Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

Area of Science:

• Biochemistry
• Glycobiology
• Chemical Biology

Background:

• O-Linked α-N-acetylgalactosamine (O-GalNAc) glycans are crucial in the human glycome.
• Studying the 20 initiating polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) isoenzymes is challenging due to their complex interactions.
• Lack of tools hinders correlation of GalNAc-T activity with biological function and substrate specificity.

Purpose of the Study:

• To develop a novel chemical reporter system for studying GalNAc-T activity and substrate specificity.
• To enable precise identification of enzyme-substrate pairs within the GalNAc-T family.
• To provide a tool for investigating GalNAc-T function in complex biological environments.

Main Methods:

• Engineered individual GalNAc-Ts with enlarged active sites ("holes").
• Synthesized 20 unique "bumped" uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs.
• Probed engineered enzymes with analogs to identify orthogonal enzyme-substrate pairs.

Main Results:

• Successfully identified specific GalNAc-T and UDP-GalNAc analog pairs.
• Demonstrated applicability to various GalNAc-T isoenzymes, including those with different substrate preferences (e.g., GalNAc-T1, -T2, -T10).
• Detailed kinetic and specificity analysis confirmed the system's robustness in reporting GalNAc-T activity.

Conclusions:

• The "bump-hole" chemical reporter system accurately reports on GalNAc-T activity.
• This approach facilitates the study of enzyme-substrate specificities for individual GalNAc-Ts.
• The developed system paves the way for future investigations in living systems.