Sequencing the Polysaccharide Component of Proteoglycans

对蛋白聚糖的多糖成分进行测序

• 批准号: 7729276
• 负责人: ROBERT J LINHARDT
• 金额: $ 28.93万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7729276
• 关键词: Acetylglucosamine; Affect; Aging; Anions; Antithrombin III; Arts; Binding Proteins; Binding Sites; Biochemical; Biological; Biology; Biomedical Engineering; Brain; Capillary Electrophoresis; Cardiovascular system; Cattle; Charge; Chemistry; Chondroitin; Chondroitin Sulfates; Complex; Core Protein; Crystallography; Data; Dermatan Sulfate; Development; Disaccharides; Disease; Dissociation; Electrons; Electrophoresis; Electrospray Ionization; Enzymatic Biochemistry; Enzymes; Extracellular Matrix; Family; Fourier transform ion cyclotron resonance; Functional disorder; Funding; Galactosamine; Galactose; Gel Chromatography; Glucosamine; Glucose; Glucuronic Acids; Glycosaminoglycans; Grant; Health; Heparan Sulfate Proteoglycan; Heparin; Heparitin Sulfate; Herpesvirus 1; High Pressure Liquid Chromatography; Human; Hyaluronic Acid; Iduronic Acid; Infection; Inorganic Sulfates; Ions; Isotope Labeling; Keratan Sulfate; Knowledge; Label; Lead; Liquid Chromatography; Liquid substance; Liver; Low-Molecular-Weight Heparin; Magnetism; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Minor; Modeling; Molecular Weight; NMR Spectroscopy; Names; Nuclear; Nuclear Magnetic Resonance; Oligosaccharides; Performance; Phase; Physiology; Play; Polyacrylamide Gel Electrophoresis; Polysaccharides; Proteoglycan; Research; Residual state; Resolution; Respiratory System; Role; SHFM1 gene; Scientist; Sequence Analysis; Serine; Skeletal system; Sodium; Son of Sevenless Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrum Analysis; Structure; Structure-Activity Relationship; Sucrose; System; Temperature; Time; Unspecified or Sulfate Ion Sulfates; Uridine Diphosphate; Variant; X-Ray Crystallography; Xylose; analytical method; animal tissue; base; bikunin; decorin; density; extracellular; fast protein liquid chromatography; improved; ion mobility; liquid chromatography mass spectrometry; member; molecular dynamics; novel strategies; polymerization; prevent; progesterone 11-hemisuccinate-(2-iodohistamine); proteoglycan core protein; public health relevance; receptor; sugar

DESCRIPTION (provided by applicant): Proteoglycans (PGs) play an important role in a variety of diseases affecting the excretory system, respiratory system, circulatory system, skeletal system, also the multisystem diseases of aging and cancer. Progress in the isolation, purification, and characterization of proteoglycans from extracellular matrix has led to the sequencing of proteoglycan core-proteins. The polysaccharide or glycosaminoglycan (GAG) components for which a proteoglycan is named dominates its chemistry and biology. Until now, the characterization of these GAG components has focused primarily on the identification of their family (i.e., chondroitin, dermatan, heparin, etc.), molecular weight, charge density and disaccharide analysis. We are developing a new approach to elevate the structural characterization of the GAG portion of the PG to the same level used on the core protein. Because of the polydispersity and structural similarity between GAGs within a given class, scientists originally viewed GAGs as molecules of undetermined structure (or sequence) and thus, GAGs were not considered informational molecules. There are structural differences between the core proteins of various PGs including members of a particular class. Thus, GAGs are not a continuum of molecules but rather are structurally distinct. The polysaccharide portion of PGs also has structurally distinctive domains. For example, defined tetrasaccharide sequences are present at the poly- saccharide's reducing end, where it attaches to its core protein. Specific residues terminate newly formed chondroitin sulfate chains. An unusual GlcN2S3S6S residue found within heparin, also marks the center of its binding site for antithrombin III. Two unusual residues, GlcN3S6S and GlcA2S, in the heparan sulfate (HS) receptor responsible for herpes simplex virus type 1 infection. The presence of such uniquely modified sugars within 10-50 kDa polysaccharide chains suggests that these molecules contain information. Biosynthetic studies have resulted in the identification of most of the enzymes involved in GAG synthesis but have not yet lead to clear understanding of what controls the placement of sequence information within a GAG. The specific aims of this focused proposal (07/09-06/14) are to: 1. Improve MS-based GAG microsequencing, including the development of new state-of-the-art and routine analytical methods widely available to biologists and chemists; 2. Elucidate secondary structure of GAGs and their complexes with GAG-binding proteins using NMR spectroscopy and X-ray crystallography; 3. Develop MS-based quantitative profiling of GAG-derived oligosaccharides using isotopically-labeled standards; 4. Sequence decorin using methods developed for bikunin; and 5. Sequence an animal tissue- derived HS using isotope-labeled bioengineered model HS and HS-oligosaccharide models. PUBLIC HEALTH RELEVANCE: The proposed effort impacts human health by developing methods to analyze and sequence the glycosaminoglycan chains of proteoglycans. Currently it is difficult to analyze and not possible to sequence these complex polysaccharides. This has prevented a firm understanding of the structure- activity relationship of this important class of biomacromolecules.

描述（由申请人提供）：蛋白聚糖（PGS）在影响排泄系统，呼吸系统，循环系统，骨骼系统的多种疾病中起着重要作用，以及衰老和癌症的多系统疾病。从细胞外基质中蛋白聚糖的隔离，纯化和表征的进展已导致蛋白聚糖核蛋白的测序。多糖或糖胺聚糖（GAG）组成的蛋白聚糖被命名为其化学和生物学。到目前为止，这些插科打数成分的表征主要集中在其家族（即软骨素，皮肤病，肝素等），分子量，电荷密度和二糖分析的鉴定上。我们正在开发一种新方法，将PG的插孔部分的结构表征提升到核心蛋白质上使用的相同水平。由于给定类别内的插科打no之间的多分散性和结构相似性，科学家最初将插科打术视为未确定结构（或序列）的分子，因此，插科打non被视为信息分子。包括特定类别的成员在内的各种PG的核心蛋白质之间存在结构差异。因此，插科打s不是分子的连续体，而是结构上的独特之处。 PGS的多糖部分也具有结构上独特的域。例如，定义的四糖序列存在于聚糖的还原端，并附着在其核心蛋白上。特定残基终止新形成的软骨素硫酸盐链。在肝素中发现的不寻常的GLCN2S3S6S残基也标志着其抗凝血酶III结合位点的中心。硫酸乙酰肝素（HS）受体中的两个不寻常的残基GLCN3S6S和GLCA2S，负责单纯疱疹病毒1型感染。在10-50 kDa多糖链中存在这种独特修饰的糖表明这些分子包含信息。生物合成研究导致鉴定出参与GAG合成的大多数酶，但尚未清楚地了解如何控制序列信息在GAG中的位置。该重点提案（07/09-06/14）的具体目的是：1。改善基于MS的GAG微钉，包括开发新的最先进的ART和常规分析方法，可为生物学家和化学家广泛使用； 2。使用NMR光谱和X射线晶体学阐明GAG的二级结构及其与GAG结合蛋白的复合物； 3。使用同位素标记的标准来开发基于MS的GAG衍生寡糖的定量分析； 4。使用用于比库宁开发的方法序列的序列； 5。使用同位素标记的生物工程模型HS和HS-寡糖模型序列的动物组织衍生的HS。公共卫生相关性：拟议的努力通过开发分析和对蛋白聚糖的糖胺聚糖链的方法来影响人类健康。目前，很难分析，无法对这些复杂的多糖进行测序。这阻止了对这一重要类型生物分子的结构活动关系的坚定理解。