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Characterization and engineering of Drosophila melanogaster β1-3-galactosyltransferase for glycoengineering
Linhan Wang1, Jichao Wei2, Fang Yuan3
1School of Biological Engineering, Dalian Polytechnic University, Dalian, 116034, China; School of Health and Life Sciences, University of Health and Rehabilitation Sciences, Qingdao, 266113, China; National Glycoengineering Research Center and Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, and State Key Laboratory of Microbial Technology, Shandong University, Qingdao, 266237, China.
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.
