Biosynthesis and transbilayer flipping of mycobacterial PIM glycolipids

分枝杆菌 PIM 糖脂的生物合成和跨双层翻转

• 批准号: 7511622
• 负责人: DELPHI CHATTERJEE
• 金额: $ 17.84万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-15 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7511622
• 关键词: Active Sites; Address; Anabolism; Animal Model; Biochemical; Biogenesis; Bioinformatics; Biology; Candidate Disease Gene; Cell Wall; Cell membrane; Cell surface; Core Assembly; Cytoplasm; Detergents; Disease; Dolichol Monophosphate Mannose; Enzymes; Event; Experimental Models; Face; Family; Fatty Acids; Future; Gap Junctions; Genus Mycobacterium; Glycoconjugates; Glycolipids; Guanosine Diphosphate Mannose; Human; Knock-out; Lecithin; Light; Lipids; Mannose; Mannosyltransferases; Membrane; Membrane Proteins; Molecular; Mycobacterium smegmatis; Mycobacterium tuberculosis; Names; Nexus (resin cement); Organism; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Phosphatidylinositols; Phospholipids; Play; Principal Investigator; Proteins; Public Health; Rate; Reaction; Risk; Role; Staging; Structure; System; Transferase; Work; cell envelope; extracellular; glycosyltransferase; inorganic phosphate; interest; lipoarabinomannan; lipomannan; macromolecule; mutant; mycobacterial; periplasm; phosphatidylinositol mannoside; programs; proteoliposomes; reconstitution; sugar nucleotide

DESCRIPTION (provided by applicant): Mycobacteria species, exemplified by M. tuberculosis and M. leprae are the cause of major diseases in humans. In this R21 `high risk/high impact' application we propose an exploratory program to define early steps in the biosynthesis of phosphatidylinositol mannosides (PIMs). PIMs are a family of mannolipids that plays a critical role in the pathogenesis of mycobacteria, because they are biosynthetic precursors in the assembly of the key cell surface glycoconjugates lipomannan (LM) and lipoarabinomannan (LAM). PIM biosynthesis occurs in the cytoplasmic membrane and there is considerable evidence to suggest that it occurs by the sequential addition of mannose residues to PI. We hypothesize that the first three mannosyltransfer reactions of PIM biosynthesis occur on the cytoplasmic face of the membrane and use GDP-mannose as the mannosyl donor; these reactions are catalyzed by the mannosyltransferases (ManTs) PimA, PimB and an undefined enzyme that we term PimC*. We further propose that subsequent mannosylation reactions use polyprenol-phosphate-mannose (PPM) as the mannosyl donor and occur on the membrane's periplasmic face. The enzyme that catalyzes the first of these periplasmically oriented reactions is unknown; we refer to it as PimD*. To accommodate the topological split in PIM biosynthesis, we propose that the trimannosylated glycolipid (AcPIM3) produced by PimC* must flip across the membrane in order to be further mannosylated. Since non-catalyzed phospholipid flipping is a rare event, we propose that AcPIM3 flipping requires a lipid translocator or flippase (referred to herein as Flp*), a protein that remains to be identified. The nexus of three steps surrounding the topological split in the PIM assembly pathway thus remains to be defined. In this "proof of principal" project evolving from close interactions between the three principal investigators over the last year, we propose two specific aims to identify the protein components required for these steps. Our studies will exploit the experimental model Mycobacterium smegmatis in conjunction with a multi-faceted approach including bioinformatics, mutational analysis, and biochemical reconstitution of transport activity. Our aims are: 1) To identify PimC* and PimD*, the ManTs that catalyze the mannosyltransfer reactions that occur immediately prior to and after the transbilayer translocation of a PIM intermediate. 2) To develop a reconstitution system that recapitulates Flp*-catalyzed PIM flip- flop and to use this system to identify Flp*, the flippase responsible for flipping AcPIM3 across the membrane. Thus the unifying theme of this proposal encompasses filling in `missing steps' in the biogenesis of PIMs, establish OUR hypothesized pathway, all in relation to identifying unique, extracellular targets. PUBLIC HEALTH RELEVANCE: Herein, two exploratory specific aims are proposed to address the vital missing gaps in the assembly of core glycolipids in Mycobacterium.

描述（由申请人提供）：结核分枝杆菌和麻风大麻分枝杆菌的分枝杆菌是人类主要疾病的原因。在此R21“高风险/高影响力”应用中，我们提出了一个探索性计划，以定义磷脂酰肌醇甘露糖苷（PIMS）的生物合成的早期步骤。 PIM是一个在分枝杆菌的发病机理中起着至关重要的作用，因为它们是钥匙细胞表面糖缀合物lipomannan（LM）和脂肪肉骨氨基氨基甲群（LAM）的生物合成前体（LAM）。 PIM生物合成发生在细胞质膜中，并且有大量证据表明，它是通过在PI中的顺序添加甘露糖残基而发生的。我们假设PIM生物合成的前三个甘露糖基转移反应发生在膜的细胞质面上，并使用GDP-甘露糖作为甘露糖基供体。这些反应是由甘露糖基转移酶（MANTS）PIMA，PIMB和我们称为PIMC*的不确定酶催化的。我们进一步提出，随后的甘露糖基化反应使用聚丙烯 - 磷酸甘露糖（PPM）作为甘露糖基供体，并出现在膜的周质面部。这些催化这些周围定向反应中的第一个酶是未知的。我们称其为PIMD*。为了适应PIM生物合成中的拓扑分裂，我们提出，PIMC*产生的三甘南糖基化的糖脂（ACPIM3）必须在整个膜上翻转，以便进一步甘露糖基化。由于非催化的磷脂翻转是罕见的事件，因此我们提出ACPIM3翻转需要脂质转运剂或Flippase（此处称为FLP*），这是一种尚待鉴定的蛋白质。因此，围绕PIM组件途径中拓扑拆分的三个步骤的联系尚待定义。在过去一年的三位主要研究人员之间的密切相互作用中，我们提出了两个具体的目的，以确定这些步骤所需的蛋白质成分。我们的研究将利用实验模型分枝杆菌与多方面的方法结合使用，包括生物信息学，突变分析和运输活性的生化重构。我们的目的是：1）识别PIMC*和PIMD*，这是催化PIM中间体的跨贝元易位之前和之后发生的甘露糖基转移反应。 2）开发一个重新概括flp*催化的pim flop的重构系统，并使用该系统识别FLP*，这是负责翻转ACPIM3的Flippase*。因此，该提案的统一主题包括PIM生物发生中“缺失步骤”的填充，建立了我们的假设途径，这都是与识别独特的细胞外靶标有关的。公共卫生相关性：在本文中，提出了两个探索性特定目标，以解决分枝杆菌核心糖脂组装中的重要缺失差距。