Vacuum-Ultraviolet Circular Dichroism Analysis of Glycosaminoglycans by Synchrotron-Radiation Spectroscopy

Matsuo, Koichi; Namatame, H.; Takeuchi, Masaki; Gekko, Kunihiko

doi:10.1271/bbb.80605

Cited by 23 publications

(19 citation statements)

References 25 publications

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“…This difference is unlikely to be contributed directly by the sugar moiety given the typically low mean residue ellipticity of glycans. 38,39 FGGn-Skp1 was indistinguishable from Gn-Skp1, whereas fully modified GGFGGn-Skp1 exhibited intermediate values also correlated with an increased level of Fbs1 binding. Secondary structure deconvolution analysis indicated that addition of α-GlcNAc results in greater α-helical content, at the partial expense of β-sheet content (Figure 3B).…”

Section: Resultsmentioning

confidence: 90%

Glycosylation of Skp1 Affects Its Conformation and Promotes Binding to a Model F-Box Protein

et al. 2014

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In the social amoeba Dictyostelium, Skp1 is hydroxylated on proline 143 and further modified by three cytosolic glycosyltransferases to yield an O-linked pentasaccharide that contributes to O2 regulation of development. Skp1 is an adapter in the Skp1/cullin1/F-box protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent proteasomal degradation. To investigate the biochemical consequences of glycosylation, untagged full-length Skp1 and several of its posttranslationally modified isoforms were expressed and purified to near homogeneity using recombinant and in vitro strategies. Interaction studies with the soluble mammalian F-box protein Fbs1/Fbg1/OCP1 revealed preferential binding to the glycosylated isoforms of Skp1. This difference correlated with the increased α-helical and decreased β-sheet content of glycosylated Skp1s based on circular dichroism and increased folding order based on small-angle X-ray scattering. A comparison of the molecular envelopes of fully glycosylated Skp1 and the apoprotein indicated that both isoforms exist as an antiparallel dimer that is more compact and extended in the glycosylated state. Analytical gel filtration and chemical cross-linking studies showed a growing tendency of less modified isoforms to dimerize. Considering that regions of free Skp1 are intrinsically disordered and Skp1 can adopt distinct folds when bound to F-box proteins, we propose that glycosylation, which occurs adjacent to the F-box binding site, influences the spectrum of energetically similar conformations that vary inversely in their propensity to dock with Fbs1 or another Skp1. Glycosylation may thus influence Skp1 function by modulating F-box protein binding in cells.

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Section: Resultsmentioning

confidence: 90%

Glycosylation of Skp1 Affects Its Conformation and Promotes Binding to a Model F-Box Protein

et al. 2014

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“…This is thought to be associated with the formation of supramolecular helical assembly of 1 upon electrostatic binding to the substrates [2b] because 1 is intrinsically achiral and the CD signals for these substrates occur at wavelengths shorter than 230 nm. [15] As compared to heparin, a more significant CD enhancement and a different CD pattern could be obtained for the corresponding concentration of ChS, while no similar CD enhancement was observed within the wavelength region of study upon addition of the same concentration of HA. Such CD results suggest that this sensing strategy could clearly discriminate heparin from ChS and HA, and the differences in the spectroscopic properties are not only governed by the distribution of the negatively charged functionalities on the polysaccharides, but also by the helical conformation of the polysaccharide-complex mixture itself.…”

mentioning

confidence: 82%

NIR‐Emissive Alkynylplatinum(II) Terpyridyl Complex as a Turn‐On Selective Probe for Heparin Quantification by Induced Helical Self‐Assembly Behaviour

Yeung

Yam

2011

Chemistry A European J

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Platinum(II) polypyridyl complexes, adopting squareplanar geometry, have exhibited interesting spectroscopic and luminescence properties, dependent on the extent of d 8 -d 8 metal-metal interaction and p-p stacking of the polypyridyl ligand. [1, 2] With the utilisation of the self-assembly and disassembly processes through such Pt···Pt and p-p interactions of the alkynylplatinum(II) terpyridyl complexes, induced by the multiple negatively charged synthetic polyelectrolytes or biopolymers, recent work by our group has demonstrated the differentiation of various single-stranded nucleic acids, [2b] detection of the conformational changes in G-quadruplexes, [2e, f] probing of enzymatic activities, [2e, g] and the quantification of important proteins and sugars [2f, g] in aqueous media by monitoring the UV/Vis absorption, nearinfrared (NIR) emission and circular dichroism (CD) spectral changes. Such NIR emission and large Stokes shift would make this label-free sensing protocol desirable as the problems of autofluorescence and light scattering arising from the biological environment could be effectively avoided. [3] Heparin, a linear unbranched polydispersed polysaccharide consisting of repeating disaccharide units of iduronic acid and glucosamine (Scheme 1), is prevalent and possesses one of the highest negative charge densities amongst the biological macromolecules due to the presence of numerous sulfate and carboxylate groups.[4] Most importantly, heparin is pharmacologically functional as an anticoagulant because of its capability to accelerate the antithrombin III (ATIII)-mediated inactivation of serine proteases in the coagulation cascade upon its electrostatic interaction with ATIII through a specific pentasaccharide sequence.[4-6] Unfractionated heparin (UFH) and the chemically or enzymatically fractionated low-molecular-weight heparin (LMWH) are the two forms of heparin in clinical use. In order to prevent the catastrophic hemorrhagic complications and heparin-induced thrombocytopenia (HIT) arising from over usage, [5,7] the control of heparin dosage levels in surgery and postoperative treatment is of pivotal importance. Therefore, various traditional clinical heparin administration assays, such as activated partial thromboplastin time (aPTT), have been established, yet these indirect methods are not accurate and suffer from potential interference from other factors.[8] Recently, a list of fluorescent, colorimetric and electrochemical methods [9] have been designed for the quantification of heparin, of which only few can measure both UFH and LMWH within the clinical range and effectively differentiate heparin from structurally similar analogues as well.[9e] Moreover, striking clinical evidence has revealed the prominent inhibiting effect of heparin treatment on tumour metastasis, [4,10] and thus there is a strong demand for the design of selective and practical detection techniques.With the utilisation of such high anionic charge density, we herein demonstrate the heparin-induced self-assembly of an N...

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“…Figure 6c shows the VUVCD spectra of three N-acetylaminosugars (N-acetylglucosamine, Nacetylgalactosamine, and N-acetylgalactosamine 4-sulfate), and of glucuronic acid down to 160 (pH 2.4) and 185 nm (pH 6.7), with the spectra of D-glucose and D-galactose included for comparison. 23 It is evident that the VUVCD spectra of GAGs and their component sugars sensitively reflect the differences in their structures, with them being highly self-similar ( Figure 6a). The differences in the CD spectra are larger in the VUV region than in the far-UV region, indicating that VUVCD spectroscopy provides much more information about the structures of GAGs in aqueous solution.…”

Section: Structural Analysis Of Saccharidesmentioning

confidence: 99%

Construction of a Synchrotron-Radiation Vacuum-Ultraviolet Circular-Dichroism Spectrophotometer and Its Application to the Structural Analysis of Biomolecules

Matsuo

Gekko

2013

Bulletin of the Chemical Society of Japan

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A vacuum-ultraviolet (VUV) circular-dichroism (CD) spectrophotometer usable down to 140 nm was constructed using synchrotron radiation and applied to the structural analyses of various saccharides, amino acids, and proteins in aqueous solutions. Most monosaccharides and disaccharides exhibited a positive CD peak around 170 nm, depending on anomeric and axial/equatorial configurations of hydroxy groups, transgauche configurations of hydroxymethyl groups, and the type of glycosidic linkage. The VUVCD spectra of glycosaminoglycans sensitively reflected the characteristic contributions of constituent functional groups in the VUV region. L-Isomers of amino acids showed two successive positive peaks around 200 and 180 nm, depending on the types of side chains. The VUVCD spectrum of alanine theoretically calculated using a time-dependent density functional theory revealed the important role of hydration for stabilizing the alanine structure. The VUVCD spectra of native proteins down to 160 nm improved the predictive accuracy for the contents, numbers of segments, and sequences of ¡-helices and ¢-strands. These secondary-structure analyses were also successfully applied to various types of nonnative proteins. The obtained results demonstrate that synchrotronradiation VUVCD spectroscopy is a powerful technique for the structural analysis of biomolecules in aqueous solution, and hence could open a new field in structural biology.

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Vacuum-Ultraviolet Circular Dichroism Analysis of Glycosaminoglycans by Synchrotron-Radiation Spectroscopy

Cited by 23 publications

References 25 publications

Glycosylation of Skp1 Affects Its Conformation and Promotes Binding to a Model F-Box Protein

Glycosylation of Skp1 Affects Its Conformation and Promotes Binding to a Model F-Box Protein

NIR‐Emissive Alkynylplatinum(II) Terpyridyl Complex as a Turn‐On Selective Probe for Heparin Quantification by Induced Helical Self‐Assembly Behaviour

Construction of a Synchrotron-Radiation Vacuum-Ultraviolet Circular-Dichroism Spectrophotometer and Its Application to the Structural Analysis of Biomolecules

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