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Rational design of N-glycosyltransferases from Aggregatibacter aphrophilus to synthesize Gal-modified glycoconjugates
Yue Yu1, Jiangyu Yang2, Yongqi Liu2
1State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, Qingdao 266237, China; College of Life Science, Shandong Normal University, Jinan 250399, China.
Abstract:
Hepatocellular carcinoma (HCC) significantly impacts human health due to its tendency to develop multi-drug resistance. Regarding the treatment of HCC, galactosylated conjugates can target drug delivery. Bacterial N-glycosyltransferases (NGTs) catalyze glucosylation or galactosylation of the Asn residue motif Asn0-X+1-Ser/Thr+2 (X ≠ P). However, the limited utilization of the sugar donor UDP-Gal by NGTs severely restricts the potential applications in the development of glycol-drugs and vaccines. In this study, we rationally designed an NGT derived from Aggregatibacter aphrophilus and generated a mutant called AaFQ, which has a catalytic activity 18.8 times greater than that of wild-type towards UDP-Gal. AaFQ could rapidly synthesize Gal-modified peptides, which have been demonstrated to possess high targeting activity for HCC cells HepG2. Moreover, AaFQ exhibited a marked affinity for sugar donors with an equatorial bond-linked hydroxyl group at the C-2 position in monosaccharides. The mutant led to a significantly enhanced binding of K440 to the hydroxyl group at the C-2 position, which might be a major factor contributing to the significantly increased ability to utilize UDP-Gal. This work provides a crucial enzymatic synthetic tool for the targeted therapy of HCC and also offers a new perspective for the engineering modification and application of NGTs.
Insights
Researchers engineered a bacterial enzyme (NGT) mutant, AaFQ, to efficiently utilize UDP-Gal for synthesizing galactosylated peptides. This breakthrough offers a novel enzymatic tool for targeted hepatocellular carcinoma (HCC) therapy.
Area of Science:
- Biochemistry
- Enzymology
- Glycobiology
Background:
- Hepatocellular carcinoma (HCC) presents a significant health challenge, often developing multi-drug resistance.
- Galactosylated conjugates show promise for targeted drug delivery in HCC treatment.
- Bacterial N-glycosyltransferases (NGTs) modify proteins with sugars, but their limited use of UDP-Gal restricts applications.
Purpose of the Study:
- To engineer a more effective NGT for UDP-Gal utilization.
- To develop a novel enzymatic tool for synthesizing galactosylated compounds for HCC therapy.
Main Methods:
- Rational design and engineering of an NGT from Aggregatibacter aphrophilus.
- Characterization of the mutant enzyme's (AaFQ) catalytic activity and substrate specificity.
- Synthesis and evaluation of Gal-modified peptides for HCC cell targeting.
Main Results:
- The engineered NGT mutant, AaFQ, demonstrated an 18.8-fold increase in catalytic activity towards UDP-Gal compared to the wild-type.
- AaFQ efficiently synthesized Gal-modified peptides with high targeting activity for HepG2 HCC cells.
- AaFQ showed enhanced affinity for sugar donors with specific hydroxyl group configurations, particularly at the C-2 position.
Conclusions:
- Engineered NGT AaFQ provides a powerful enzymatic tool for synthesizing galactosylated peptides, crucial for targeted HCC therapy.
- The study offers insights into NGT engineering for improved UDP-Gal utilization, expanding applications in glycol-drug and vaccine development.
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