{"title":"利用体外 GT 阵列(i-GT-ray)平台对 β-聚糖合成酶的新认识","authors":"","doi":"10.1016/j.plaphy.2024.109052","DOIUrl":null,"url":null,"abstract":"<div><p>Cellulose and hemicellulose are the major structural β-glycan polysaccharides in cell walls of land plants. They are characterized by a backbone of β-(1,3)- and/or β-(1,4)-linked sugars such as glucose, mannose, or xylose. The backbones of these polymers are produced by processive glycosyltransferases (GTs) called synthases having multiple transmembrane domains anchoring them to the membrane. Thus, they are among the most difficult membrane proteins to test <em>in vitro</em> and to purify. Recently, we developed an <em>in vitro</em> GT-array (<em>i</em>-GTray) platform and showed that non-processive type II membrane GTs could be produced <em>via</em> cell-free system in a soluble and active form and tested in this platform. To determine whether <em>i</em>-GT-ray platform is adequate for the production and testing of β-glycan synthases, we tested five synthases involved in cellulose, xyloglucan, (gluco)mannan, and β-(1,3)(1,4)-mixed-linkage glucan synthesis. Our results revealed unsuspected features of these enzymes. For example, all these synthases could be produced in a soluble and active form and are active in the absence of detergent or membrane lipids, and none of them required a primer for initiation of synthesis. All synthases produced ethanol-insoluble products that were susceptible to the appropriate hydrolases (i.e., cellulase, lichenase, mannanase). Using this platform, we showed that <em>At</em>CslC4 and <em>At</em>XXT1 interact directly to form an active xyloglucan synthase that produced xylosylated cello-oligosaccharides (up to three xylosyl residues) when supplied with UDP-Glc and UDP-Xyl. <em>i</em>-GTray platform represents a simple and powerful functional genomics tool for discovery of new insights of synthase activities and can be adapted to other enzymes.</p></div>","PeriodicalId":20234,"journal":{"name":"Plant Physiology and Biochemistry","volume":null,"pages":null},"PeriodicalIF":6.1000,"publicationDate":"2024-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0981942824007204/pdfft?md5=ef92edfbe81567f9f648dae6420d70f5&pid=1-s2.0-S0981942824007204-main.pdf","citationCount":"0","resultStr":"{\"title\":\"New insights on β-glycan synthases using in vitro GT-array (i-GT-ray) platform\",\"authors\":\"\",\"doi\":\"10.1016/j.plaphy.2024.109052\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Cellulose and hemicellulose are the major structural β-glycan polysaccharides in cell walls of land plants. They are characterized by a backbone of β-(1,3)- and/or β-(1,4)-linked sugars such as glucose, mannose, or xylose. The backbones of these polymers are produced by processive glycosyltransferases (GTs) called synthases having multiple transmembrane domains anchoring them to the membrane. Thus, they are among the most difficult membrane proteins to test <em>in vitro</em> and to purify. Recently, we developed an <em>in vitro</em> GT-array (<em>i</em>-GTray) platform and showed that non-processive type II membrane GTs could be produced <em>via</em> cell-free system in a soluble and active form and tested in this platform. To determine whether <em>i</em>-GT-ray platform is adequate for the production and testing of β-glycan synthases, we tested five synthases involved in cellulose, xyloglucan, (gluco)mannan, and β-(1,3)(1,4)-mixed-linkage glucan synthesis. Our results revealed unsuspected features of these enzymes. For example, all these synthases could be produced in a soluble and active form and are active in the absence of detergent or membrane lipids, and none of them required a primer for initiation of synthesis. All synthases produced ethanol-insoluble products that were susceptible to the appropriate hydrolases (i.e., cellulase, lichenase, mannanase). Using this platform, we showed that <em>At</em>CslC4 and <em>At</em>XXT1 interact directly to form an active xyloglucan synthase that produced xylosylated cello-oligosaccharides (up to three xylosyl residues) when supplied with UDP-Glc and UDP-Xyl. <em>i</em>-GTray platform represents a simple and powerful functional genomics tool for discovery of new insights of synthase activities and can be adapted to other enzymes.</p></div>\",\"PeriodicalId\":20234,\"journal\":{\"name\":\"Plant Physiology and Biochemistry\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":6.1000,\"publicationDate\":\"2024-08-17\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S0981942824007204/pdfft?md5=ef92edfbe81567f9f648dae6420d70f5&pid=1-s2.0-S0981942824007204-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Plant Physiology and Biochemistry\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0981942824007204\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"PLANT SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Plant Physiology and Biochemistry","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0981942824007204","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PLANT SCIENCES","Score":null,"Total":0}
New insights on β-glycan synthases using in vitro GT-array (i-GT-ray) platform
Cellulose and hemicellulose are the major structural β-glycan polysaccharides in cell walls of land plants. They are characterized by a backbone of β-(1,3)- and/or β-(1,4)-linked sugars such as glucose, mannose, or xylose. The backbones of these polymers are produced by processive glycosyltransferases (GTs) called synthases having multiple transmembrane domains anchoring them to the membrane. Thus, they are among the most difficult membrane proteins to test in vitro and to purify. Recently, we developed an in vitro GT-array (i-GTray) platform and showed that non-processive type II membrane GTs could be produced via cell-free system in a soluble and active form and tested in this platform. To determine whether i-GT-ray platform is adequate for the production and testing of β-glycan synthases, we tested five synthases involved in cellulose, xyloglucan, (gluco)mannan, and β-(1,3)(1,4)-mixed-linkage glucan synthesis. Our results revealed unsuspected features of these enzymes. For example, all these synthases could be produced in a soluble and active form and are active in the absence of detergent or membrane lipids, and none of them required a primer for initiation of synthesis. All synthases produced ethanol-insoluble products that were susceptible to the appropriate hydrolases (i.e., cellulase, lichenase, mannanase). Using this platform, we showed that AtCslC4 and AtXXT1 interact directly to form an active xyloglucan synthase that produced xylosylated cello-oligosaccharides (up to three xylosyl residues) when supplied with UDP-Glc and UDP-Xyl. i-GTray platform represents a simple and powerful functional genomics tool for discovery of new insights of synthase activities and can be adapted to other enzymes.
期刊介绍:
Plant Physiology and Biochemistry publishes original theoretical, experimental and technical contributions in the various fields of plant physiology (biochemistry, physiology, structure, genetics, plant-microbe interactions, etc.) at diverse levels of integration (molecular, subcellular, cellular, organ, whole plant, environmental). Opinions expressed in the journal are the sole responsibility of the authors and publication does not imply the editors'' agreement.
Manuscripts describing molecular-genetic and/or gene expression data that are not integrated with biochemical analysis and/or actual measurements of plant physiological processes are not suitable for PPB. Also "Omics" studies (transcriptomics, proteomics, metabolomics, etc.) reporting descriptive analysis without an element of functional validation assays, will not be considered. Similarly, applied agronomic or phytochemical studies that generate no new, fundamental insights in plant physiological and/or biochemical processes are not suitable for publication in PPB.
Plant Physiology and Biochemistry publishes several types of articles: Reviews, Papers and Short Papers. Articles for Reviews are either invited by the editor or proposed by the authors for the editor''s prior agreement. Reviews should not exceed 40 typewritten pages and Short Papers no more than approximately 8 typewritten pages. The fundamental character of Plant Physiology and Biochemistry remains that of a journal for original results.