Impact of Glycosylation on the Comparability of the Higher-Order Structures in Idursulfase by Hydrogen–Deuterium Exchange Mass Spectrometry

Abstract

Characterization of the higher-order structures in idursulfase (iduronate-2-sulfatase, I2S) has been accomplished through the use of hydrogen−deuterium exchange mass spectrometry (HDX-MS). The method has over 97% sequence coverage, including seven of the eight glycosylation sites, and has been used to study the impact of glycosylation on backbone proton exchange. In addition, the method adapted a well-used biophysical spectra comparison method (similarity scoring) to define quantitative acceptance criteria for analytical comparability of different batches of drug substance as well as samples with modulated glycans. Differences in the HDX profile were induced by enzymatic removal of terminal sialic and phosphate groups on negatively charged glycans. These differences were mapped to the crystal structure and demonstrated synergistic HDX changes focused around the N221 and N255 glycosylation sites, which contain mannose-6-phosphate motifs important for I2S uptake into cells.

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

“…In some systems, HDX-MS showed mixed effects with some areas becoming stabilized and other regions showing increased accessibility upon glycan removal. 548 − 551 In other glycoproteins, the only observed changes were increased dynamics through the protein upon glycan removal. 541 , 552 Some studies observed only very minor changes in HDX kinetics despite known effects on overall thermal stability upon glycan removal.…”

“…Even with limited sequence coverage, HDX-MS has been a reliable tool for mapping binding interfaces and tracking structural changes upon protein–protein and protein–ligand interactions involving serum glycoproteins ,,− and receptors. ,,, Several studies have also used HDX-MS to assess the influence of glycosylation itself on the overall structure of serum glycoproteins with some interesting variability that reveals a complex relationship between a protein’s glycosylation state and its conformational dynamics. In some systems, HDX-MS showed mixed effects with some areas becoming stabilized and other regions showing increased accessibility upon glycan removal. − In other glycoproteins, the only observed changes were increased dynamics through the protein upon glycan removal. , Some studies observed only very minor changes in HDX kinetics despite known effects on overall thermal stability upon glycan removal . The available studies have thus far revealed a complex and very context-dependent relationship between glycosylation and structural dynamics.…”

Subsecond Time-Resolved Mass Spectrometry in Dynamic Structural Biology

“…HDX-MS has an important role in understanding conformation/dynamics changes driven by post-translational modification. , To date, there are two examples of subsecond time-resolved HDX-MS being used specifically to examine changes in conformational dynamics due to post-translational modification. In the first, Zhu, Wilson, and co-workers examined the impact of phosphorylation on the binding specificity of the intrinsically disordered N-terminus of the apoptosis modulator protein (and key cancer target) p53 .…”

HDX-MS: An Analytical Tool to Capture Protein Motion in Action

“…HDX-MS can also be used to detect how different glycosylations can impact the overall structure of a protein. One recent study was able to distinguish the subtle changes that occur in the idursulfase enzyme when glycosylation sites were modulated [ 71 ]. This can have significant applications in the pharmaceutical industry, as monoclonal antibodies produced by different manufacturing processes often have differing glycosylation patterns.…”