Mucus forms the first line of the host immune system by forming a hydrogel that overlays epithelia. However, mucus disorder is found in many mucosal diseases, such as chronic obstructive pulmonary disease and adenocarcinoma. The polymeric glycoprotein mucin determines the mucus biology, where the mucin-type O-glycans are terminated with an α-GlcNAc residue installed by the glycosyltransferase α-1,4-N-acetylglucosaminyltransferase (A4GNT). Our study seeks to understand the biosynthesis and regulation of α-GlcNAc-terminated glycans on mucosal proteins and how this impacts mucus biology in health and disease.
The catalytic domain of human A4GNT was recombinantly produced in insect cells, and the recombinant protein was then used for enzymology studies. The substrate preference of A4GNT was revealed by in vitro enzyme activity assays using a range of acceptor sugar substrates. The Michaelis-Menten kinetics were then determined for each substrate. Our data showed that A4GNT has a high specificity towards β-linked Gal against the α-linked one. The enzyme displayed a preference towards a β-1,3-linked Gal rather than a β-1,4-linked one, while similar catalytic efficiencies were observed for the linear and branched substrates. To understand how A4GNT functions in vivo, we also performed confocal microscopy studies with A4GNT-transfected HT29 cells. Our immunochemistry data demonstrated that A4GNT localizes at the trans-Golgi compartment towards the end of the O-glycan biosynthesis pathway.
To promote A4GNT crystallisation, seven nanobodies were generated with the WEHI antibody facility. Bio-layer interferometry assays determined that all the nanobodies bound to A4GNT at Kd of 1.2 to 16.2 nM. Only one nanobody abrogated A4GNT enzyme activity, while the other six maintained enzyme activity and stabilized A4GNT to different extents. Protein crystals were successfully generated by co-crystallising the A4GNT-nanobody complex with UDP and the enzyme cofactor MnCl2. The crystallisation conditions are currently under optimisation.
Our enzymology, coupled with imaging studies, showed that α-GlcNAc biosynthesis occurs to a range of sugar substrates while the enzyme preference matches the local substrate concentration. Such a mechanism highlights the importance of enzyme localization and subcellular compartmentation. The ongoing structural biology will provide insight into substrate recognition and catalytic mechanisms at a molecular level.