An Integrated Mass Spectrometry-Based Glycomics-Driven Glycoproteomics Analytical Platform to Functionally Characterize Glycosylation Inhibitors

Abstract

Cancer progression is linked to aberrant protein glycosylation due to the overexpression of several glycosylation enzymes. These enzymes are underexploited as potential anticancer drug targets and the development of rapid-screening methods and identification of glycosylation inhibitors are highly sought. An integrated bioinformatics and mass spectrometry-based glycomics-driven glycoproteomics analysis pipeline was performed to identify an N-glycan inhibitor against lung cancer cells. Combined network pharmacology and in silico screening approaches were used to identify a potential inhibitor, pictilisib, against several glycosylation-related proteins, such as Alpha1-6FucT, GlcNAcT-V, and Alpha2,6-ST-I. A glycomics assay of lung cancer cells treated with pictilisib showed a significant reduction in the fucosylation and sialylation of N-glycans, with an increase in high mannose-type glycans. Proteomics analysis and in vitro assays also showed significant upregulation of the proteins involved in apoptosis and cell adhesion, and the downregulation of proteins involved in cell cycle regulation, mRNA processing, and protein translation. Site-specific glycoproteomics analysis further showed that glycoproteins with reduced fucosylation and sialylation were involved in apoptosis, cell adhesion, DNA damage repair, and chemical response processes. To determine how the alterations in N-glycosylation impact glycoprotein dynamics, modeling of changes in glycan interactions of the ITGA5–ITGB1 (Integrin alpha 5-Integrin beta-1) complex revealed specific glycosites at the interface of these proteins that, when highly fucosylated and sialylated, such as in untreated A549 cells, form greater hydrogen bonding interactions compared to the high mannose-types in pictilisib-treated A549 cells. This study highlights the use of mass spectrometry to identify a potential glycosylation inhibitor and assessment of its impact on cell surface glycoprotein abundance and protein–protein interaction.

High-Throughput Mass Spectrometry Analysis of N-Glycans and Protein Markers after FUT8 Knockdown in the Syngeneic SW480/SW620 Colorectal Cancer Cell Model

“…In this sense, it has recently been described that deregulated fucosylation can affect many intracellular proteins, including the ribosomal protein S3, which participates in nuclear DNA repair . Indeed, the fucosylation and sialylation inhibitor pictilisib affected the DNA repair capacity of lung cancer cells . The histone H2AFJ (cluster e) is usually expressed in luminal epithelial cells, although its role is still poorly understood .…”

Comparative proteomics reveals anticancer compounds from Lansium domesticum against NSCLC cells target mitochondrial processes

“…Comparative LC‐MS/MS proteomics was done as described previously 11 . A549 cells were grown in T75 flasks until it reached approximately 80% confluency.…”

Integrating Computational Methods in Network Pharmacology and In Silico Screening to Uncover Multi-targeting Phytochemicals against Aberrant Protein Glycosylation in Lung Cancer

“…Network pharmacology was performed to identify potential interactions of phytochemicals with glycosylation-related proteins. 32 Protein–drug interactions of the 161 phytochemicals with 260 glycosylation-related proteins were predicted using BindingDB ( ), Chemical Toxicogenomics Database ( ), 33 DGIdb ( ), 34 STITCH ( ), 35 and SWISS Target Prediction ( ). The interactions were mapped out using Cytoscape 3.8.2.…”

“…Targeting glycoproteins is an underexploited strategy for the development of cancer therapeutics. Carbohydrate-active enzymes such as glycosidases and glycosyltransferases are attractive drug targets due to their involvement in the biosynthesis of glycan structures. , Relatively few studies have investigated potential small molecule inhibitors with drug-like properties against these enzymes. − These include plant alkaloids that inhibit glycosidases by inducing inhibition of trimming reactions after the attachment of Glc 3 Man 9 GlcNAc 2 oligosaccharide. An example of a plant alkaloid is swainsonine from an Astralagus species known as locoweeds.…”