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Using High-Throughput Experiments To Screen N-Glycosyltransferases with Altered Specificities
Liang Lin1,2,3, Weston Kightlinger2,4, Katherine F Warfel2,4
1Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States.
ACS Synthetic Biology
|March 25, 2024
Summary
Researchers developed a new method to engineer glycosyltransferases for precise protein modification. This advances synthetic glycosylation for protein therapeutics, improving their efficacy and expanding glycoengineering applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Protein glycosylation is crucial for therapeutic protein activity and efficacy.
- Current methods for site-specific protein glycosylation are limited, especially in vitro and in bacteria.
- Engineering glycosyltransferases is key to overcoming these limitations.
Purpose of the Study:
- To develop an efficient and systematic method for screening glycosyltransferases.
- To engineer a prokaryotic N-glycosyltransferase (NGT) for broad substrate specificity.
- To expand the scope of glycoengineering for protein therapeutics.
Main Methods:
- Utilized cell-free protein synthesis and mass spectrometry of self-assembled monolayers.
- Screened 26 pools of site-saturated NGT libraries against 1052 unique peptides.
- Characterized 122 NGT mutants under 52,894 reaction conditions.
Main Results:
- Identified key residues determining polypeptide specificity for NGTs.
- Defined a panel of 14 NGTs modifying 93% of canonical eukaryotic glycosylation sequences.
- Developed a panel for noncanonical sequences, including those with non-S/T amino acids at X+2.
- Successfully applied engineered NGTs to enhance glycosylation efficiency in three protein therapeutics.
Conclusions:
- The developed screening method enables efficient engineering of glycosyltransferases.
- The engineered NGT panels significantly broaden substrate specificity for glycoengineering.
- This work advances in vitro and bacterial glycoengineering, with implications for protein therapeutic development.
