Analysis of substrate specificity of oligosaccharyltransferases (OSTs) by functional expression of domain-swapped chimeras in yeast.

-Linked protein glycosylation is an essential and highly conserved post-translational modification in eukaryotes. The transfer of a glycan from a lipid-linked oligosaccharide (LLO) donor to the asparagine residue of a nascent polypeptide chain is catalyzed by an oligosaccharyltransferase (OST) in the lumen of the endoplasmic reticulum (ER). encodes three paralogue single-protein OSTs called STT3A, STT3B, and STT3C that can functionally complement the OST, making it an ideal experimental system to study the fundamental properties of OST activity. We characterized the LLO and polypeptide specificity of all three OST isoforms and their chimeric forms in the heterologous expression host where we were able to apply yeast genetic tools and newly developed glycoproteomics methods. We demonstrated that STT3A accepted LLO substrates ranging from ManGlcNAc to ManGlcNAc In contrast, STT3B required more complex precursors ranging from ManGlcNAc to GlcManGlcNAc structures, and STT3C did not display any LLO preference. Sequence differences between the isoforms cluster in three distinct regions. We have swapped the individual regions between different OST proteins and identified region 2 to influence the specificity toward the LLO and region 1 to influence polypeptide substrate specificity. These results provide a basis to further investigate the molecular mechanisms and contribution of single amino acids in OST interaction with its substrates.