Hanee Kim
New York University
Engineering Bifunctional Enzymes for the Synthesis of Molecular Probes to Elucidate Microbial Glycan Recognition
Pathogenic bacteria utilize "rare" deoxysugars, such as L-colitose (L-Col), to construct surface glycans that mimic host structures and evade immune detection. Characterizing host carbohydrate-binding proteins (CBPs) that recognize these microbial sugars has been hindered by difficulties in synthesizing nucleoside diphosphate (NDP)-sugar donors and a lack of defined molecular probes. To address this, we developed a chemoenzymatic strategy to synthesize diverse activated sugars, including GDP-β-L-Col, by exploiting and engineering the promiscuous bifunctional enzyme Fkp. Using these donors, we leveraged a human fucosyltransferase FUT2 to install terminal L -sugars onto a glycoprotein precursor (BSA-LNT), generating a unique panel of precisely defined glycoconjugate probes. Screening human serum with these probes revealed previously uncharacterized anti-Col IgA antibodies. Unlike prior studies using heterogeneous glycolipids that suffer from cross-reactivity, our structurally defined ligands confirmed a highly specific human immune response to single rare sugars. This work overcomes critical bottlenecks in NDP-sugar availability and provides a robust molecular blueprint for elucidating host-pathogen glycan recognition, facilitating the discovery of novel CBPs and the design of targeted therapeutics.
Pathogenic bacteria utilize "rare" deoxysugars, such as L-colitose (L-Col), to construct surface glycans that mimic host structures and evade immune detection. Characterizing host carbohydrate-binding proteins (CBPs) that recognize these microbial sugars has been hindered by difficulties in synthesizing nucleoside diphosphate (NDP)-sugar donors and a lack of defined molecular probes. To address this, we developed a chemoenzymatic strategy to synthesize diverse activated sugars, including GDP-β-L-Col, by exploiting and engineering the promiscuous bifunctional enzyme Fkp. Using these donors, we leveraged a human fucosyltransferase FUT2 to install terminal L -sugars onto a glycoprotein precursor (BSA-LNT), generating a unique panel of precisely defined glycoconjugate probes. Screening human serum with these probes revealed previously uncharacterized anti-Col IgA antibodies. Unlike prior studies using heterogeneous glycolipids that suffer from cross-reactivity, our structurally defined ligands confirmed a highly specific human immune response to single rare sugars. This work overcomes critical bottlenecks in NDP-sugar availability and provides a robust molecular blueprint for elucidating host-pathogen glycan recognition, facilitating the discovery of novel CBPs and the design of targeted therapeutics.