Novel Reagents for Monitoring Sulfation Patterns in Heparin and Heparan Sulfate
Novel Reagents for Monitoring Sulfation Patterns in Heparin and Heparan Sulfate
批准号:
9909150
负责人:
Sheng-Cheng Wu
金额:
$22.5万
依托单位国家:
美国
项目类别:
财政年份:
2020
资助国家:
美国
项目状态:
已结题
起止时间:
2020-03-01 至 2022-02-28
关键词:
AdhesionsAffinityAffinity ChromatographyAntibodiesAnticoagulantsAntithrombinsBindingBinding ProteinsBiochemical ReactionBiologicalBiological AssayBiological MarkersCarbohydratesCell surfaceCellsCompetitive BindingComplexComputer AnalysisComputer SimulationCrystallizationDetectionDirected Molecular EvolutionDiseaseDisease MarkerDistalEnzymesEventFutureGene LibraryHealthHeparan Sulfate BiosynthesisHeparinHeparitin SulfateHeparitin sulfotransferaseHistological TechniquesImageIn SituLettersLibrariesLocationMalignant neoplasm of prostateMediatingMonitorMutagenesisNeoplasm MetastasisOligonucleotidesOligosaccharidesParentsPathogenicityPathologicPathway interactionsPatternPoint MutationPolysaccharidesPositioning AttributeProcessPropertyProtein AnalysisProtein IsoformsReagentReportingScaffolding ProteinSignal TransductionSiteSpecificityStructureStructure-Activity RelationshipSulfateTechniquesTherapeuticTimeTissuesVariantVertebral columnViralWestern BlottingYeastsangiogenesisaxon guidancebasecombinatorialcommercial applicationdesigndisease diagnosisheparan-sulfate 2-sulfotransferasehistological stainsimprovedin vivo imaginginsightmigrationmutantneoplastic cellnovelpathogenpreferencereceptorresponsescaffoldscreeningsulfotransferasesulfurtransferase
中文摘要
项目总结
寡糖和多糖参与基本的分子相互作用及其在细胞上的定位
表面使它们成为各种疾病状态的理想生物标记物。硫酸乙酰肝素(HS)是一种
高度硫酸盐化的线性多糖,参与一系列细胞信号事件,
无论是有益的还是致病的,取决于沿着多糖链的硫酸盐化模式。
高亲和力结合探针,如抗体,是识别分子相互作用的有效手段
在细胞内;然而,很少有试剂具有足够的特异性来原位监测HS的硫化模式。
该项目将开发一类新的试剂,能够识别特定的硫酸盐化模式
通过利用酶(磺基转移酶)的固有专一性来调节HS链
在HS生物合成过程中将硫酸盐基团转移到多糖上。蛋白质支架将被创造出来
通过催化使磺基转移酶失活,支架的亲和力将通过
计算引导的单点突变,以及定向进化。通过有选择地瞄准
酶反应的底物或产物,每个失活的磺基转移酶都有可能结合到
两个特定的HS序列。
本项目将重点研究磺基转移酶HS2-O-磺基转移酶(HS2ST)和3-O-磺基转移酶
异构体1(HS3ST-1);然而,有许多来自生物合成的各种酶的异构体
HS的途径可能是未来研究的靶点。这两种酶都与具有生物学意义的
硫酸盐化模式。例如,HS2ST参与了多种功能,包括血管生成,
轴突引导和前列腺癌。HS3ST-1酶直接作用于抗凝剂
肝素的性质,通过增加HS对抗凝血酶的亲和力。这些试剂将会是巨大的
用于通过典型的抗体介导的分析跟踪硫酸盐化模式的变化,包括亲和力
纯化、免疫印迹、原位组织学染色和活体成像。
英文摘要
PROJECT SUMMARY
Oligo- and polysaccharides participate in fundamental molecular interactions, and their location on cell
surfaces makes them ideal candidates as biomarkers for various disease states. Heparan sulfate (HS) is a
highly sulfated linear polysaccharide that participates in an assortment of cellular signaling events that are
either advantageous or pathogenic, depending upon the sulfation pattern along the polysaccharide chain.
High-affinity binding probes, such as antibodies, are an effective means for discerning molecular interactions
within cells; however, few reagents are sufficiently specific for monitoring the sulfation patterns of HS in situ.
This project will develop a new class of reagents that is capable of recognizing specific sulfation patterns within
the HS chain by exploiting the inherent specificity of enzymes (sulfotransferases) that are responsible for
transferring the sulfate groups to the polysaccharide during HS biosynthesis. Protein scaffolds will be created
by catalytically inactivating the sulfotransferases, and the affinity of the scaffolds will be enhanced via
computationally-guided single point mutations, as well as by directed evolution. By selectively targeting either
the substrate or product of the enzymatic reaction, each inactive sulfotransferase has the potential to bind to
two specific sequences of HS.
This project will focus on sulfotransferases HS 2-O-Sulfotransferase (HS2ST) and 3-O-Sulfotransferase
Isoform 1 (HS3ST-1); however, there are numerous isoforms of various enzymes from the biosynthetic
pathway of HS that could be targeted in future studies. Both enzymes interact with biologically significant
sulfation patterns. For example, HS2ST has been implicated in a variety of functions including angiogenesis,
axon guidance, and prostate cancer. The HS3ST-1 enzyme is directly responsible for the anticoagulant
properties of heparin, by increasing the affinity of HS for antithrombin. These reagents would be immensely
useful for tracking changes in sulfation patterns via typical antibody-mediated assays, including affinity
purification, western blotting, in situ histological staining, and in vivo imaging.
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批准号:10761111
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项目类别:
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Novel Reagents for Monitoring Sulfation Patterns in Heparin and Heparan Sulfate
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批准号:10087944
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项目类别:
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资助金额:$22.28万
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财政年份:2020
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负责人:Sheng-Cheng Wu
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依托单位:
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批准号:9883115
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项目类别:
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资助金额:$74.06万
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财政年份:2020
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负责人:Sheng-Cheng Wu
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依托单位:
海外基金