PhD Scientific Days 2025

Poster Session III. - H: Pharmaceutical Sciences and Health Technologies

Proteomic, N-glycoproteomic and Chondroitin-sulfate Profiling of A549 and BEAS-2B Cell Line-derived Small Extracellular Vesicles

Name of the presenter

Balbisi Mirjam

Institute/workplace of the presenter

MTA-HUN-REN TTK Lendület (Momentum) Glycan Biomarker Research Group, HUN-REN Research Centre for Natural Sciences

Authors

Mirjam Balbisi¹, Tamás Langó², Virág Nikolett Horváth¹, Domonkos Pál¹, Gitta Schlosser³, Gábor Kecskeméti⁴, Zoltán Szabó⁴, Lilla Turiák¹

1: MTA-HUN-REN TTK Lendület (Momentum) Glycan Biomarker Research Group, HUN-REN Research Centre for Natural Sciences
2: Institute of Molecular Life Sciences, HUN-REN Research Centre for Natural Sciences
3: MTA-ELTE Lendület (Momentum) Ion Mobility Mass Spectrometry Research Group, ELTE Eötvös Loránd University
4: Department of Medical Chemistry, Albert Szent-Györgyi Medical School, University of Szeged

Text of the abstract

Introduction: Small extracellular vesicles (sEVs) are <200 nm particles secreted by cells that mediate intercellular communication and carry diverse biomolecules, including proteins. While protein content has been widely studied, post-translational modifications such as glycosylation remain less explored. Among glycosylated proteins, proteoglycans - composed of a core protein and glycosaminoglycan (GAG) chains like chondroitin sulfate (CS) - form a distinct class.
Aims: This study aimed to compare the proteomic, N-glycoproteomic, and CS GAG profiles of sEVs from A549 lung adenocarcinoma and BEAS-2B non-tumorigenic epithelial cell lines.
Methods: A549 and BEAS-2B cells were cultured for 3 days in F12 and BEGM media, respectively. sEVs were isolated by size-exclusion chromatography (n=6 per group), followed by solvent exchange and lysis. One part of each sample underwent tryptic digestion and N-glycopeptide enrichment via acetone precipitation. The other part was treated with chondroitinase ABC to release CS disaccharides. Peptides, N-glycopeptides, and CS disaccharides were analyzed by nanoUHPLC-MS(/MS).
Results: Of 945 proteins analyzed, 408 showed differential expression, including five CS proteoglycan core proteins. Versican and testican-1 were upregulated in A549, while aggrecan, syndecan-4 and CSPG4 were downregulated. Among the 301 N-glycoforms tested, 176 differed significantly between the sample groups. Dysregulated glycoforms included those of EV markers (e.g., galectin-3-binding protein) and proteoglycans (e.g., versican). CS disaccharide content was 3.4-fold higher in A549 sEVs, and the 6S/4S monosulfated disaccharide ratio decreased, suggesting altered sulfotransferase activity. Principal component analysis clearly separated tumor and non-tumor sEVs in all datasets, emphasizing that tumor and non-tumor sEVs not only differ at the protein level, but also have substantially different glycosylation patterns.
Conclusion: We demonstrated that A549 and BEAS-2B sEVs show distinct proteomic, N-glycoproteomic, and CS profiles, which calls for further research to understand the role of EV glycosylation in cancer.
Funding: Lendület (Momentum) Program of the Hungarian Academy of Sciences, Semmelweis 250+ Excellence PhD Scholarship