Biosynthesis of Polysaccharides

多糖的生物合成

• 批准号: 8990010
• 负责人: Peng George Wang
• 金额: $ 29.6万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-03 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8990010
• 关键词: Accounting; Adjuvant; Aftercare; Anabolism; Antibiotic Resistance; Biochemical; Biomedical Research; Cell Wall; Cell surface; Characteristics; Chemicals; Complex; Core Assembly; Cytoplasm; Diagnosis; Diphosphates; Distal; Endotoxins; Enzymes; Escherichia coli; Escherichia coli Infections; Evaluation; Event; Fluorine; Foundations; Funding; Gram-Negative Bacteria; Grant; Health; Hospitalization; Human; Immune; Immunology; In Vitro; Individual; Infection; Investigation; Kidney Diseases; Length; Lipid A; Lipids; Lipopolysaccharide Biosynthesis Pathway; Lipopolysaccharides; Medical; Membrane; Modification; Mono-S; Morbidity - disease rate; Natural Immunity; Natural Resources; Nature; O Antigens; Oligosaccharides; Pathway interactions; Patients; Polymerase; Polymers; Polysaccharides; Prevalence; Prevention; Process; Proteins; Reaction; Recurrence; Research; Rest; Roentgen Rays; Side; Structure; TLR4 gene; Testing; Time; Toll-Like Receptor Pathway; Tube; Urinary tract infection; Urologic Diseases; Uropathogenic E. coli; Vaccine Adjuvant; Vaccine Design; Vaccines; Woman; analog; antimicrobial; base; biochemical tools; drug candidate; glycosyltransferase; men; microbial; mortality; novel; novel vaccines; periplasm; polymerization; programs; reconstitution; resistance mechanism; resistant strain; standard care; sugar; tool; vaccine candidate; vaccine development

DESCRIPTION (provided by applicant): This proposed program is a competitive renewal of grant R01 GM085267 titled "Biosynthesis of Polysaccharides", funded from 7/30/2009 - 7/29/2013. This program is our continuing efforts to use both chemical and biochemical tools to elucidate the mechanism of polysaccharide biosynthesis and to produce promising drug candidates for biomedical evaluation. Lipopolysaccharides (LPS) are characteristic components of cell walls of Gram-negative bacteria, localize in the outer leaflet of asymmetric outer membrane (OM) and expose on the cell surface. LPS typically consists of a hydrophobic domain known as Lipid A (or endotoxin), a nonrepeating core oligosaccharide (including both the inner core and outer core) and a distal polysaccharide (O-antigen or O-PS). The biosynthetic pathway to LPS consists of independent biosynthesis of O-antigen and Core-Lipid A, respectively, and combination of these two parts to make LPS, and then transport to OM. Thus, total biosynthesis of LPS in vitro includes three Milestone events: 1) assembly of O-PS; 2) assembly of Core-Lipid A; 3) finally assembly of LPS. The first Milestone has already been completed in the last funding period of this grant. Biosynthesis of O-antigen of LPS is a wzy-dependent pathway: the individual repeating oligosaccharide unit is synthesized in the cytoplasm by the sequential action of specific glycosyltransferases. The repeating unit is then transported to the periplasmic side of the membrane by Wzx where it is polymerized into a polysaccharide by the polymerase Wzy. The chain length of the polymer is regulated by an unknown mechanism that involves Wzz protein. We used purified enzyme Wzy and Wzz to reconstitute such polymerization process, and for the first time, achieved the synthesis of polysaccharides in a test tube! Moreover, the enzyme WaaL (which transfers the polysaccharide from its diphosphate-lipid precursor to core-lipid A) was found to accept almost any structures of sugar-diphosphate-lipid donors. These results lay out an excellent foundation for accomplishing the remaining two milestones. For Milestone 2 of assembly of Core-lipid A, we will chemically synthesize a number of Lipid A molecules. The glycosyltransferases involved in the biosynthesis of core oligosaccharide will be over-expressed and used for in vitro sequential assembly of Core-Lipid A. The synthesized chemically defined Lipid A and Core-Lipid A molecules are not only useful research tools for studying LPS biosynthesis, but also potential vaccine adjuvants. Finally, Milestone 3 will be achieved by transferring the O-PS to the Core-Lipid A by WaaL to produce full LPS. Based on the understanding of Lipid A-TLR4/MD2 complex, many Lipid A molecules and Lipid A analogs have been found to have strong immune activities and can be used as adjuvant either alone or with other adjuvants in a variety of vaccine formulations. Thus, we hypothesize that our chemo-enzymatically constructed E. coli Inner Core-Lipid A and Core-Lipid A conjugates would represent as a novel set of wide- spectrum and stand-alone vaccine candidates with defined structures for prevention of urinary tract infection (UTI). Specifically, he program includes the following aims: Milestone 1: Our efforts in the biosynthesis of O-PS will include the synthesis of O-PS from two most commonly used uropathogenic E. coli (UPEC) strain CFT073 (O6:K2:H1) and UTI89 (O18:K1), and X-ray structure determination of WaaL and Wzy. Milestone 2: Chemically synthesize both E. coli di- and mono-phosphorylated lipid A with either tetraacylated or hexaacylated lipids, or with fluorine-containing hexaacylated lipid (total f 9 lipid A compounds), then transfer either E. coli R3 or R1 core oligosaccharides to these lipid A structures by following the biosynthetic pathway using sequential glycosyltransferase-catalyzed reactions. Milestone 3: Total assembly of E. coli O86 LPS, and other natural and chimeric LPS structures by WaaL catalyzed transformation. Successful execution of this research program should provide two unmet biomedical needs. Although LPS is one of widely used biochemicals in immunology and other biomedical research, there is essentially no pure LPS available. All the commercial LPS coming from isolation and purification from natural resources are inevitably a mixture. This program will achieve the total chemo-enzymatic synthesis of LPS, and open the field to investigate the structure-activity relation of LPS with its TLR4-MD2 complex. Moreover, the reconstituted, synthetic core-lipid A structures are novel wide-spectrum and stand-alone vaccine candidates against urinary tract infection caused by uropathogenic E. coli.

描述（由申请人提供）：该拟议计划是一个竞争性的赠款R 01 GM 085267更新，题为“多糖的生物合成”，从2009年7月30日至2013年7月29日资助。该计划是我们持续努力使用化学和生物化学工具来阐明多糖生物合成的机制，并产生有前途的候选药物进行生物医学评价。 脂多糖（Lipopolysaccharides，LPS）是革兰氏阴性菌细胞壁的特征性成分，定位于不对称外膜（asymmetric outer membrane，OM）的外叶，暴露于细胞表面。LPS通常由称为脂质A（或内毒素）的疏水结构域、非重复核心寡糖（包括内核和外核）和远端多糖（O-抗原或O-PS）组成。LPS的生物合成途径包括O-抗原和核心脂质A的独立生物合成，以及这两个部分的结合产生LPS，然后转运到OM。因此，体外LPS的总生物合成包括三个里程碑事件：1）O-PS的组装; 2）核心-脂质A的组装; 3）LPS的最终组装。 第一个里程碑已经在本补助金的最后一个资助期内完成。LPS的O-抗原的生物合成是一个wzy依赖性途径：通过特异性糖基转移酶的顺序作用，在细胞质中合成单个重复寡糖单元。然后重复单元通过Wzx转运到膜的周质侧，在那里它通过聚合酶Wzy聚合成多糖。聚合物的链长由涉及Wzz蛋白的未知机制调节。我们利用纯化的Wzy和Wzz酶对这一聚合过程进行了重组，首次实现了试管内多糖的合成。此外，发现酶WaaL（其将多糖从其二磷酸-脂质前体转移到核心-脂质A）接受糖-二磷酸-脂质供体的几乎任何结构。这些成果为实现其余两个里程碑奠定了良好的基础。 对于核心-脂质A组装的里程碑2，我们将化学合成许多脂质A分子。参与核心寡糖生物合成的糖基转移酶将过表达并用于核心脂质A的体外顺序组装。合成的化学上确定的Lipid A和Core-Lipid A分子不仅是研究LPS生物合成的有用工具，而且是潜在的疫苗佐剂。最后，里程碑3将通过WaaL将O-PS转移到核心脂质A以产生完全LPS来实现。 基于对Lipid A-TLR 4/MD 2复合物的理解，已经发现许多Lipid A分子和Lipid A类似物具有强免疫活性，并且可以单独或与其他佐剂一起在各种疫苗制剂中用作佐剂。因此，我们假设我们的化学-酶法构建的E。大肠杆菌内核-脂质A和内核-脂质A缀合物将代表一组新的广谱和独立的候选疫苗，其具有用于预防尿路感染（UTI）的确定的结构。 具体而言，该计划包括以下目标：里程碑1：我们在O-PS生物合成方面的努力将包括从两种最常用的致尿病大肠杆菌合成O-PS。大肠杆菌（UPEC）菌株CFT 073（O 6：K2：H1）和UTI 89（O 18：K1）的结构，并对WaaL和Wzy进行了X射线结构测定。里程碑2：化学合成E. coli二磷酸化和单磷酸化的脂质A与四酰化或六酰化的脂质或与含氟的六酰化的脂质（总f 9脂质A化合物），然后将E. coli R3或R1核心寡糖与这些脂质A结构通过使用连续糖基转移酶催化反应的生物合成途径。里程碑3：E. coli O 86 LPS和其它天然和嵌合LPS结构。 这项研究计划的成功执行应提供两个未满足的生物医学需求。虽然LPS是免疫学和其他生物医学研究中广泛使用的生物化学品之一，但基本上没有纯的LPS可用。所有来自天然资源的分离和纯化的商业LPS不可避免地是混合物。本研究实现了LPS的化学-酶法全合成，为进一步研究LPS及其TLR 4-MD 2复合物的构效关系奠定了基础。此外，重组的合成核心脂质A结构是针对由尿路致病性大肠杆菌引起的尿路感染的新的广谱和独立的疫苗候选物。杆菌