Characterizing the Effects of N-linked Glycosylations on the IL-33/ST2 Axis through Experiment and Simulation

The role and effects of the IL-33/ST2 pathway in disease is still unknown. The work described here centers upon two important members of the IL-33/ST2 axis: pro-IL-33 and the sST2 receptor. The first portion of the work focuses on the ligand pro-IL-33, an important signaling and transcriptional regulator for the IL-33/ST2 system whose structure and biophysical characteristics have yet to be elucidated due to lack of a high expression system. This need was addressed through the iterative testing and eventual purification of an Eschericia coli based expression system utilizing a His-SUMO construct coupled with autoinduction. The second portion of the work centers upon the role of glycosylations on the sST2 receptor. To address the role of glycosylations in the IL-33/ST2 axis, site specific occupancy was analyzed on the sST2 receptor revealing occupancy of 7 of 8 possible sites present. Subsequent enzymatic based characterization of these sites revealed a mixed population of tri- and tetra- antennary complex glycans present. The effects of these glycans were probed through the implementation of a novel add-on to the computational processing program SMOG2 to enable interpretation and simulation using GROMACS. Analysis of the trajectories revealed that N-linked glycosylations reduce dynamics present in sST2 in a global and local manner; and stabilize the structure of an unstructured loop present from Tyr34 to Val65 of the sST2 receptor when bound to IL-33. As such, this work lays the foundation for further exploration into the function and internal regulation abilities of the IL-33/ST2 axi