. From the FP analyses, the dissociation constants (K(d)) of Ru(bpy-2Gal)(3) to PNA and Ru(bpy-2Glc)(3) to ConA were calculated to be ca. 6.1 x 10(-6) M and 1.8 x 10(-5) M. Furthermore, the FP analyses proved specific binding of Ru(bpy-2Gal)(3) to TCF.

Self-catalyzing functions of DNA.

We already reported that 281 bp DNA was degraded to 5'-dNMP by treatment at 70 degrees C and pH 7.5 for 1 h in the presence of 10 mM Mn ions, and the detailed results are published on Biosci. Biotechnol. Biochem., Vol. 71, 2670-2679 (2007). The degradation was accelerated by 100 mM NaCl. More than 80 bp DNA prepared by PCR using human ZNF 219 cDNA as the template were degraded into 5'- dNMP. Fifty bp DNA prepared by PCR us- ing the synthetic F and R primers of 22 mer was degraded into unknown material besides dNMP. Single-strand 281 b DNA prepared by Strandase (Novagen) was suggested not to be degraded into dNMP but to be degraded into the unknown material. Only double-strand DNA is presumed to be degraded into dNMP, therefore the double-strand structure is considered to be necessary for the degradation into dNMP. Furthermore, the unknown material was found in the ppt. fraction after centrifugation of the reaction mixture in the case of 34mer only G oligomer, while 5'-dGMP were found not to be degraded into any material. The m/z of the unknown material prepared from 34mer only G oligomer was determined to be 266 by LC-TOFMS. The elucidation of the conversion mechanism is under investigation.

Degradation of DNA into 5'-monodeoxyribonucleotides in the presence of Mn(2+) ions.

DNA is known to be aggregated by metal ions including Mn(2+) ions, but analysis of the aggregation process from a chemical viewpoint, which means identification of the product yielded during the process, has not been performed yet. On examination of the kinds of degraded materials that were in the supernatant obtained on centrifugation of a DNA mixture aggregated under conditions of 10 mM Mn(2+) ions ([Mn]/[P] = 46.3) at 70 degrees C for 1 h, the degradation products were found to be dAMP, dCMP, dGMP, and TMP. These dNMPs were purified by HPLC on TSKgel ODS-80Ts and identified by LC-TOF/MS. The degradation activity was lost on pretreatment of the DNA with a phenol-chloroform mixture, and the activity was recovered by pretreatment with a mixture of DMSO and a buffer containing surfactants. Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), and Cd(2+), as transition element metal ions, were effective as to the degradation into dNMP. Mg(2+), Ca(2+), Sr(2+), and Ba(2+), as alkali earth element metal ions, were not effective as to the degradation. Monovalent anions such as Cl(-), CH(3)OO(-), and NO(3)(-) were found to increase the degradation rate. Sixty mug of the 120 mug of the starting DNA in 450 mul was degraded into dNMP on reaction for 1 h in the presence of 100 mM NaCl and 10 mM Mn(2+) ions. In this process, aggregation did not occur, and thus was not considered to be necessary for degradation. The degradation was found not to occur at pH 7.0, and to be very sensitive to pH. The OH(-) ion should have a critical role in cleavage of the phosphodiester linkages in this case. The dNMP obtained in the degradation process was found to be only 5'-NMP, based on the H(1)NMR spectra. This prosess should prove to be a new process for the production of 5'-dNMP in addtion to the exonuclease.

DNA detection system using molecularly imprinted polymer as the gel matrix in electrophoresis.

To develop a simple and inexpensive method for DNA detection, we prepared a molecularly imprinted polymer (MIP) for recognizing a specific double-stranded DNA (dsDNA) sequence and used it in an electrophoretic gel matrix. The MIP gel has many binding sites that are complementary in size, shape, and arrangement of functional groups of the target dsDNA sequence. During MIP gel electrophoresis (MIPGE), migration of the target dsDNA should be hindered by the capture effect of the binding sites in the MIP gel. This was confirmed by observation of deviations from the linear relationship between the migration distances of the DNA standard size markers in the polyacrylamide gel and those in the MIP gel. The migration distances of nontarget dsDNA maintained a linear relationship, however. In addition, the sequence selectivity of dsDNA in this method was investigated by using the Ha-ras gene and its point mutants. Except for A.T to T.A base pair substitution, mutant dsDNA (for example, substitution from A.T to C.G and from G.C to T.A) could be distinguished from the target (wild-type) dsDNA. Although some improvement in A.T (T.A) base pair distinction is still needed, this study is the first to demonstrate detection of a specific dsDNA sequence with MIPs and, as such, opens up a new realm for practical applications of MIPs.

Self-cleavage of DNA in the presence of metal ions.

DNA is well known to be aggregated by metal ions including Mn ions, however, analysis of the aggregation process from a chemical aspect, which means identification of the product yielded during the process, has not been performed yet. On determination of what kinds of degraded materials were in the supernatant obtained on centrifugation of a DNA mixture aggregated under the conditions of 10 mM Mn ions ([Mn]/[P]=46.3) at 70 degrees C for 1 h, dAMP, dCMP, dGMP, and TMP produced through self-cleavage of DNA were found in the water-soluble part. These mononucleotides were purified by HPLC using TSKgel ODS-80Ts, and identified by LC-TOF/MS. The self-cleavage was effectively occurred under the conditions of more than 5 mM Mn ions, a reaction temperature of more than 70 degrees C, a reaction time of more than 30 min, and the use of DNA with a molecular weight of more than 140 bp. The self-cleavage was affected by the molecular size of the DNA.

Detection of a specific DNA sequence by electrophoresis through a molecularly imprinted polymer.

To develop a simple and inexpensive DNA detection method, we prepared a molecularly imprinted polymer (MIP) gel for recognizing a specific double-stranded DNA (dsDNA) target sequence in MIP gel electrophoresis (MIPGE). During MIPGE, migration of the target sequence of dsDNA should be hindered by the capture effect of the binding sites in the MIP gel. This migration hindrance of target dsDNA was determined by plotting the relationship between the migration distance in the MIP gel and that in polyacrylamide gel, commonly used in gel electrophoresis. Using this plot, detection of a target dsDNA from a mixture of different-sized dsDNA fragments was achieved. Moreover, we found the detection method successfully distinguished between a target and its base-pair substitutes. These results suggest that MIPGE could be employed for detection of a target dsDNA sequence.

Potentiometric Cr(VI) selective electrode based on novel ionophore-immobilized PVC membranes.

For the determination of Cr(VI) concentrations with a potentiometric ion-selective electrode (ISE), ionophore-immobilized membranes were prepared by ultraviolet (UV)-induced graft polymerization followed by chemical treatment. Novel ionophores comprising various amine structures were immobilized onto poly(vinyl chloride) (PVC) matrixes, and these were examined to determine Cr(VI) selectively. Of the three ionophores examined in this study, the membranes with N,N,N,N-tetrakis(3-aminopropyl)-1,4-butanediamine (DABAm4) exhibited the highest Cr(VI) ion selectivity in both extraction and potentiometry experiments. The plasticizer in the membrane was optimized as 1.0ml o-nitrophenyl octyl ether (NPOE)/g PVC to form diffusible channels. The potentiometric studies revealed that the performance of DABAm4-immobilized PVC was equivalent to that of mobile ionophores in supported liquid membranes (SLMs). A reproducible response of Cr(VI) was attained within a response time of 1s in the range of 2.16x10(-6) to 0.1M, using the membrane prepared in this study. The selectivity for the Cr(VI) ion against the other interfering ions was compared reasonably between a solvent extraction and potentiometry. The long-term response of the Cr(VI) ISE showed slight deterioration over a continuous operation for 6 months, while the detection limit slightly decreased due to the leaching-out of the plasticizer. The ISE along with the DABAm4 immobilized membrane showed a higher Cr(VI) ion selectivity and more stable response under long-term usage than ISEs with typical SLMs.

Imprinted polymer layer for recognizing double-stranded DNA.

A method of preparing a thin polymer layer able to recognize double-stranded DNA (dsDNA) was developed by using 2-vinyl-4,6-diamino-1,3,5-triazine (VDAT) as a functional monomer for creating a DNA-imprinted polymer. The formation of hydrogen bonds between VDAT and A-T base pairs in dsDNA was confirmed by measuring the effects of VDAT on the melting point and the NMR and CD spectra of dsDNA. An imprinted polymer that can recognize dsDNA of the verotoxin gene was prepared by polymerizing VDAT, acrylamide, a crosslinking agent, and the template verotoxin dsDNA on a silanized glass surface. The specificity of this polymer layer for binding verotoxin dsDNA was investigated by using fluorescent-labelled dsDNAs. The fluorescence intensity of the polymer layer after binding verotoxin dsDNA was twice as high as after binding oligo(dG)-oligo(dC), indicating that verotoxin dsDNA was preferentially bound to the polymer imprinted with verotoxin dsDNA. The kinetics of verotoxin dsDNA binding to the imprinted polymer were analyzed by surface plasmon resonance measurements. The dissociation constant (KD) was low, of the order of 10(-9)M.

Synthetic potential of molluscan sulfatases for the library synthesis of regioselectively O-sulfonated D-galacto-sugars.

The substrate specificities of three molluscan sulfatases (E.C. 3.1.6.1; snail, abalone, and limpet origins) were investigated with assorted p-nitrophenyl (pNP) di-O-sulfonated beta-D-galactopyranosides and beta-lactosides [3,6-SO(3) Gal (1), 3',6'-SO(3) Lac (2), 4, 6SO(3) Gal (3), 2,6-SO(3) Gal (4), 3,4-SO(3) Gal (5), and 3,6-SO(3) GalNAc (6); Ac, acetyl; Gal, galactose; Lac, lactose] together with mono-O-sulfonated beta-D-galactopyranoside [pNP 3SO(3)-Gal (7)] and tri-O-sulfonated alpha-D-galactopyranoside [2,3,6-SO(3)-alpha-Gal (11)]. Some notable differences between the substrate specificity of the three sulfatases were disclosed; snail sulfatase hydrolyzed the 3O- and 2O-sulfo groups of 1 and 4, respectively, to afford 6SO(3) Gal (9) in high yields, while the abalone enzyme did not act on 4. Only the limpet enzyme could cleave the 3O-sulfo groups of 7 to give pNP beta-galactoside. In contrast, every enzyme could utilize 11 as a good substrate to afford a mixture of 6SO(3)-alpha-Gal (13) and 2,6-SO(3) alpha-Gal (12). None of the enzymes could cleave the O-sulfo groups of 5 and 6, which indicates that a primary 6O-sulfo group tends to promote the enzymatic hydrolysis of O-sulfo groups at the secondary positions.

Chemoenzymatic synthesis of the sialyl-alpha-(2-->3')-lactosamine trisaccharide with a 3-aminopropyl group as a spacer at the reducing end.

The trisaccharide, 3-aminopropyl 5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid-(2-->3)-beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside has been synthesized chemoenzymatically for the first time. First, the acceptor, 3-aminopropyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside was synthesized in a conventional chemical manner, and then it was coupled with CMP-sialic acid using alpha-(2-->3)-(N)-sialyltransferase to afford the desired trisaccharide by an enzymatically stereocontrolled manner.

Synthesis of 6'-sulfodisaccharides by beta-N-acetylhexosaminidase-catalyzed transglycosylation.

Presulfated N-acetylglucosaminyl donor (pNP beta-D-6-SO3-GlcNAc) was applied for the synthesis of sulfosugars using the beta-N-acetylhexosaminidase-catalyzed transglycosylation, to afford the critically stereocontrolled sulfodisaccharides carrying the 6-sulfo GlcNAc residue at the non-reducing sides in one step.

Enzymatic degradation behavior of porous silk fibroin sheets.

We investigated the degradation behavior of porous silk fibroin sheets by in vitro enzymatic experiments with alpha-chymotrypsin, collagenase IA, and protease XIV. With 1.0 U/ml protease XIV, 70% of a silk fibroin sheet was degraded within 15 days at 37 degrees C. When the fibroin sheet was exposed to collagenase IA, the amount of Silk II crystalline structure in the sheets decreased slightly, and a small amount of Silk I crystalline structure was formed. When protease XIV was used, almost all Silk II disappeared, but the crystallinity increased overall because the amount of Silk I increased. During digestion with protease XIV, the pore size of the fibroin sheets increased with increasing degradation time, until the sheets finally collapsed and became totally shapeless. The average molecular weight of the products after degradation with the three enzymes followed the order protease XIV < collagenase IA < alpha-chymotrypsin. More than 50% of the products resulting from degradation with protease XIV were free amino acids.

A quartz crystal microbalance method for rapid detection and differentiation of shiga toxins by applying a monoalkyl globobioside as the toxin ligand.

A simple globobiosyl (Gb2) ceramide mimic carrying a monoalkyl chain (C18) was applied for a monolayer Langmuir-Blodgett (L-B) technique to detect Shiga toxins (Stxs) by a quartz crystal microbalance (QCM) method. The artificial glycolipid, synthesized from penta-O-acetyl-D-galactopyranose via a conventional glycosidation pathway, was developed at the air-water surface for the formation of the monolayer film. Then, the film was transferred onto a QCM cell surface modified with alkanethiols. Upon the addition of each of Stx-1 and Stx-2, the decrease of frequency reached saturation within 45 min at a few nanogram order per quartz cell. Binding constants (Ka) estimated for each of Stx-1 and Stx-2 showed little difference between the two toxins. On the other hand, in the presence of an artificial acrylamido Gb2 copolymer as a competitive inhibitor, the two toxins showed a large difference in the binding behavior to the L-B monolayer.