Characterization and engineering of Drosophila melanogaster β1-3-galactosyltransferase for glycoengineering applications

IF 3.4 3区化学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Carbohydrate Research Pub Date : 2026-02-01 Epub Date: 2025-12-12 DOI:10.1016/j.carres.2025.109791

Linhan Wang , Jichao Wei , Fang Yuan , Yongheng Rong , Wanqian Du , Wenjing Shi , Xintan Wang , Mei Wang , Yankang Wang , Anran Liu , Junqiao Zhao , Yun Kong , Na Sun , Wenzhu Tang , Shengjun Wang

{"title":"Characterization and engineering of Drosophila melanogaster β1-3-galactosyltransferase for glycoengineering applications","authors":"Linhan Wang , Jichao Wei , Fang Yuan , Yongheng Rong , Wanqian Du , Wenjing Shi , Xintan Wang , Mei Wang , Yankang Wang , Anran Liu , Junqiao Zhao , Yun Kong , Na Sun , Wenzhu Tang , Shengjun Wang","doi":"10.1016/j.carres.2025.109791","DOIUrl":null,"url":null,"abstract":"<div><div>β1-3-Galactosyltransferase from <em>Drosophila melanogaster</em> (<em>Dm</em>C1GalT1) plays a key role in O-glycan synthesis and holds considerable potential for glycoengineering applications. In this study, we achieved high-yield expression and purification of <em>Dm</em>C1GalT1 in <em>Escherichia coli,</em> obtaining over 5 mg of protein per liter of culture. Similar with the enzyme expressed from <em>Pichia pastoris</em>, this enzyme exhibited strict donor specificity towards UDP-Gal and efficiently galactosylated human CD74-derived Tn-glycopeptides. Structural analysis identified key residues involved in substrate binding and catalysis. Site-directed mutagenesis of active-site residues yielded variants with altered activity and stability profiles. Notably, the N108G and Y325W mutants retained high activity towards UDP-Gal but lost the ability to utilize UDP-GalNH<sub>2</sub>, while N108A mutants retained 61.77 % of its activity toward UDP-Gal but maintained similar activity levels towards UDP-GalNH<sub>2</sub>, while gaining weak but detectable activity toward UDP-Glc. These findings highlight the critical roles of residues N108 and Y325 in donor recognition. Thermal stability predictions using ProStab indicated that certain mutations affected enzyme stability without compromising activity. This work establishes an efficient platform for the expression, characterization, and engineering of <em>Dm</em>C1GalT1, facilitating the development of glycosyltransferase variants with tailored properties for synthetic glycobiology.</div></div>","PeriodicalId":9415,"journal":{"name":"Carbohydrate Research","volume":"560 ","pages":"Article 109791"},"PeriodicalIF":3.4000,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Carbohydrate Research","FirstCategoryId":"92","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0008621525004173","RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/12/12 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}

引用次数: 0

Abstract

β1-3-Galactosyltransferase from Drosophila melanogaster (DmC1GalT1) plays a key role in O-glycan synthesis and holds considerable potential for glycoengineering applications. In this study, we achieved high-yield expression and purification of DmC1GalT1 in Escherichia coli, obtaining over 5 mg of protein per liter of culture. Similar with the enzyme expressed from Pichia pastoris, this enzyme exhibited strict donor specificity towards UDP-Gal and efficiently galactosylated human CD74-derived Tn-glycopeptides. Structural analysis identified key residues involved in substrate binding and catalysis. Site-directed mutagenesis of active-site residues yielded variants with altered activity and stability profiles. Notably, the N108G and Y325W mutants retained high activity towards UDP-Gal but lost the ability to utilize UDP-GalNH₂, while N108A mutants retained 61.77 % of its activity toward UDP-Gal but maintained similar activity levels towards UDP-GalNH₂, while gaining weak but detectable activity toward UDP-Glc. These findings highlight the critical roles of residues N108 and Y325 in donor recognition. Thermal stability predictions using ProStab indicated that certain mutations affected enzyme stability without compromising activity. This work establishes an efficient platform for the expression, characterization, and engineering of DmC1GalT1, facilitating the development of glycosyltransferase variants with tailored properties for synthetic glycobiology.

Abstract Image

查看原文本刊更多论文

黑胃果蝇β1-3-半乳糖基转移酶的表征与工程应用。

黑腹果蝇β1-3-半乳糖转移酶（DmC1GalT1）在o -聚糖合成中起着关键作用，在糖工程方面具有相当大的应用潜力。在本研究中，我们在大肠杆菌中实现了DmC1GalT1的高产表达和纯化，每升培养液中蛋白含量超过5 mg。与毕赤酵母中表达的酶类似，该酶对UDP-Gal具有严格的供体特异性，并能有效地半乳糖化人cd74衍生的n-糖肽。结构分析确定了参与底物结合和催化的关键残基。活性位点残基的定点诱变产生了具有改变的活性和稳定性的变异。值得注意的是，N108G和Y325W突变体对UDP-Gal保持了高活性，但失去了利用UDP-GalNH2的能力，而N108A突变体对UDP-Gal保持了61.77%的活性，但对UDP-GalNH2保持了相似的活性水平，而对UDP-Glc的活性较弱，但可检测到。这些发现突出了N108和Y325残基在供体识别中的关键作用。使用ProStab进行热稳定性预测表明，某些突变会影响酶的稳定性，但不会影响酶的活性。这项工作为DmC1GalT1的表达、表征和工程建立了一个有效的平台，促进了糖基转移酶变体的开发，这些变体具有适合合成糖生物学的特性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Carbohydrate Research 化学-生化与分子生物学

CiteScore

5.00

自引率

3.20%

发文量

183

审稿时长

3.6 weeks

期刊介绍： Carbohydrate Research publishes reports of original research in the following areas of carbohydrate science: action of enzymes, analytical chemistry, biochemistry (biosynthesis, degradation, structural and functional biochemistry, conformation, molecular recognition, enzyme mechanisms, carbohydrate-processing enzymes, including glycosidases and glycosyltransferases), chemical synthesis, isolation of natural products, physicochemical studies, reactions and their mechanisms, the study of structures and stereochemistry, and technological aspects. Papers on polysaccharides should have a "molecular" component; that is a paper on new or modified polysaccharides should include structural information and characterization in addition to the usual studies of rheological properties and the like. A paper on a new, naturally occurring polysaccharide should include structural information, defining monosaccharide components and linkage sequence. Papers devoted wholly or partly to X-ray crystallographic studies, or to computational aspects (molecular mechanics or molecular orbital calculations, simulations via molecular dynamics), will be considered if they meet certain criteria. For computational papers the requirements are that the methods used be specified in sufficient detail to permit replication of the results, and that the conclusions be shown to have relevance to experimental observations - the authors'' own data or data from the literature. Specific directions for the presentation of X-ray data are given below under Results and "discussion".