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High-Throughput Mass Spectrometry Analysis of N-Glycans and Protein Markers after FUT8 Knockdown in the Syngeneic
Rubén López-Cortés1, Laura Muinelo-Romay2, Almudena Fernández-Briera3
1Doctoral Program in Methods and Applications in Life Sciences, Faculty of Biology, Universidade de Vigo, Campus Lagoas-Marcosende, 36310 Vigo, Pontevedra (Galicia), Spain.
Abstract:
Disruption of the glycosylation machinery is a common feature in many types of cancer, and colorectal cancer (CRC) is no exception. Core fucosylation is mediated by the enzyme fucosyltransferase 8 (FucT-8), which catalyzes the addition of α1,6-l-fucose to the innermost GlcNAc residue of N-glycans. We and others have documented the involvement of FucT-8 and core-fucosylated proteins in CRC progression, in which we addressed core fucosylation in the syngeneic CRC model formed by SW480 and SW620 tumor cell lines from the perspective of alterations in their N-glycosylation profile and protein expression as an effect of the knockdown of the FUT8 gene that encodes FucT-8. Using label-free, semiquantitative mass spectrometry (MS) analysis, we found noticeable differences in N-glycosylation patterns in FUT8-knockdown cells, affecting core fucosylation and sialylation, the Hex/HexNAc ratio, and antennarity. Furthermore, stable isotopic labeling of amino acids in cell culture (SILAC)-based proteomic screening detected the alteration of species involved in protein folding, endoplasmic reticulum (ER) and Golgi post-translational stabilization, epithelial polarity, and cellular response to damage and therapy. This data is available via ProteomeXchange with identifier PXD050012. Overall, the results obtained merit further investigation to validate their feasibility as biomarkers of progression and malignization in CRC, as well as their potential usefulness in clinical practice.
Insights
Altering core fucosylation in colorectal cancer (CRC) cells by reducing FucT-8 enzyme activity changed N-glycosylation patterns and protein expression. These changes may offer new biomarkers for CRC progression and clinical use.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Disrupted glycosylation is common in cancer, including colorectal cancer (CRC).
- Core fucosylation, mediated by fucosyltransferase 8 (FucT-8), plays a role in CRC progression.
- Previous studies highlight FucT-8 and core-fucosylated proteins in CRC development.
Purpose of the Study:
- To investigate alterations in N-glycosylation and protein expression in CRC cells with reduced FucT-8 activity.
- To explore the potential of these changes as biomarkers for CRC progression and malignization.
Main Methods:
- Utilized a syngeneic CRC model (SW480 and SW620 cell lines) with FUT8 gene knockdown.
- Employed label-free, semiquantitative mass spectrometry (MS) for N-glycosylation profiling.
- Conducted stable isotopic labeling of amino acids in cell culture (SILAC)-based proteomic screening.
Main Results:
- FUT8 knockdown significantly altered N-glycosylation patterns, including core fucosylation, sialylation, Hex/HexNAc ratio, and antennarity.
- Proteomic screening revealed changes in proteins related to protein folding, ER/Golgi stabilization, epithelial polarity, and cellular stress response.
- Data is publicly available via ProteomeXchange (identifier PXD050012).
Conclusions:
- Reduced FucT-8 activity profoundly impacts the N-glycosylation profile and proteome of CRC cells.
- Identified alterations warrant further investigation as potential biomarkers for CRC progression and clinical applications.
- Findings suggest a role for core fucosylation modulation in CRC pathobiology.
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