Biosynthesis of Polysaccharides

描述（由申请人提供）：该项目是R01 GM085267“多糖生物合成”项目的竞争性续期，资助时间为2009年7月30日至2013年7月29日。这个项目是我们继续努力使用化学和生化工具来阐明多糖生物合成的机制，并生产有希望的生物医学评价候选药物。脂多糖（lipopolaccharides， LPS）是革兰氏阴性菌细胞壁的特征成分，定位于不对称外膜（asymmetric outer membrane， OM）的外小叶，并暴露在细胞表面。LPS通常由疏水结构域脂质a（或内毒素）、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。脂多糖的生物合成途径是由o抗原和core -脂质A分别独立生物合成，并将两者结合合成脂多糖，然后转运到OM。因此，LPS的体外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)最后组装LPS。第一个里程碑项目已经在本次资助的最后一个资助期完成。脂多糖o抗原的生物合成是一种依赖于wzy的途径：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的低聚糖单元。然后，重复单元通过Wzx被运送到膜的质周侧，在那里它被聚合酶Wzy聚合成多糖。这种聚合物的链长受一种未知的机制调控，其中包括Wzz蛋白。我们用纯化的酶Wzy和Wzz对这种聚合过程进行了重构，首次在试管中实现了多糖的合成！此外，WaaL酶（将多糖从二磷酸脂前体转移到核心脂质A）被发现几乎可以接受糖二磷酸脂供体的任何结构。这些结果为完成剩下的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，我们将化学合成一些脂质A分子。参与核心寡糖生物合成的糖基转移酶将被过表达并用于体外序列组装核心脂质A。合成的化学定义的脂质A和核心脂质A分子不仅是研究LPS生物合成的有用研究工具，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以产生完整的LPS。基于对脂质A- tlr4 /MD2复合物的了解，许多脂质A分子和脂质A类似物已被发现具有很强的免疫活性，可以单独或与其他佐剂一起用作多种疫苗配方中的佐剂。因此，我们假设我们的化学酶构建的大肠杆菌内核-脂质A和核心-脂质A偶联物将代表一组新的广谱和独立的候选疫苗，具有明确的结构，用于预防尿路感染（UTI）。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尿路致病性大肠杆菌（UPEC）菌株CFT073 （O6:K2:H1）和UTI89 （O18:K1）中合成O-PS，以及确定WaaL和Wzy的x射线结构。里程碑2：用四酰基化或六酰基化脂质，或含氟六酰基化脂质（共9种脂质A化合物）化学合成大肠杆菌二磷酸化和单磷酸化脂质A，然后通过连续的糖基转移酶催化反应，通过生物合成途径将大肠杆菌R3或R1核心低聚糖转移到这些脂质A结构中。里程碑3：通过WaaL催化转化完全组装大肠杆菌O86 LPS和其他天然和嵌合LPS结构。该研究计划的成功实施应提供两个未满足的生物医学需求。虽然脂多糖是一种广泛应用于免疫学和其他生物医学研究的生化物质，但基本上没有纯脂多糖。所有从自然资源中分离提纯出来的商业LPS都不可避免地是一种混合物。本项目将实现LPS的全化学-酶合成，为研究LPS与其TLR4-MD2复合物的构效关系开辟领域。此外，重组的合成核心脂质A结构是抗尿路致病性大肠杆菌引起的尿路感染的新型广谱和独立的候选疫苗。

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.