Mechanism of Beta-Glucan Biosynthesis in Higher Plants

在高等植物中，(1,3)- b -葡聚糖的生物合成是由质膜结合酶胼胝质合成酶（UDP-Glc: (1,3)- b -葡聚糖合成酶）催化的。胼胝质合酶活性在各种植物的分离膜组分中普遍存在，并且被假设为纤维素合酶的一种不受调节的形式。因此，为了了解细胞壁多糖生物生成和创面愈合调控的生化基础，有必要对愈伤组织合酶等多糖合酶的多肽亚基及其编码基因进行鉴定和表征。虽然胼胝质合酶已被证明是一种难以通过标准蛋白质分离技术纯化到同质性的酶，但在从各种来源鉴定胼胝质合酶多肽成分方面取得了快速进展。将我们的系统、Beta vulgaris L.和其他植物的多肽谱进行比较，发现了共同多肽谱的出现。在过去的两年中，通过光亲和标记，改进富集程序和最近的多肽消耗，我们已经确定了57-，31-和29-kD的udp - glc结合亚基，以及一组92,83,43和27 kD的内在疏水性多肽。我们的工作假设，在此基础上提出的实验是预测，是一个或多个这些多肽是一个多亚基胼胝质合成酶复合物的一部分。这个项目将沿着两条路线进行。首先，我们将继续努力明确地识别胼胝质合成酶亚基，并将包括多肽耗尽实验，使用确定边长的囊泡进行地形分析，使用叠氮探针进行光标记研究和有限的蛋白质水解。我们将继续培养针对单个酶亚基的抗体。其次，我们计划克隆一个编码亚基的结构基因并对其进行测序，该亚基是酶活性所必需的。胼胝体合酶亚基的鉴定和克隆将为解决（1,3）和（1,4）b链葡聚糖是由单一酶还是由不同的酶合成的长期问题铺平道路，每种酶产生不同的连锁类型。纤维素合酶亚基氨基酸序列和cDNA的可用性将揭示与已知细菌纤维素合酶基因是否存在序列同源性，从而确定胼胝质和纤维素合酶在结构上的相关程度。本研究也将为细胞壁生物发生过程中基因表达的研究铺平道路。纤维素（1,4- b - d -葡聚糖）是世界上最丰富的大分子，估计每年生物合成1011吨。它是植物细胞壁的主要组成部分，提供结构完整性和对入侵植物病原体的保护。胼胝质是纤维素的(1,3)- b链对应物，在生理上很重要，因为它的合成是在受伤和感染的反应中诱导的。胼胝质也存在于植物结构中，如花粉管、筛子植物和一些单子叶植物的细胞壁中。从植物和酵母中提取的b -葡聚糖由于其独特的农业、生理和营养特性而具有重要的经济价值。最近的研究表明，燕麦中的混合链b -葡聚糖(（1,3),(1,4)- b -葡聚糖）和酵母中的(1,3)- b -葡聚糖在饮食中加入显著水平时具有降低高胆固醇个体胆固醇的能力。制药业对真菌葡聚糖合成酶很感兴趣，因为它们代表了治疗全身真菌感染所需的新药的潜在分子靶点。我们对产生葡聚糖的生化机制知之甚少。本项目的重点是扩大我们对(1,3)- b -葡聚糖合成酶（俗称胼胝质合成酶）的结构和功能的了解，这是一种普遍存在于高等植物中的细胞壁生物合成酶复合物。近期，一个目标是继续应用基础蛋白质化学，在明确鉴定CS的蛋白质成分方面取得进一步进展。此外，我们对CS复合物的了解已经达到了分子生物学成分得到保证的程度。因此，第二个目标是克隆CS复合物的选定组分，以获得推断出的CS多肽的氨基酸序列。然后，我们将确定是否存在相似之处，从纤维素生产微生物纤维素合酶的已知序列。这将表明纤维素和CSs在结构上是否相关。这项研究也将有助于更好地理解胼胝质沉积作为伤口反应的一部分。研究植物源性生物聚合物的生物合成具有重要的科学和经济意义。美国的实验室继续在这些努力中发挥重要作用是至关重要的。

The biosynthesis of (1,3)-B-linked glucan in higher plants is catalyzed by the plasma membrane-bound enzyme callose synthase (UDP-Glc: (1,3)-B-glucan synthase). Callose synthase activity is ubiquitous in isolated membrane fractions from a wide variety of plants, and has been hypothesized to be a deregulated form of cellulose synthase. Therefore, to understand the biochemical basis for the regulation of cell wall polysaccharide biogenesis and wound healing, it is necesary to identify and characterize the polypeptide subunits of polysaccharide synthases such as calloses synthase, and the genes which encode them. Although callose synthase has proven to be a stubborn enzyme to purify to homogeneity by standard protein fractionation techniques, rapid progress has been made towards identifying the polypeptide components of callose synthases from various sources. Comparisons of polypeptide profiles between our system, Beta vulgaris L., and other plants show the emergence of common polypeptide profiles. During the previous two years, through photoaffinity labeling, improved enrichment procedures and most recently polypeptide depletion, we have identified UDP-Glc-binding subunits of 57-,31- and 29-kD, as well as a group of instrinsic hydrophobic polypeptides of 92, 83, 43 and 27 kD. Our working hypothesis, upon which the proposed experiments are predicated, is that the one or more of these polypeptidesare part of a multi-subunit callose synthase complex. This project will proceed along two lines. The first will be a continuation of our efforts to unambiguously identify callose synthase subunits, and will consist of polypeptide-depletion experiments, topographic analysis using vesicles of defined sidedness, photolabeling studies using azido-probes and limited proteolysis. We will continue to raise antibodies against individual enzyme subunits. Second, we plan to clone and sequence a structural gene(s) encoding subunit shown to be required for enzyme activity. Identification and cloning of callose synthase subunits should pave the way for resolving the longstanding question of whether (1,3) and (1,4) B-linked glucans are biosynthesized by a single enzyme or distinct enzymes where each produces a distinct linkage type. The availability of amino acid sequences and cDNA for callose synthase subunits will reveal whether sequence homologies exist with known bacterial cellulose synthase genes, and therefore define the extent to which callose and cellulose synthases are structurally related. This research will also help pave the way to study gene expression during cell wall biogenesis. %%% Cellulose (1,4-B-D-glucan) is the world's most abundant macromolecule, with an estimated 1011 tons biosynthesized per year. It is a major component of plant cell walls providing structural integrity and protection against invading plant pathogens. Callose, the (1,3)-B-linked counterpart of cellulose, is physiologically important since its synthesis is induced in response to wounding and infection. Callose is also found in plant structures such as pollen tubes, sieve plants and in the cell walls of some monocots. B-glucans from plants and yeast are are economically important due to their unique agrilcultural, physiological and nutritional properties. Recent studies show that B-Glucans of mixed linkage ((1,3),(1,4)-B-glucan) from oats and (1,3)-B-glucan from yeast have the ability to lower cholesterol in hypercholesteremic individuals when significant levels are incorporated into the diet. The pharmaceutical industry is interested in fungal glucan synthases because they represent potential molecular targets for new drugs which are needed to treat systemic fungal infections. Very little is known about the biochemical mechanism by which glucans are produced. This project focuses on expanding our knowledge of the structure and function of (1,3)-B-glucan synthase, commonly known as callose synthase, a cell wall biosynthetic enzyme complex ubiquitous in higher plants. In the near-term, one objective is continued application of basic protein chemistry to make further progress towards the unambiguous identification of the protein components of CS. In addition, our knowledge of the CS complex has reached the point where a molecular biology component is warranted. Thus, a second objective is to clone selected components of the CS complex in order to obtain deduced amino acid sequences for CS polypeptides. We will then determine whether similarities exist with known sequences of cellulose synthase from cellulose-producing microorganisms. This would indicate whether cellulose and CSs are structurally related. This research will also help to gain an improved understanding of callose deposition as part of the wound response. Research on the biosynthesis of plant-derived biopolymers has important scientific and economic implications. It is critical for U.S.-based labs to continue to play a significant role in these efforts.