STRUCTURAL ANALYSIS OF SULF2 PROCESSING OF EXTRACELLULAR HEPARAN SULFATE

胞外硫酸乙酰肝素 SULF2 加工的结构分析

基本信息

  • 批准号:
    8365551
  • 负责人:
  • 金额:
    $ 1.23万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2011
  • 资助国家:
    美国
  • 起止时间:
    2011-06-01 至 2012-08-09
  • 项目状态:
    已结题

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The recently discovered Sulf2 enzyme acts to modify heparan sulfate (HS) associated with cell surface and extracellular matrix proteoglycans. This remodeling affects cellular recognition of growth factor families including transforming growth factors, vascular endothelial growth factors, and fibroblast growth factors. Detailed structural information on the HS domain context required for Sulf2 activity is lacking due to analytical challenges. Such information is essential to understanding the functional roles of Sulf2 enzymes as a function of tissue location and temporal changes in the growth environment (development and disease). Over the past year we have use d the LC/MS platform to profile changes to HS structure mediated by recombinant Sulf2 enzyme. Data were collected by digesting HS from different bovine organs with Sulf2 and then treating with lyase enzymes. Exhaustive depolymerization followed by LC/MS extended the information on the disaccharide sites recognized by Sulf enzymes to include 2 sites not previously identified. This work was published recently (1). In continuing work, porcine intestine mucosa HS was partially digested by heparin lyases I and III. HS oligosaccharides of degree of polymerization of 6-8 (dp6-8) were fractionated by SEC and collected. Part of the faction was treated with recombinant HSulf2 provided by Shire Pharmaceuticals. An equal quantity was treated with digestion buffer only as control. Both HSulf2 treated and control dp6-8 fractions were analyzed by a custom packed hydrophilic interaction chromatography HPLC-chip with pulsed make-up flow online with an Agilent 6520 QTOF. Those oligosaccharides exhibiting pronounced change with HSulf2 treatment were subjected to LC-MS/MS, with sulfolane pulsed at the corresponding retention time to elevate their charge states for better tandem MS. Sulfs act on a subset of 6O-sulfates within HS chains. Our previously study of HSulf2 treatment followed by exhaustive heparin lyase depolymerization has shown that HSulf2 cleaves 6O-sulfate in the non-reducing end as well. However, due to the lack of structurally defined HS oligosaccharides, the elucidation of structural specificity of HSulf2 remains a challenge. In this study, we used oligosaccharides with reasonable lengths, of which isomeric profiles were simplified by lyase specificity, to discern the substrate structural preferences of HSulf2. HSulf2 acts preferentially on highly sulfated dp6-8 with 0-1 acetate group. At the same time, the abundance of relatively low sulfated dp6-8 increased as the products by HSulf2. The average sulfation degree decreased more for dp6-8 with zero acetate than for those with one acetate group. Although dp6-8 with 2 acetate groups were highly sulfated and contain 6O-sulfation, HSulf2 did not change the profiles of dp6-8 with 2 acetate groups, suggesting its preference for few acetates and highly sulfated oligosaccharides. Since only 6O-sulfation is cleaved by HSulf2, only certain isomers were affected and this was reflected by the chromatographic peak shape change of the extracted ion chromatograms (EICs). Those dp6-8 that were suspected to experience these changes were chosen as targets for tandem MS. A customized pulsed make-up flow HPLC-chip was used to pulsed sulfolane during the elution time of the target precursors to enhance their charge states for more informative tandem mass spectra. Although these tandem mass spectra seem to look similar for entire EIC peak range and entire collision energy range, by careful selection of retention time windows and collision energy, the spectra differences were made more evident, suggesting a subset of the oligosaccharides have been processed and HSulf2. These tandem mass spectra have the potential to provide more structural information of the substrate specificity of HSulf2. 1. Staples, G. O., Shi, X., and Zaia, J. (2011) Glycomics Analysis of Mammalian Heparan Sulfates Modified by the Human Extracellular Sulfatase HSulf2, PLoS ONE 6, e16689.
这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的, 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量, 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 最近发现的Sulf2酶作用于与细胞表面和细胞外基质蛋白多糖相关的硫酸乙酰肝素(HS)。这种重构影响细胞对生长因子家族的识别,包括转化生长因子、血管内皮生长因子和成纤维细胞生长因子。由于分析方面的挑战,缺乏关于Sulf2活动所需的HS领域背景的详细结构信息。这些信息对于理解作为生长环境(发育和疾病)中组织位置和时间变化的功能的Sulf2酶的功能作用是必不可少的。 在过去的一年里,我们利用LC/MS平台研究了重组Sulf2酶对HS结构的影响。通过用硫磺消化不同牛器官的HS,然后用裂解酶处理来收集数据。彻底解聚后的LC/MS扩展了关于Sulf酶识别的双糖位置的信息,包括两个以前没有确定的位置。这项工作发表于最近(1)。 在后续工作中,用肝素裂解酶I和III部分消化猪肠粘膜HS,用SEC分离和收集聚合度为6-8(dp6-8)的HS寡糖。部分派系接受了夏尔制药提供的重组HSulf2治疗。等量只用消化缓冲液处理作为对照。HSulf2处理组分和对照Dp6-8组分均采用定制填充式亲水作用色谱柱进行分析,芯片采用Agilent 6520 QTOF在线脉冲补流。那些随着HSulf2处理而发生显著变化的低聚糖被用LC-MS/MS进行处理,并在相应的保留时间用环丁砜脉冲以提高它们的荷电状态以获得更好的串联MS。 硫磺作用于HS链上的一部分60-硫酸盐。我们先前对HSulf2处理后肝素裂解酶彻底解聚的研究表明,HSulf2在非还原末端也能裂解60-硫酸盐。然而,由于缺乏结构上定义的HS寡糖,对HSulf2结构特异性的阐明仍然是一个挑战。在这项研究中,我们使用长度合理的低聚糖,通过裂解酶专一性简化了低聚糖的异构体图谱,以区分HSulf2的底物结构偏好。 HSulf2优先作用于具有0-1乙酸酯基团的高硫酸盐化DP6-8。同时,相对较低的硫酸盐化dp6-8的丰度随着HSulf2产物的增加而增加。添加零乙酸酯的DP6-8的平均硫酸盐化程度比添加单乙酸酯的DP6-8降低得更多。虽然含有2个乙酸酯基团的dp6-8是高度硫酸盐化的,并且含有60-O-硫酸盐化,但HSulf2没有改变含有2个醋酸酯基团的dp6-8的构型,这表明它更倾向于少量的醋酸酯和高度硫酸盐化的寡糖。 由于HSulf2只裂解6O-硫酸,所以只影响某些异构体,这从萃取离子色谱图(EICS)的峰形变化中可以看出。选择那些怀疑经历这些变化的dp6-8作为串联MS的目标,在目标前体的洗脱时间使用定制的脉冲补流高效液相色谱芯片来脉冲环丁砜,以增强其电荷状态,以获得更多信息的串联质谱图。尽管这些串联质谱图在整个EIC峰范围和整个碰撞能量范围内看起来都很相似,但通过仔细选择保留时间窗口和碰撞能量,光谱差异变得更加明显,这表明低聚糖的子集已经被处理和HSulf2。这些串联质谱图有可能提供HSulf2底物专一性的更多结构信息。 1.Staples,G.O.,Shii,X.,and Zaia,J.(2011)人胞外硫酸酯酶HSulf2修饰的哺乳动物肝素硫酸盐的糖组分分析,PLoS one 6,e16689。

项目成果

期刊论文数量(0)
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科研奖励数量(0)
会议论文数量(0)
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JOSEPH ZAIA其他文献

JOSEPH ZAIA的其他文献

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{{ truncateString('JOSEPH ZAIA', 18)}}的其他基金

Methods for measuring matrisome molecule similarity during disease processes
测量疾病过程中基质体分子相似性的方法
  • 批准号:
    10582128
  • 财政年份:
    2022
  • 资助金额:
    $ 1.23万
  • 项目类别:
Methods for measuring matrisome molecule similarity during disease processes
测量疾病过程中基质体分子相似性的方法
  • 批准号:
    10580774
  • 财政年份:
    2022
  • 资助金额:
    $ 1.23万
  • 项目类别:
Methods for measuring matrisome molecule similarity during disease processes
测量疾病过程中基质体分子相似性的方法
  • 批准号:
    10330789
  • 财政年份:
    2022
  • 资助金额:
    $ 1.23万
  • 项目类别:
Methods for determination of glycoprotein glycosylation similarities among disease states
确定疾病状态之间糖蛋白糖基化相似性的方法
  • 批准号:
    10194553
  • 财政年份:
    2019
  • 资助金额:
    $ 1.23万
  • 项目类别:
An open-source software suite for processing glycomics and glycoproteomics mass spectral data
用于处理糖组学和糖蛋白质组学质谱数据的开源软件套件
  • 批准号:
    9391486
  • 财政年份:
    2017
  • 资助金额:
    $ 1.23万
  • 项目类别:
A Thermo-Fisher Scientific Q-Exactive HF Mass Spectrometry System
Thermo-Fisher Scientific Q-Exactive HF 质谱系统
  • 批准号:
    9075665
  • 财政年份:
    2016
  • 资助金额:
    $ 1.23万
  • 项目类别:
Software for automated interpretation of heparan sulfate tandem mass spectra
用于自动解释硫酸乙酰肝素串联质谱的软件
  • 批准号:
    9337106
  • 财政年份:
    2015
  • 资助金额:
    $ 1.23万
  • 项目类别:
Software for automated interpretation of heparan sulfate tandem mass spectra
用于自动解释硫酸乙酰肝素串联质谱的软件
  • 批准号:
    9144851
  • 财政年份:
    2015
  • 资助金额:
    $ 1.23万
  • 项目类别:
Software for automated interpretation of heparan sulfate tandem mass spectra
用于自动解释硫酸乙酰肝素串联质谱的软件
  • 批准号:
    8984998
  • 财政年份:
    2015
  • 资助金额:
    $ 1.23万
  • 项目类别:
Quantitative profiling of glycosaminoglycans from breast tumor tissue arrays
乳腺肿瘤组织阵列中糖胺聚糖的定量分析
  • 批准号:
    9079438
  • 财政年份:
    2014
  • 资助金额:
    $ 1.23万
  • 项目类别:

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