Global analysis of glycoprotein chaperones reveals a role for intracellular lectins in regulating ER stress and unfolded protein response

Introduction:: Introduction: Glycosylation is a complex post-translational modification that is ubiquitously observed on almost all cell surface glycoproteins and protein therapeutics. Besides having biological and regulatory functions, glycans also play a critical role in protein folding due to molecular interactions with intracellular glycan binding proteins or lectins, which act as glycoprotein-folding chaperones. Such interactions commonly occur in the cellular endoplasmic reticulum (ER), with misfolded proteins being removed via the endoplasmic-reticulum associated protein degradation (ERAD) pathway (Fig. 1A). Besides the above, our studies demonstrate a critical role for these intracellular lectins during the processing of viral glycoproteins like the SARS-COV-2 Spike protein [1]. At the current time, while selected biochemical investigations have been performed to understand the role of these glycoprotein chaperones, a global systems level dissection of the underlying pathways has not been conducted. In this paper, we performed systems level analysis using a panel of cells lacking selected glycoprotein folding chaperones using both high-throughput glycomics and proteomics profiling. Our studies reveal a critical role for ER chaperones in regulating cell-stress and unfolded protein response (UPR).


Materials and Methods:: Materials and Methods: We developed four isogenic knockout cell lines on the HEK239T (Human embryonic kidney) background that lack either the intracellular-chaperone Calnexin (CANX), Calreticulin (CALR), Mannosyl-oligosaccharide glucosidase (MOGS), or a dual knockout lacking both CANX and CALR. Western blotting and next-generation sequencing (NGS) was performed to validate the knockout lines, with respect to the wild-type control. Glycomics profiling was performed using mass spectrometry using nano-LC separation followed by LTQ-Fusion Orbitrap analysis. Additionally, each of the cells were stained with a panel of 23 fluorescent carbohydrate binding lectins to quantify cell-surface glycan changes. Quantitative LC-MS proteomics profiling was performed using a recently developed IonStart proteomics workflow, also on the LTQ-Fusion Orbitrap instrument [2]. This enabled label-free quantification of individual proteins in complex mixtures (Fig. 1B). Pathway enrichment analysis on the systems-level data were used to generate hypothesis. These were tested using western blot analysis and using fluorescence lentiviral reporters (pLVx-ATF4 and pLVx-XBP) in an Incucyte SX-5 instrument to monitor the time-course of ER stress response [3].


Results, Conclusions, and Discussions:: Results: CRISPR-Cas9 technology was used to develop a panel of cells lacking MOGS-, CANX-, CALR-, and a CANX-/CALR- dual knockout. These knockouts were validated using western blot analysis and genomic DNA sequencing. When profiled for cell surface glycans using a panel of lectins, most lectin binding levels were similar to wild-type, except for ~50% change in binding of VVA, RCA and AAL to cells lacking either CALR or the dual CANX-/CALR-KOs. Thus, chaperone related pathways only have a minor role in regulating cell surface carbohydrate expression, and glycoproteins likely bypass this quality control step. Proteomics profiling revealed the expression of ~4000 proteins in each cell-type, with several proteins being differentially expression. Pathway enrichment analysis further predicted that many of these differentially expressed proteins may regulate the UPR (unfolded protein response) with the highest effect being expected for CALR-KO (FDR=0), followed by CANX-KO (FDR=0.029) and MOGS-KO (FDR=0.035). To test this hypothesis, we measured the cellular expression of the CHOP protein whose expression is induced upon ER stress. We also stably expressed two fluorescence reporters of unfolded protein response (UPR), pLVx-ATF4 and pLVx-XBP1 in each of the knockouts. Here, addition of thapsigargin, a potent SERCA inhibitor, caused a 2500-fold increase in pLVx-ATF4 reporter red fluorescence after 24 hours (Fig. 1C) along with CHOP expression increase (Fig. 1D). Signal increase from the pLVx-XBP1 reporter was noted, albeit at lower levels. Similar to this, cells lacking the chaperone proteins (MOGS, CALR, CANX) also displayed increased pLVx-ATF4 reporter signal and CHOP expression (Fig. 1 C-D). The study reveals that the intracellular lectin-chaperones regulate ER stress.

Conclusions: The systems-level study of knockout cells shows that while the lectin chaperones have only a minor effect on cell surface glycan expression, they profoundly impact UPR and ER stress response. Among the players, Calreticulin, in particular, exerted a substantial effect. Overall, the study provides a basic science understanding of cell function from a systems perspective, that has implications for our understanding of normal physiology and viral diseases where chaperone proteins play key roles. This understanding is also necessary for the development of protein biotherapeutics as they are commonly glycosylated.


Acknowledgements (Optional): : Supported by grants from the National Institutes of Health

References (Optional): : 1. Yang et al., ‘Role for N-glycans and calnexin-calreticulin chaperones in SARS-CoV-2 Spike maturation and viral infectivity ’. Science Advances, 8:eabq8678, 2022.
2. Shen et al., ‘IonStar enables high-precision, low-missing-data proteomics quantification in large biological cohorts’. PNAS USA, 115:E4767-76, 2018.
3. Nougarède, A., et al., ‘Improved IRE1 and PERK Pathway Sensors for Multiplex Endoplasmic Reticulum Stress Assay Reveal Stress Response to Nuclear Dyes Used for Image Segmentation.’ Assay Drug Dev Technol, 16: p. 350-60, 2018.