Change Of Protein & Cell Functions By Heparin/heparan su

蛋白质的变化

• 批准号: 6541159
• 负责人: AUDREY L STONE
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6541159
• 关键词: antiAIDS agent; anticoagulants; antiviral agents; bacterial polysaccharides; bioassay; biomimetics; chemical models; chemical structure function; heparan sulfate; heparin; human immunodeficiency virus 1; infrared spectrometry; nuclear magnetic resonance spectroscopy; oligosaccharides; protein structure function

This project elucidates the fundamental molecular design of the heparin/heparan sulfate class of biological regulatory polysaccharides (H/HS) and studies how the high degree of diversity of its sulfated structures relates to its parallel, multi-functional capacity. This capacity is exerted through specific binding to, and thus modulating the functional activity of, many different protein partners in normal and disease processes (e.g., cell growth, secretion, multi-cell reactions in development, blood coagulation, physiological stability, and in viral and other infections). Established and/or newly devised biological, biochemical, and physical methods are utilzed. Previously: To study putative unique structures required for specific H/HS functions, and focussing on its anti-viral capacities against HIV-1, we started with a pharmaceutical heparin-mimetic sulfated xylan to devise a simpler, model system composed of a family of heparin-mimetic sulfated xylan oligosaccharides (S-oligoS). A macro combinatorial strategy with five in vitro target bioassays (anti-thrombin, anti-Xa, anti-cytotoxicity and -syncytium-forming infectivity of HIV-1, and clinical aPTT) was used and demonstrated that distinct functional Components were separable and isolated by low pressure liquid chromatography based on structural differences. These differential functional capacities among the S-oligoS in HIV-1 inhibition and other heparin functions indicated that, like those in the H/HS family, each was governed by a degree of structural specificity. This established the usefulness of our model system and we thereby isolated and characterized CpF-PkII, a potent anti-HIV-1 agent which lacks anti-thrombin activity. We developed an upscaled method to produce sufficient CpF-PkII for Phase I testing, and preparation continued this year. Details of results were given in previous reports. [HD 01315-01-06]. Structural features of S-oligoS are studied by analytical, chemical, sugar and specroscopic-FTIR, NMR, dye-coupling analysis, and by titration. Early findings showed that S-oligoS must contain a tetrasaccharide motif (beta-1,4-linked trixyloside with one alpha 1,2-linked branch) [HD 01315-01-05] In addition, FTIR spectra unexpectedly revealed the presence of axial sulfate moieties as well as the expected equatorial sulfates; heparin also contains both types of sulfate substituents. This finding indicated a structural basis for the heparin-mimetic capacity of S-oligoS for the first time. [HD 01315-03-04] Moreover, the relative proportions of these multiple forms differred among Components with different functional specificities. [HD 01313-05]. We reasoned that axial and equatorial sulfates must originate from different ring conformations of the glycopyranose moieties, i.e., an alternate and normal chair, resp., and that the corresponding equatorial and axial anomeric hydrogens would be seen in proton NMR spectra of the S-oligoS. Proton NMR spectra of CpF-PkII and several other S-oligoS last year were consistent with the interpretation that both beta-D Xyl and alpha-D Glc had tendency towards alternate conformation upon the sulfation of xylan, but the data were not conclusive because unambiguous assignment of the proton peaks of heavily sulfated oligoS was difficult. Further studies were required. [HD 01313-06] Because of the complex diversity of sulfated oligosaccharide structures within H/HS the structure-function relations of all but a few well- characterized HS-protein partners remains to be clarified. Findings from the S-oligoS family may contribute to this end. This year, continued production of CpF-PkII as a potential anti-AIDS agent, and the structure function studies of various other functionally distinct S-oligoS were pursued, using the same and more sophisticated methods. Two significant advances were made. In one, the macro combinatorial strategy above was extended to elucudate the anti-malaria capacity of S-oligoS and H/HS [See HD 008733-01]. In the second, compelling NMR evidence confirmed our results from FTIR spectroscopy. CpF-PkII was analyzed by two-dimensional NMR proton-13C -correlation spectroscopy using an inverted probe in a 600 MHz spectrometer at 60 degrees and the heteronuclear multi-dimensional quantum coherences (HMQC) method. In that study, If a given hydrogen were bonded to a sugar carbon that existed in two distinct conformations, the hydrogen would exhibit two complex interactions with that carbon. The correlation spectrum displayed two pairs of 13C-satellite signals about 71 ppm on the carbon spectrum, demonstrating that at least one sugar moiety existed in two conformations in CpF-PkII. The mid-points between these satellites were at 3.75 ppm and 4.65 ppm on the proton spectrum. This provides a basis for the peak assignments for the hydrogen and hence the position of the carbon the ring. Experimental data for the proton-NMR peaks of the sugars in similar S-oligoS indicates that the peaks of the 13C-satellite pairs originated from splitting by the proton bound to the 13C3 of a glycopyranose, that the sugar existed in normal chair and in an alternate form. and that the CpF-PkII structure contained axial sulfates on the C3 position of a portion of one of its sugars. This finding is important because it raises the idea that specific sugar conformation of individual sugars might be an additional governing parameter in structural specificity of the protein-binding sugar sequences of H/HS. This work will be reported at the November meeting of the Society for Glycobiology and the abstract published: MO Longas and AL Stone "Structure-function relations of heparin-mimetic sulfated oligosaccharides: Study of alternate chair conformations by NMR spectroscopy" Glycobiology vol. 11, 2001. Similar and/or different satellite pairs might be displayed by S-oligoS having different functional capacities. These studies will be continued next year.

该项目阐明了肝素/肝素硫酸盐类别的生物调节多糖（H/HS）的基本分子设计，并研究其硫酸化结构的高度多样性如何与其平行的多功能能力相关。这种能力是通过特异性结合与正常和疾病过程中许多不同蛋白质伴侣的功能活性（例如细胞生长，分泌，发育中的多细胞反应，血液凝结，生理稳定性以及病毒和其他感染中的多细胞反应）来调节功能活性的。建立和/或新设计的生物学，生化和物理方法已实用。以前：为了研究特定H/HS功能所需的假定独特结构，并专注于其针对HIV-1的抗病毒能力，我们开始使用药物肝素模拟硫化的Xylan，以设计一个更简单的模型系统，由肝素模拟硫酸盐硫酸盐硫酸盐寡素寡糖（S-S-Oligos）组成。具有五个体外靶向生物测定的宏观组合策略（抗凝血组，抗XA，抗XA，抗胞毒性和-HIV-1和临床APTT的 - 合成的感染性），并证明了基于结构差异的低压力液体色体可分离并通过低压力液体化学分离。 HIV-1抑制和其他肝素功能的S-Oligos之间的这些差异功能能力表明，像H/HS家族中的甲基甲基蛋白功能一样，每个功能都受到一定程度的结构特异性的控制。这确立了我们的模型系统的有用性，从而孤立并表征了CPF-PKII，这是一种有效的抗HIV-1药物，缺乏抗凝血酶活性。我们开发了一种高扫描的方法来生产足够的CPF-PKII进行I期测试，并在今年继续进行准备。结果的详细信息已在先前的报告中给出。 [HD 01315-01-06]。通过分析，化学，糖和Specroscopic-FTIR，NMR，染料偶联分析和滴定，研究了S-Oligos的结构特征。早期发现表明，S-Oligos必须含有四糖基序（β-1,4-链接的曲二酰胺，具有一个Alpha 1,2连接的分支）[HD 01315-01-05]此外，FTIR光谱出现了，FTIR光谱出乎意料地揭示了轴向硫酸盐含量的硫酸盐含量以及预期的均等赤道硫酸属性；肝素还含有两种类型的硫酸盐取代基。这一发现表明第一次S-Oligos肝素模拟能力的结构基础。 [HD 01315-03-04]此外，这些多种形式的相对比例在具有不同功能特异性的组件之间有所不同。 [HD 01313-05]。我们认为，轴向和赤道硫酸盐必须起源于糖吡喃糖部分的不同环构象，即替代和正常椅子，分别是替代和正常椅，并且在S-橄榄石的Proton NMR光谱中可以看到相应的赤道和轴向异源水元。去年CPF-PKII和其他几个S-Oligos的Proton NMR光谱与β-D Xyl和Alpha-D GLC的解释是一致的，该解释在Xylan硫酸盐时具有交替构象的趋势，但是数据并不是结论性，因为纯硫酸盐的Proton sulfated Oligos的Proton峰毫无意义。需要进一步的研究。 [HD 01313-06]由于H/HS中硫酸化的寡糖结构的复杂多样性，除少数少数表征良好的HS蛋白伴侣外，所有所有人的结构 - 功能关系仍然待阐明。 S-Oligos家族的发现可能有助于这一目的。 今年，使用相同且更复杂的方法，追求了CPF-PKII作为潜在的抗AIDS剂的继续生产，并继续进行其他各种功能上不同的S-Oligos的结构函数研究。取得了两个重大进展。在一个中，上面的宏观组合策略扩展了以阐明S-Oligos和H/HS的抗马拉里亚能力[见HD 008733-01]。在第二个中，引人注目的NMR证据证实了我们从FTIR光谱法的结果。通过在60度的600 MHz光谱仪中，使用二维NMR Proton-13C - 相关光谱分析CPF-PKII，并在60度的600 MHz光谱仪中分析CPF-PKII和异核多核多维量子量子共生（HMQC）方法。在该研究中，如果将给定的氢粘合到以两个不同构象中存在的糖碳键合，则氢将与该碳表现出两种复杂的相互作用。相关光谱在碳光谱上显示了两对13C - 卫星信号，表明CPF-PKII中的两个构象中至少存在一个糖部分。这些卫星之间的中点为3.75 ppm，质子光谱为4.65 ppm。这为氢的峰值分配提供了基础，并因此是碳的位置。类似S-Oligos中糖的质子-NMR峰的实验数据表明，13C-卫星对的峰源自质子与糖吡喃糖的13c3分裂，以糖糖的13c3，糖以正常椅子和替代形式存在。并且CPF-PKII结构在其一部分糖的一部分的C3位置包含轴向硫酸盐。这一发现很重要，因为它提出了这样一个想法，即单个糖的特定糖构象可能是H/HS蛋白质结合糖序列的结构特异性的附加参数。这项工作将在11月的血糖学会和摘要的11月会议上报告：Mo Longas和Al Stone“肝素模拟硫化寡糖的结构 - 功能关系：NMR光谱的替代椅子构象的研究” Glycobiology vol。 11，2001。通过具有不同功能能力的S-Oligos可以显示相似和/或不同的卫星对。这些研究将于明年继续。