In-Depth Analysis of the N-Glycome of Colorectal Cancer Cell Lines

Abstract

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer deaths worldwide. A well-known hallmark of cancer is altered glycosylation. Analyzing the N-glycosylation of CRC cell lines may provide potential therapeutic or diagnostic targets. In this study, an in-depth N-glycomic analysis of 25 CRC cell lines was conducted using porous graphitized carbon nano-liquid chromatography coupled to electrospray ionization mass spectrometry. This method allows for the separation of isomers and performs structural characterization, revealing profound N-glycomic diversity among the studied CRC cell lines with the elucidation of a number of 139 N-glycans. A high degree of similarity between the two N-glycan datasets measured on the two different platforms (porous graphitized carbon nano-liquid chromatography electrospray ionization tandem mass spectrometry (PGC-nano-LC-ESI-MS) and matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS)) was discovered. Furthermore, we studied the associations between glycosylation features, glycosyltransferases (GTs), and transcription factors (TFs). While no significant correlations between the glycosylation features and GTs were found, the association between TF CDX1 and (s)Le antigen expression and relevant GTs FUT3/6 suggests that CDX1 contributes to the expression of the (s)Le antigen through the regulation of FUT3/6. Our study provides a comprehensive characterization of the N-glycome of CRC cell lines, which may contribute to the future discovery of novel glyco-biomarkers of CRC.

(Sialyl)Lewis Antigen Expression on Glycosphingolipids, N-, and O-Glycans in Colorectal Cancer Cell Lines is Linked to a Colon-Like Differentiation Program

“…Our recent studies provided in-depth comprehensive glycomic profiling ( N -, O -glycans, and GSL glycans) of CRC cell lines ( 21 , 22 , 23 ). Glycomics data for CRC cell lines were retrieved from GlycoPOST ( 29 ): GPST000239 (GSL glycans) ( 21 ), GPST000035 ( O -glycan) ( 23 ), and GPST000302 ( N -glycan) ( 22 ). An overview of the included CRC cell lines is provided in supplemental Table S1 .…”

“…We combined the N -, O -glycomic, and GSL glycomic data which all have been measured by porous graphitized carbon nanoliquid chromatography-MS/MS for the same set of CRC cell lines ( 21 , 22 , 23 ). More information about glycomic data can be found in supplemental Tables S2–S5 .…”

“…On the basis of analysis of mutations and RNA and protein expression, CRC cell lines have been classified into two groups, which are colon-like cell lines with expression of gastrointestinal differentiation markers and undifferentiated cell lines characterized by upregulation of genes linked to epithelial–mesenchymal transition (EMT) ( 20 ). Mass spectrometry (MS) is a powerful tool to perform in-depth characterization of glycomic profiles and has been widely used to study the role of glycosylation in cancer, including CRC ( 21 , 22 , 23 ). Just recently, we examined the protein N - and O -glycosylation ( 22 , 23 ) as well as glycosphingolipids (GSLs) glycosylation profiles ( 21 ) of CRC cell lines, using porous graphitized carbon nanoliquid chromatography-MS/MS.…”

“…Mass spectrometry (MS) is a powerful tool to perform in-depth characterization of glycomic profiles and has been widely used to study the role of glycosylation in cancer, including CRC ( 21 , 22 , 23 ). Just recently, we examined the protein N - and O -glycosylation ( 22 , 23 ) as well as glycosphingolipids (GSLs) glycosylation profiles ( 21 ) of CRC cell lines, using porous graphitized carbon nanoliquid chromatography-MS/MS. Striking differences were found between colon-like and undifferentiated cell lines for all three glycan classes.…”

“…With regard to O -glycosylation, colon-like cell lines showed high expression of I-branched and sLe A/X epitope-carrying glycans, while undifferentiated cell lines were characterized by high prevalence of truncated α2-6 core sialylated glycans, and some undifferentiated cell lines expressed high abundances of glycans with blood group antigens (A, B, and H) ( 23 ). As for N -glycans, colon-like cell lines presented a high expression of sulfation, (s)Le A/X , Le B/Y , antenna fucosylation, oligomannosidic, and hybrid-type N- glycans, while undifferentiated cell lines highly expressed phosphorylation, bisection, and α2-3 sialylation, as well as paucimannosidic N -glycans and N -glycans carrying (fucosylated) LacdiNAc (GalNAcβ1-4GlcNAc) ( 22 ). When it comes to GSL glycans, high expression of (s)Le A/X and Le B/Y antigens was found for colon-like cell lines while undifferentiated cell lines showed higher abundances of glycans with all blood group antigens (A, B, and H) ( 21 ).…”