Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity

Area of Science:

• Microbiology and Molecular Biology
• Biochemistry and Glycobiology
• Synthetic Biology and Protein Engineering

Background:

• The Campylobacter jejuni protein glycosylation locus (pgl) is the model system for bacterial asparagine-linked (N-linked) glycosylation.
• Transferring this machinery to Escherichia coli allows for detailed mechanistic studies in a well-characterized host.
• The oligosaccharyltransferase PglB enzyme exhibits specificity for glycan acceptor sites with a negative charge at the -2 position.

Purpose of the Study:

• To identify sequence determinants within PglB that govern its acceptor site specificity.
• To develop a high-throughput screening method for analyzing N-linked glycosylation phenotypes.
• To engineer PglB variants with altered substrate specificity for novel biocatalytic applications.

Main Methods:

• Development of the glycosylation of secreted N-linked acceptor proteins (glycoSNAP) assay in E. coli.
• High-throughput screening of PglB variants using the glycoSNAP assay to identify altered substrate specificity.
• Characterization of PglB variants capable of glycosylating noncanonical acceptor sequences.

Main Results:

• The glycoSNAP assay successfully facilitated high-throughput screening of bacterial N-linked glycosylation phenotypes.
• Several C. jejuni PglB variants were isolated that exhibit altered specificity, glycosylating noncanonical acceptor sites.
• One engineered PglB variant demonstrated the ability to glycosylate a site within a eukaryotic N-glycoprotein.

Conclusions:

• The glycoSNAP assay is a powerful tool for investigating N-linked glycosylation mechanisms and engineering biocatalysts.
• Sequence determinants in PglB can be modified to alter its acceptor site specificity.
• This work enables the development of designer N-linked glycosylation systems for diverse biotechnological applications.