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

Linhan Wang ¹, Jichao Wei ², Fang Yuan ³

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.
²Department of Hepatobiliary Surgery, Qingdao Hospital, University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital), Qingdao, 266011, China.
³National 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.
⁴School 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.
⁵Shandong Seprosin Biotechnology Co., Ltd, Jinan, 250032, China.
⁶School of Health and Life Sciences, University of Health and Rehabilitation Sciences, Qingdao, 266113, China.
⁷School of Biological Engineering, Dalian Polytechnic University, Dalian, 116034, China.

⁰School 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.

Carbohydrate Research +

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December 14, 2025

View abstract on PubMed

Summary

High-yield expression of Drosophila melanogaster β1-3-Galactosyltransferase (DmC1GalT1) was achieved. This enzyme is crucial for O-glycan synthesis and engineering, with key residues identified for substrate specificity.

Area Of Science

Biochemistry
Glycobiology
Enzymology

Background

β1-3-Galactosyltransferase from Drosophila melanogaster (DmC1GalT1) is vital for O-glycan synthesis.
DmC1GalT1 has significant potential in glycoengineering applications.

Purpose Of The Study

To achieve high-yield expression and purification of DmC1GalT1 in Escherichia coli.
To characterize the enzyme's substrate specificity and identify key residues for activity and stability.
To engineer DmC1GalT1 variants for synthetic glycobiology.

Main Methods

High-yield expression and purification of DmC1GalT1 in E. coli.
Enzyme activity assays with various glycopeptides and nucleotide sugars.
Site-directed mutagenesis and structural analysis.
Thermal stability predictions using ProStab.

Main Results

Over 5 mg/L of purified DmC1GalT1 was obtained.
The enzyme showed strict donor specificity for UDP-Gal and galactosylated human CD74-derived Tn-glycopeptides.
Mutagenesis studies identified residues N108 and Y325 as critical for donor recognition and specificity.
Engineered variants exhibited altered activity, stability, and substrate utilization profiles.

Conclusions

An efficient platform for DmC1GalT1 expression, characterization, and engineering was established.
Key residues influencing donor recognition were identified, enabling targeted engineering.
This work facilitates the development of tailored glycosyltransferase variants for synthetic glycobiology.

Keywords:

Mutagenesis O-Glycosylation Protein engineering Substrate specificity Thermal stability β1-3-galactosyltransferase