The Experts below are selected from a list of 1476 Experts worldwide ranked by ideXlab platform
Robert J. Linhardt - One of the best experts on this subject based on the ideXlab platform.
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antithrombin iii binding site analysis of low molecular weight heparin fractions
Journal of Pharmaceutical Sciences, 2018Co-Authors: Yin Chen, Fuming Zhang, Jing Zhao, Yanlei Yu, Robert J. LinhardtAbstract:Abstract Low-molecular-weight heparins (LMWHs) are widely used as clinical anticoagulant drugs. LMWHs are heterogeneous and highly negatively charged glycans prepared by chemical or enzymatic depolymerization of unfractionated heparin. The detailed structural analysis of a LMWH is essential for the drug quality control. In this study, an LMWH, enoxaparin sodium (a generic version of Lovenox) was separated into different molecular weight fractions by a Superdex peptide column. The disaccharide compositions, 3- O -Sulfo Group–containing tetrasaccharides composition, and antithrombin III-binding affinity of the fractions from this LMWH were analyzed. The results showed that all the fractions had very similar disaccharide and 3- O -Sulfo Group–containing tetrasaccharide compositions, but the fraction containing larger-sized chains had higher antithrombin III-binding affinity.
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Glycan Determinants of Heparin-Tau Interaction
Biophysical Journal, 2017Co-Authors: Jing Zhao, Robert J. Linhardt, Isabelle Huvent, Guy Lippens, David Eliezer, Anqiang Q. Zhang, Peter Tessier, Fuming M. Zhang, Chunyu Y. WangAbstract:Tau aggregates into paired helical filaments within neurons, a pathological hallmark of Alzheimer's disease. Heparin promotes tau aggregation and recently has been shown to be involved in the cellular uptake of tau aggregates. Although the tau-heparin interaction has been extensively studied, little is known about the glycan determinants of this interaction. Here, we used surface plasmon resonance (SPR) and NMR spectroscopy to characterize the interaction between two tau fragments, K18 and K19, and several polysaccharides, including heparin, heparin oligosaccharides, chemically modified heparin, and related glycans. Using a heparin-immobilized chip, SPR revealed that tau K18 and K19 bind heparin with a K-D of 0.2 and 70 mu M, respectively. In SPR competition experiments, N-desulfation and 2-O-desulfation had no effect on heparin binding to K18, whereas 6-O-desulfation severely reduced binding, suggesting a critical role for 6-O-sulfation in the tau-heparin interaction. The tau-heparin interaction became stronger with longer-chain heparin oligosaccharides. As expected for an electrostatics-driven interaction, a moderate amount of salt (0.3 M NaCl) abolished binding. NMR showed the largest chemical-shift perturbation (CSP) in R2 in tau K18, which was absent in K19, revealing differential binding sites in K18 and K19 to heparin. Dermatan sulfate binding produced minimal CSP, whereas dermatan disulfate, with the additional 6-O-Sulfo Group, induced much larger CSP. 2-O-desulfated heparin induced much larger CSP in K18 than 6-O-desulfated heparin. Our data demonstrate a crucial role for the 6-O-Sulfo Group in the tau-heparin interaction, which to our knowledge has not been reported before.
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investigating changes in the gas phase conformation of antithrombin iii upon binding of arixtra using traveling wave ion mobility spectrometry twims
Analyst, 2015Co-Authors: Yuejie Zhao, Robert J. Linhardt, Arunima Singh, Lingyun Li, Yongmei Xu, Robert J Woods, Jonathan I AmsterAbstract:We validate the utility of ion mobility to measure protein conformational changes induced by the binding of glycosaminoglycan ligands, using the well characterized system of Antithrombin III (ATIII) and Arixtra, a pharmaceutical agent with heparin (Hp) activity. Heparin has been used as a therapeutic anticoagulant drug for several decades through its interaction with ATIII, a serine protease inhibitor that plays a central role in the blood coagulation cascade. This interaction induces conformational changes within ATIII that dramatically enhance the ATIII-mediated inhibition rate. Arixtra is the smallest synthetic Hp containing the specific pentasaccharide sequence required to bind with ATIII. Here we report the first travelling wave ion mobility mass spectrometry (TWIMS) investigation of the conformational changes in ATIII induced by its interaction with Arixtra. Native electrospray ionization mass spectrometry allowed the gentle transfer of the native topology of ATIII and ATIII–Arixtra complex. IM measurements of ATIII and ATIII–Arixtra complex showed a single structure, with well-defined collisional cross section (CCS) values. An average 3.6% increase in CCS of ATIII occurred as a result of its interaction with Arixtra, which agrees closely with the theoretical estimation of the change in CCS based on protein crystal structures. A comparison of the binding behavior of ATIII under both denaturing and non-denaturing conditions confirmed the significance of a folded tertiary structure of ATIII for its biological activity. A Hp oligosaccharide whose structure is similar to Arixtra but missing the 3-O Sulfo Group on the central glucosamine residue showed a dramatic decrease in binding affinity towards ATIII, but no change in the mobility behavior of the complex, consistent with prior studies that suggested that 3-O sulfation affects the equilibrium constant for binding to ATIII, but not the mode of interaction. In contrast, nonspecific binding by a Hp tetrasaccharide showed more complex mobility behavior, suggesting more promiscuous interactions with ATIII. The effect of collisional activation of ATIII and ATIII–Arixtra complex were also assessed, revealing that the binding of Arixtra provided ATIII with additional stability against unfolding. Overall, our results validate the capability of TWIMS to retain the significant features of the solution structure of a protein–carbohydrate complex so that it can be used to study protein conformational changes induced by the binding of glycosaminoglycan ligands.
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Analysis of 3-O-Sulfo Group-containing heparin tetrasaccharides in heparin by liquid chromatography-mass spectrometry.
Analytical biochemistry, 2014Co-Authors: Bo Yang, Fuming Zhang, Changhu Xue, Robert J. LinhardtAbstract:Abstract Complete heparin digestion with heparin lyase 2 affords a mixture of disaccharides and resistant tetrasaccharides with 3- O -Sulfo Group-containing glucosamine residues at their reducing ends. Quantitative online liquid chromatography–mass spectrometric analysis of these resistant tetrasaccharides is described in this article. The disaccharide and tetrasaccharide compositions of seven porcine intestinal heparins and five low-molecular-weight heparins were analyzed by this method. These resistant tetrasaccharides account for from 5.3 to 7.3 wt% of heparin and from 6.2 to 8.3 wt% of low-molecular-weight heparin. Because these tetrasaccharides are derived from heparin’s antithrombin III-binding sites, we examined whether this method could be applied to estimate the anticoagulant activity of heparin. The content of 3- O -Sulfo Group-containing tetrasaccharides in a heparin correlated positively ( r = 0.8294) to heparin’s anticoagulant activity.
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Characterization of interactions between heparin/glycosaminoglycan and adeno-associated virus.
Biochemistry, 2013Co-Authors: Fuming Zhang, J. Javier Aguilera, Julie M. Beaudet, Qing Xie, Thomas F. Lerch, Omar Davulcu, Wilfredo Colón, Michael S. Chapman, Robert J. LinhardtAbstract:Adeno-associated virus (AAV) is a key candidate in the development of gene therapy. In this work, we used surface plasmon resonance spectroscopy to study the interaction between AAV and heparin and other glycosaminoglycans (GAGs). Surface plasmon resonance results revealed that heparin binds to AAV with an extremely high affinity. Solution competition studies showed that binding of AAV to heparin is chain length-dependent. AAV prefers to bind full chain heparin. All Sulfo Groups (especially N-Sulfo and 6-O-Sulfo Groups) on heparin are important for the AAV–heparin interaction. Higher levels of Sulfo Group substitution in GAGs enhance their binding affinities. Atomic force microscopy was also performed to image AAV-2 in a complex with heparin.
Yunhui Dong - One of the best experts on this subject based on the ideXlab platform.
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The Label-Free Immunosensor Based on rhodium@palladium nanodendrites/Sulfo Group Functionalized Multi-Walled Carbon Nanotubes for the Sensitive Analysis of Carcino Embryonic Antigen
Analytica chimica acta, 2017Co-Authors: Xiaobo Zhang, Ping Wang, Zengqiang Gao, Jinhui Feng, Yunhui DongAbstract:Abstract In this work, bimetallic core-shell rhodium@palladium nanodendrites (Rh@Pd NDs) loaded on Sulfo Group functionalized multi-walled carbon nanotubes (MWCNTs-SO3H) were combined to form Rh@Pd NDs/MWCNTs-SO3H nanocomposites. And the composites were used to construct a simple and label-free electrochemical immunosensor for carcino embryonic antigen (CEA) detection using differential pulse voltammetry (DPV). Rh@Pd NDs with dendritic nanostructure not only provide abundant catalytically active sites, but also increase the loading of antibody, which could improve the analytical performance and result in high sensitivity. In addition, the MWCNTs-SO3H could further enhance electrochemical properties due to the excellent conductivity, good solubility and high surface area. Taking advantages of both Rh@Pd NDs and MWCNTs-SO3H, the proposed immunosensor showed a broad linear range from 25 fg mL−1 to 100 ng mL−1 for CEA detection and a low detection limit of 8.3 fg mL−1 (signal-to-noise ratio of 3) under optimal experimental conditions. Moreover, the expected immunosensor exhibited good reproducibility and high sensitivity, which could achieve excellent analysis of CEA in human serum with satisfactory results. Therefore, the Rh@Pd NDs/MWCNTs-SO3H nanocomposites may be considered as a sensing platform for fabrication of simple, ultrasensitive and label-free electrochemical immunosensor.
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the label free immunosensor based on rhodium palladium nanodendrites Sulfo Group functionalized multi walled carbon nanotubes for the sensitive analysis of carcino embryonic antigen
Analytica Chimica Acta, 2017Co-Authors: Xiaobo Zhang, Ping Wang, Zengqiang Gao, Jinhui Feng, Yunhui DongAbstract:Abstract In this work, bimetallic core-shell rhodium@palladium nanodendrites (Rh@Pd NDs) loaded on Sulfo Group functionalized multi-walled carbon nanotubes (MWCNTs-SO3H) were combined to form Rh@Pd NDs/MWCNTs-SO3H nanocomposites. And the composites were used to construct a simple and label-free electrochemical immunosensor for carcino embryonic antigen (CEA) detection using differential pulse voltammetry (DPV). Rh@Pd NDs with dendritic nanostructure not only provide abundant catalytically active sites, but also increase the loading of antibody, which could improve the analytical performance and result in high sensitivity. In addition, the MWCNTs-SO3H could further enhance electrochemical properties due to the excellent conductivity, good solubility and high surface area. Taking advantages of both Rh@Pd NDs and MWCNTs-SO3H, the proposed immunosensor showed a broad linear range from 25 fg mL−1 to 100 ng mL−1 for CEA detection and a low detection limit of 8.3 fg mL−1 (signal-to-noise ratio of 3) under optimal experimental conditions. Moreover, the expected immunosensor exhibited good reproducibility and high sensitivity, which could achieve excellent analysis of CEA in human serum with satisfactory results. Therefore, the Rh@Pd NDs/MWCNTs-SO3H nanocomposites may be considered as a sensing platform for fabrication of simple, ultrasensitive and label-free electrochemical immunosensor.
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an ultrasensitive sandwich type electrochemical immunosensor based on the signal amplification strategy of mesoporous core shell pd pt nanoparticles amino Group functionalized graphene nanocomposite
Biosensors and Bioelectronics, 2017Co-Authors: Mingdang Li, Faying Li, Yueyun Li, Ping Wang, Yunhui DongAbstract:Abstract Herein, a novel and sensitive sandwich-type electrochemical immunosensor was fabricated for quantitative monitoring of prostate specific antigen (PSA). The Sulfo Group functionalized multi-walled carbon nanotubes (MWCNTs-SO 3 H) were used as substrate material to increase the specific surface area and enhance the conductivity of the glassy carbon electrode. Gold nanoparticles (Au NPs) were introduced to enhance the load capacity of the substrate material for primary antibodies (Ab 1 ) and accelerate the electron transfer on the electrode interface. The mesoporous core-shell Pd@Pt nanoparticle loaded by amino Group functionalized graphene (M-Pd@Pt/NH 2 -GS) with high specific surface area, high indexed facets, and good biocompatibility was not only as the carriers of secondary antibodies (Ab 2 ) but also catalyzed the reduction of hydrogen peroxide (H 2 O 2 ), which effectually amplified the current signal in detection of PSA. The as-proposed immunosensor exhibited high sensitivity and stability on the detection of PSA. A linear relationship between current signals and the concentrations of PSA was obtained in the range from 10 fg/mL to 50 ng/mL and the detection limit of PSA was 3.3 fg/mL (signal-to-noise ratio of 3). Furthermore, the as-proposed immunosensor showed excellent performance in detection of human serum samples. The results suggest that the proposed immunosensor will be promising in the diagnostics application for accurately quantitative detection of PSA.
Jian Liu - One of the best experts on this subject based on the ideXlab platform.
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structure based substrate specificity analysis of heparan sulfate 6 o Sulfotransferases
ACS Chemical Biology, 2017Co-Authors: Andrea F Moon, Jian Liu, Juno M Krahn, Lars C. PedersenAbstract:Heparan sulfate (HS) is a sulfated polysaccharide exhibiting essential physiological functions. HS 6-O-Sulfotransferase (6-OST) transfers a Sulfo Group to the 6-OH position of glucosamine units to confer a variety of HS biological activities. There are three different isoforms of 6-OST in the human genome. Here, we report crystal structures of the ternary complex of 6-OST with the Sulfo donor analog 3′-phosphoadenosine 5′-phosphate and three different oligosaccharide substrates at 1.95 to 2.1 A resolutions. Structural and mutational analyses reveal amino acid residues that contribute to catalysis and substrate recognition of 6-OST. Unexpectedly, the structures reveal 6-OST engages HS in a completely different orientation than other HS Sulfotransferases and sheds light on the basic HS requirements for specificity. These findings also contribute structural information to understand mutations in human 6-OST isoform 1 associated with the human genetic disease idiopathic hypogonadotropic hypogonadism character...
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Structure Based Substrate Specificity Analysis of Heparan Sulfate 6‑O‑Sulfotransferases
2016Co-Authors: Andrea F Moon, Jian Liu, Juno M Krahn, Lars C. PedersenAbstract:Heparan sulfate (HS) is a sulfated polysaccharide exhibiting essential physiological functions. HS 6-O-Sulfotransferase (6-OST) transfers a Sulfo Group to the 6-OH position of glucosamine units to confer a variety of HS biological activities. There are three different isoforms of 6-OST in the human genome. Here, we report crystal structures of the ternary complex of 6-OST with the Sulfo donor analog 3′-phosphoadenosine 5′-phosphate and three different oligosaccharide substrates at 1.95 to 2.1 Å resolutions. Structural and mutational analyses reveal amino acid residues that contribute to catalysis and substrate recognition of 6-OST. Unexpectedly, the structures reveal 6-OST engages HS in a completely different orientation than other HS Sulfotransferases and sheds light on the basic HS requirements for specificity. These findings also contribute structural information to understand mutations in human 6-OST isoform 1 associated with the human genetic disease idiopathic hypogonadotropic hypogonadism characterized by incomplete or lack of puberty
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Structurally Informative Tandem Mass Spectrometry of Highly Sulfated Natural and Chemoenzymatically Synthesized Heparin and Heparan Sulfate Glycosaminoglycans
Molecular & cellular proteomics : MCP, 2013Co-Authors: Muchena J. Kailemia, Robert J. Linhardt, Jian Liu, I. Jonathan AmsterAbstract:Heparin (Hp)1 and heparan sulfate (HS) are linear, polydisperse, and highly sulfated glycosaminoglycans (GAGs), with a repeating disaccharide building block composed of a 1–4-linked glucosamine and a uronic acid residue (1). The saccharide residues may have a variety of modifications, and these are usually heterogeneous due to the nontemplate nature of their biosynthesis (2). Glucosamine residues may be substituted with N-Sulfo or N-acetyl and 3- and/or 6-O-Sulfo Groups. Uronic acid residues can be either glucuronic or iduronic acid and substituted with 2-O-Sulfo Groups (3, 4). These structural features are thought to control Hp and HS biological activity, e.g. their interactions with proteins, and so the structural characterization of GAGs is an important target for chemical analysis (1, 5, 6). A particularly well known example of a GAG-protein interaction is the role of Hp as an antithrombin III activator. A pentasaccharide unit with a very specific pattern of modification interacts with antithrombin III causing it to undergo a conformational change that increases the anticoagulation activity of antithrombin III by more than 3 orders of magnitude (7, 8). Contamination of pharmaceutical Hp was a major issue recently, i.e. associated with over 70 fatalities worldwide (9–12). This problem highlights the need for rapid, robust, and sensitive analytical methods for the analysis of heparin and for identifying contaminants of similar composition (11). Although nuclear magnetic resonance spectroscopy is often the method of choice for determining the structure of GAGs, such as Hp and HS, it requires substantial sample preparation to obtain pure samples, relatively large amounts of sample, and time-consuming interpretation. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) offer high sensitivity and specificity and are often used for the analysis of complex mixtures. For these reasons, MS and MS/MS have been explored by a number of researchers as tools for the structural analysis of GAGs (13–31). Recently, the sequence of intact full-length chondroitin sulfate GAG chains from bikunin was elucidated (32). However, these were sparsely sulfated compared with typical HS/Hp GAGs, averaging less than 0.5 sulfate modifications per disaccharide repeat unit. Hp in particular is highly sulfated, making it extremely difficult to deduce composition and other structural details from intact samples. Controlled enzymatic digestion (heparin lyases I, II, and III) (33) and chemical methods (using nitrous acid) (34) can depolymerize Hp and HS to oligosaccharides of sizes that can be analyzed using the current instrumentation. The resulting products occur as a mixture of different sizes, compositions, isomers, and epimers, making their characterization extremely challenging. Use of MS to analyze highly sulfated HS oligosaccharides, with two or more Sulfo Groups per disaccharide repeating unit, is difficult due to the loss of labile SO3 with mild ion activation (27). Negative mode electrospray ionization (ESI) is commonly used to analyze GAGs due to its ability to preserve the Sulfo Groups during the ionization process and its propensity to form multiply charged anions from these acidic molecules (35). Information about the composition and the length of the molecule can be achieved by the first step MS but cannot provide structural details on the monosaccharide residue connectivity and locations of various GAG modifications, i.e. Sulfo Groups, acetyl Groups, and uronic acid C5 epimers. MS/MS is required to obtain these structural details. Fragmentation of glycosidic bonds produce ion products that determine the composition of individual residues, although cross-ring cleavages are useful for assigning the sites of modification within a monosaccharide residue. For Hp and HS oligosaccharides, previous studies have shown that threshold ion activation methods such as low energy collision-induced dissociation (CID) or infrared multiphoton dissociation produce a relatively low number of structurally useful fragments, due to the preference for loss of SO3 rather than glycosidic bond fragmentation or cross-ring cleavage (13, 25, 36). Nevertheless, CID has been used to differentiate 6-O-Sulfo and 3-O-Sulfo Groups in Hp disaccharide units, due to differences in their multidimensional tandem mass spectrometry (MSn) fragments (37). Metal cationization has also been used for the MS/MS analysis of carbohydrates (38–42), and it has been found that increasing the charge state and metal-hydrogen exchange in these biomolecules increases the density of fragments and reduces SO3 loss (17, 25, 43, 44). Hp and HS GAGs have been characterized by their MSn with CID activation, for both multiply charged ions cationized by Ca2+ (and Na+ to a lesser extent), ranging from a trisaccharide with four Sulfo Groups to a pentasaccharide with eight Sulfo Groups. Although this work resulted in more glycosidic and cross-ring cleavages, there were still too few fragment ions to provide detailed structural information about the sites of Sulfo Group modifications (25). However, this work established that the presence of metal cations in these molecules increased the stability of the Sulfo Groups, leading to more useful fragment ions (45). In recent years, electron-based methods, especially electron detachment dissociation, have proved to be very useful in both locating the Sulfo Groups and determining the uronic acid C-5 stereochemistry of undersulfated HS-derived tetrasaccharides, but the efficiency of electron detachment dissociation decreases as the number of Sulfo Groups per disaccharide unit increases (13–16, 18, 20, 22). Sodium metal cation/proton exchange has been investigated for electron detachment dissociation and infrared multiphoton dissociation of sparsely sulfated dermatan sulfate oligosaccharides (one Sulfo Group per disaccharide) (17). This work shows that increasing sodium cationization so that all Sulfo Groups in the molecule are deprotonated greatly reduces the number of peaks due to SO3 loss, but this approach also resulted in a reduction of the number of both glycosidic and cross-ring cleavages (17). This study demonstrates a new method to stabilize Sulfo Groups during MS/MS of Hp and HS oligosaccharides, while producing much more extensive and structurally informative fragmentation. This is achieved by the exhaustive deprotonation of all ionizable sites in Hp and HS oligomers, using sodium hydroxide to cationize or deprotonate every acidic Group in the precursor ion. This approach has recently been shown in our laboratory to be effective for the highly sulfated heparin-like pentasaccharide drug, Arixtra® (46). Here, we show that this approach is generally applicable to Hp and HS oligosaccharides from pentasulfated tetrasaccharides up to undecasulfated octasaccharide Hp, derived from natural sources, as well as chemoenzymatically synthesized HS oligomers up to 12 residues in length.
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Substrate specificity of 6-O-endosulfatase (Sulf-2) and its implications in synthesizing anticoagulant heparan sulfate.
Glycobiology, 2012Co-Authors: Elizabeth H. Pempe, Tanya C Burch, Courtney J Law, Jian LiuAbstract:Heparan sulfate (HS) 6-O-endosulfatase (Sulf) catalyzes the hydrolysis of 6-O-Sulfo Groups from HS polysaccharides. The resultant HS has reduced sulfation levels and displays altered biological activities. The Sulfs have been associated with several cancers and developmental problems and could function as a tool for editing specific HS structures. Here, we characterize the substrate specificity of human Sulf-2 using site-specifically radiolabeled synthetic polysaccharides. The enzyme was expressed and harvested from the conditioned medium of Chinese hamster ovary cells transfected with Sulf-2 expression plasmids. The uniquely [ 35 S]sulfated polysaccharides were prepared using purified recombinant HS biosynthetic enzymes. We found that Sulf-2 is particularly effective in removing the 6-O-Sulfo Group residing in the trisulfated disaccharide repeating unit comprising 2-O-sulfated uronic acid and N-sulfated 6-O-Sulfo glucosamine, but can also hydrolyze Sulfo Groups from N- and 6-O-sulfated disaccharides. In addition, we found that Sulf-2 treatment significantly decreases HS’s ability to bind to platelet factor 4 (PF4), a chemokine, while binding to antithrombin is maintained. Because HS–PF4 complexes are the initiating cause of heparin-induced thrombocytopenia, this finding provides a promising strategy for developing heparin therapies with reduced side effects. Further understanding of Sulf-2 activity will help elucidate HS structure–function relationships and provide a valuable tool in tailoring HSbased anticoagulant drugs.
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Mutational Study of Heparan Sulfate 2-O-Sulfotransferase and Chondroitin Sulfate 2-O-Sulfotransferase
Journal of Biological Chemistry, 2007Co-Authors: Danyin Song, Lars C. Pedersen, Jian LiuAbstract:Abstract Heparan sulfate (HS) and chondroitin sulfate (CS) are highly sulfated polysaccharides with a wide range of biological functions. Heparan sulfate 2-O-Sulfotransferase (HS-2OST) transfers the Sulfo Group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the 2-OH position of the hexauronic acid that is adjacent to N-sulfated glucosamine, whereas chondroitin sulfate 2-O-Sulfotransferase (CS-2OST) transfers the Sulfo Group to the hexauronic acid that is adjacent to N-acetylated galactosamine. Here we report a systematic mutagenesis study of HS-2OST and CS-2OST based on their structural homology to estrogen Sulfotransferase and HS 3-O-Sulfotransferase isoform 3 (3-OST3), for which crystal structures exist. We have identified six residues possibly involved in binding to PAPS. HS-2OST carrying mutations of these residues lacks Sulfotransferase activity and the ability to bind 3′-phosphoadenosine 5′-phosphate, a PAPS analogue, as determined by isothermal titration calorimetry. Similar residues involved in binding to PAPS were also identified in CS-2OST. Additional residues that participate in carbohydrate substrate binding were also identified in both enzymes. Mutations at these residues led to the loss of Sulfotransferase activity but maintained the ability to bind to phosphoadenosine 5′-phosphate. The catalytic function of HS-2OST appears to involve two histidine residues (His140 and His142), whereas only one histidine (His168) of CS 2-OST is likely to be critical. This unique feature of HS 2-OST catalytic residues directed us to characterize the Drosophila heparan sulfate 2-O-Sulfotransferase. The results from this study provide insight into the differences and similarities various residues play in the biological roles of the HS-2OST and CS-2OST enzymes.
Ping Wang - One of the best experts on this subject based on the ideXlab platform.
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The Label-Free Immunosensor Based on rhodium@palladium nanodendrites/Sulfo Group Functionalized Multi-Walled Carbon Nanotubes for the Sensitive Analysis of Carcino Embryonic Antigen
Analytica chimica acta, 2017Co-Authors: Xiaobo Zhang, Ping Wang, Zengqiang Gao, Jinhui Feng, Yunhui DongAbstract:Abstract In this work, bimetallic core-shell rhodium@palladium nanodendrites (Rh@Pd NDs) loaded on Sulfo Group functionalized multi-walled carbon nanotubes (MWCNTs-SO3H) were combined to form Rh@Pd NDs/MWCNTs-SO3H nanocomposites. And the composites were used to construct a simple and label-free electrochemical immunosensor for carcino embryonic antigen (CEA) detection using differential pulse voltammetry (DPV). Rh@Pd NDs with dendritic nanostructure not only provide abundant catalytically active sites, but also increase the loading of antibody, which could improve the analytical performance and result in high sensitivity. In addition, the MWCNTs-SO3H could further enhance electrochemical properties due to the excellent conductivity, good solubility and high surface area. Taking advantages of both Rh@Pd NDs and MWCNTs-SO3H, the proposed immunosensor showed a broad linear range from 25 fg mL−1 to 100 ng mL−1 for CEA detection and a low detection limit of 8.3 fg mL−1 (signal-to-noise ratio of 3) under optimal experimental conditions. Moreover, the expected immunosensor exhibited good reproducibility and high sensitivity, which could achieve excellent analysis of CEA in human serum with satisfactory results. Therefore, the Rh@Pd NDs/MWCNTs-SO3H nanocomposites may be considered as a sensing platform for fabrication of simple, ultrasensitive and label-free electrochemical immunosensor.
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the label free immunosensor based on rhodium palladium nanodendrites Sulfo Group functionalized multi walled carbon nanotubes for the sensitive analysis of carcino embryonic antigen
Analytica Chimica Acta, 2017Co-Authors: Xiaobo Zhang, Ping Wang, Zengqiang Gao, Jinhui Feng, Yunhui DongAbstract:Abstract In this work, bimetallic core-shell rhodium@palladium nanodendrites (Rh@Pd NDs) loaded on Sulfo Group functionalized multi-walled carbon nanotubes (MWCNTs-SO3H) were combined to form Rh@Pd NDs/MWCNTs-SO3H nanocomposites. And the composites were used to construct a simple and label-free electrochemical immunosensor for carcino embryonic antigen (CEA) detection using differential pulse voltammetry (DPV). Rh@Pd NDs with dendritic nanostructure not only provide abundant catalytically active sites, but also increase the loading of antibody, which could improve the analytical performance and result in high sensitivity. In addition, the MWCNTs-SO3H could further enhance electrochemical properties due to the excellent conductivity, good solubility and high surface area. Taking advantages of both Rh@Pd NDs and MWCNTs-SO3H, the proposed immunosensor showed a broad linear range from 25 fg mL−1 to 100 ng mL−1 for CEA detection and a low detection limit of 8.3 fg mL−1 (signal-to-noise ratio of 3) under optimal experimental conditions. Moreover, the expected immunosensor exhibited good reproducibility and high sensitivity, which could achieve excellent analysis of CEA in human serum with satisfactory results. Therefore, the Rh@Pd NDs/MWCNTs-SO3H nanocomposites may be considered as a sensing platform for fabrication of simple, ultrasensitive and label-free electrochemical immunosensor.
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an ultrasensitive sandwich type electrochemical immunosensor based on the signal amplification strategy of mesoporous core shell pd pt nanoparticles amino Group functionalized graphene nanocomposite
Biosensors and Bioelectronics, 2017Co-Authors: Mingdang Li, Faying Li, Yueyun Li, Ping Wang, Yunhui DongAbstract:Abstract Herein, a novel and sensitive sandwich-type electrochemical immunosensor was fabricated for quantitative monitoring of prostate specific antigen (PSA). The Sulfo Group functionalized multi-walled carbon nanotubes (MWCNTs-SO 3 H) were used as substrate material to increase the specific surface area and enhance the conductivity of the glassy carbon electrode. Gold nanoparticles (Au NPs) were introduced to enhance the load capacity of the substrate material for primary antibodies (Ab 1 ) and accelerate the electron transfer on the electrode interface. The mesoporous core-shell Pd@Pt nanoparticle loaded by amino Group functionalized graphene (M-Pd@Pt/NH 2 -GS) with high specific surface area, high indexed facets, and good biocompatibility was not only as the carriers of secondary antibodies (Ab 2 ) but also catalyzed the reduction of hydrogen peroxide (H 2 O 2 ), which effectually amplified the current signal in detection of PSA. The as-proposed immunosensor exhibited high sensitivity and stability on the detection of PSA. A linear relationship between current signals and the concentrations of PSA was obtained in the range from 10 fg/mL to 50 ng/mL and the detection limit of PSA was 3.3 fg/mL (signal-to-noise ratio of 3). Furthermore, the as-proposed immunosensor showed excellent performance in detection of human serum samples. The results suggest that the proposed immunosensor will be promising in the diagnostics application for accurately quantitative detection of PSA.
Lars C. Pedersen - One of the best experts on this subject based on the ideXlab platform.
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structure based substrate specificity analysis of heparan sulfate 6 o Sulfotransferases
ACS Chemical Biology, 2017Co-Authors: Andrea F Moon, Jian Liu, Juno M Krahn, Lars C. PedersenAbstract:Heparan sulfate (HS) is a sulfated polysaccharide exhibiting essential physiological functions. HS 6-O-Sulfotransferase (6-OST) transfers a Sulfo Group to the 6-OH position of glucosamine units to confer a variety of HS biological activities. There are three different isoforms of 6-OST in the human genome. Here, we report crystal structures of the ternary complex of 6-OST with the Sulfo donor analog 3′-phosphoadenosine 5′-phosphate and three different oligosaccharide substrates at 1.95 to 2.1 A resolutions. Structural and mutational analyses reveal amino acid residues that contribute to catalysis and substrate recognition of 6-OST. Unexpectedly, the structures reveal 6-OST engages HS in a completely different orientation than other HS Sulfotransferases and sheds light on the basic HS requirements for specificity. These findings also contribute structural information to understand mutations in human 6-OST isoform 1 associated with the human genetic disease idiopathic hypogonadotropic hypogonadism character...
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Structure Based Substrate Specificity Analysis of Heparan Sulfate 6‑O‑Sulfotransferases
2016Co-Authors: Andrea F Moon, Jian Liu, Juno M Krahn, Lars C. PedersenAbstract:Heparan sulfate (HS) is a sulfated polysaccharide exhibiting essential physiological functions. HS 6-O-Sulfotransferase (6-OST) transfers a Sulfo Group to the 6-OH position of glucosamine units to confer a variety of HS biological activities. There are three different isoforms of 6-OST in the human genome. Here, we report crystal structures of the ternary complex of 6-OST with the Sulfo donor analog 3′-phosphoadenosine 5′-phosphate and three different oligosaccharide substrates at 1.95 to 2.1 Å resolutions. Structural and mutational analyses reveal amino acid residues that contribute to catalysis and substrate recognition of 6-OST. Unexpectedly, the structures reveal 6-OST engages HS in a completely different orientation than other HS Sulfotransferases and sheds light on the basic HS requirements for specificity. These findings also contribute structural information to understand mutations in human 6-OST isoform 1 associated with the human genetic disease idiopathic hypogonadotropic hypogonadism characterized by incomplete or lack of puberty
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Mutational Study of Heparan Sulfate 2-O-Sulfotransferase and Chondroitin Sulfate 2-O-Sulfotransferase
Journal of Biological Chemistry, 2007Co-Authors: Danyin Song, Lars C. Pedersen, Jian LiuAbstract:Abstract Heparan sulfate (HS) and chondroitin sulfate (CS) are highly sulfated polysaccharides with a wide range of biological functions. Heparan sulfate 2-O-Sulfotransferase (HS-2OST) transfers the Sulfo Group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the 2-OH position of the hexauronic acid that is adjacent to N-sulfated glucosamine, whereas chondroitin sulfate 2-O-Sulfotransferase (CS-2OST) transfers the Sulfo Group to the hexauronic acid that is adjacent to N-acetylated galactosamine. Here we report a systematic mutagenesis study of HS-2OST and CS-2OST based on their structural homology to estrogen Sulfotransferase and HS 3-O-Sulfotransferase isoform 3 (3-OST3), for which crystal structures exist. We have identified six residues possibly involved in binding to PAPS. HS-2OST carrying mutations of these residues lacks Sulfotransferase activity and the ability to bind 3′-phosphoadenosine 5′-phosphate, a PAPS analogue, as determined by isothermal titration calorimetry. Similar residues involved in binding to PAPS were also identified in CS-2OST. Additional residues that participate in carbohydrate substrate binding were also identified in both enzymes. Mutations at these residues led to the loss of Sulfotransferase activity but maintained the ability to bind to phosphoadenosine 5′-phosphate. The catalytic function of HS-2OST appears to involve two histidine residues (His140 and His142), whereas only one histidine (His168) of CS 2-OST is likely to be critical. This unique feature of HS 2-OST catalytic residues directed us to characterize the Drosophila heparan sulfate 2-O-Sulfotransferase. The results from this study provide insight into the differences and similarities various residues play in the biological roles of the HS-2OST and CS-2OST enzymes.