System-wide functional glycoproteomics approaches

Introduction

Protein glycosylation is a highly diverse post-translational modification, playing key roles in the regulation of cellular processes such as cell signaling, adhesion and cell-cell interactions. A major obstacle in our understanding of the role of glycosylation in health and disease, along with the molecular mechanisms underlying the modulation of glycoprotein functions, has been the lack of systematic methods for assessing the impact of glycosylation alteration on protein function.

Methods

We recently developed a glycoproteomics workflow enabling in-depth quantification of glycosylation. Building on this advancement, we now aim to study glycoforms within a functional context. First, we have devised methods to differentiate surface-exposed, mature glycoforms from intermediate glycosylated proteoforms by treating intact HEK293 cells with either proteinase K or PNGase F. To improve our understanding of how glycosylation alteration affects protein states, we developed techniques to systematically measure the effect of glycosylation alteration on biophysical properties of proteins in vivo, such as thermal stability or solubility. Such approaches have recently been used to assess phosphorylation sites functionality, and rely on the fact that changes in protein states (interaction, localization,...) can be associated with changes in protein biophysical properties.

Results

We could successfully differentiate mature, surface-exposed glycoforms from intermediate intracellular species, addressing a longstanding question in the field. We demonstrate that high-mannose glycoforms are in general less surface exposed, although this tendency is site-dependent and related to structural features. Conversely, most of the fucosylated/sialylated glycoforms are, at least partially, surface-exposed. We observed good correlation between the enzymatic treatments, both at the glycopeptide level and glycan compositions level, and could identify compositions predominantly surface-exposed. Next, we measured the solubility and thermal stability of thousands of glycoforms in the mouse brain. Our results illustrate significant variability in biophysical behaviors among different glycoproteoforms, allowing us to establish connections between glycan compositions or structural features of glycoproteins and their thermal stability and solubility.

Conclusions

Here we present a method that offers a system-wide, site-specific view of mature and immature glycans in a human cell line. We believe this approach has the potential to enhance understanding of the biological functions of glycosylation and contribute to the development of therapies by informing on which glycosylation events to target. Our biophysical glycoproteomics approaches constitute an initial step towards systematically understanding the impact of glycoform modulation on protein function, providing a framework for future research in this area.