STRUCTURAL ANALYSIS OF SULF2 PROCESSING OF EXTRACELLULAR HEPARAN SULFATE

该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。次级项目的主要支助 子项目的主要研究者可能是由其他来源提供的， 包括其它NIH来源。 为子项目列出的总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 最近发现的Sulf 2酶用于修饰与细胞表面和细胞外基质蛋白聚糖相关的硫酸乙酰肝素（HS）。 这种重塑影响细胞对包括转化生长因子、血管内皮生长因子和成纤维细胞生长因子在内的生长因子家族的识别。 由于分析挑战，缺乏Sulf 2活性所需的HS结构域背景的详细结构信息。 这些信息对于理解Sulf 2酶作为组织位置和生长环境（发育和疾病）中时间变化的函数的功能作用至关重要。 在过去的一年中，我们已经使用d的LC/MS平台，以配置文件的变化，HS结构介导的重组Sulf 2酶。通过用Sulf 2消化来自不同牛器官的HS，然后用裂解酶处理来收集数据。 详尽的解聚，然后LC/MS扩展了信息的二糖网站识别的硫酸酶，包括2个网站以前没有确定。 这篇文章最近发表（1）。 在继续的工作中，猪肠粘膜HS被肝素裂解酶I和III部分消化。通过SEC分级分离并收集聚合度为6-8（dp 6 -8）的HS寡糖。用Shire Pharmaceuticals提供的重组HSulf 2处理部分细胞。等量仅用消化缓冲液处理作为对照。HSulf 2处理的和对照dp 6 -8级分均通过定制填充的亲水相互作用色谱HPLC芯片与Agilent 6520 QTOF在线脉冲补充流进行分析。对那些在HSulf 2处理下表现出明显变化的寡糖进行LC-MS/MS，环丁砜在相应的保留时间脉冲以提高其电荷状态，以获得更好的串联MS。 硫作用于HS链内的6 O-硫酸盐的子集。我们先前对HSulf 2处理后进行彻底肝素裂解酶解聚的研究表明，HSulf 2也在非还原端切割6 O-硫酸盐。 然而，由于缺乏结构确定的HS寡糖，HSulf 2的结构特异性的阐明仍然是一个挑战。在这项研究中，我们使用的寡糖具有合理的长度，其中异构体的配置文件被简化的裂解酶的特异性，辨别底物结构的偏好HSulf 2。 HSulf 2优先作用于具有0-1个乙酸酯基团的高度硫酸化的dp 6 -8。 同时，相对低的硫酸化dp 6 -8的丰度随着HSulf 2的产物而增加。与具有一个乙酸酯基团的那些相比，具有零乙酸酯的dp 6 -8的平均硫酸化程度降低更多。虽然具有2个乙酸酯基团的dp 6 -8高度硫酸化并且含有6 O-硫酸化，但HSulf 2不改变具有2个乙酸酯基团的dp 6 -8的概况，表明其偏好少量乙酸酯和高度硫酸化的寡糖。 由于HSulf 2仅裂解6 O-硫酸化，因此仅某些异构体受到影响，这通过提取离子色谱图（EIC）的色谱峰形状变化反映出来。选择那些怀疑经历这些变化的dp 6 -8作为串联MS的目标。在目标前体的洗脱时间期间，使用定制的脉冲补给流HPLC芯片来脉冲环丁砜，以增强它们的电荷状态，以获得更多信息的串联质谱。尽管这些串联质谱在整个EIC峰范围和整个碰撞能量范围内看起来相似，但通过仔细选择保留时间窗口和碰撞能量，使光谱差异更加明显，表明寡糖的一个子集已被处理，并且HSulf 2。 这些串联质谱有可能提供更多的HSulf 2底物特异性的结构信息。 1. 斯台普斯，G. O.，施，X.，和Zaia，J.（2011）Glycomics Analysis of Mammalian Heparan Sulfates Modified by the Human Extracellular Sulfatase HSulf2，PLoS ONE 6，e16689.

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.